Proteins Hydrolysis

Study of the agglomeration, degree of hydrolysis and peptide pattern

Tatiana Rodrigues Almeida

Thesis to obtain the Master of Science Degree in

Chemical Engineering

Supervisors: Prof. Dr. Marília Mateus, Dr. Fred van de Velde

Examination Committee

Chairperson: Prof. Maria Matilde Marques

Supervisor: Prof. Marília Mateus

Members of the committee: Dr. Filipe de Carvalho

November 2018

ii

Acknowledgments

I would like to thank in first place to my professor Marília Mateus for giving me the opportunity to

know about NIZO, to apply for this internship, and for guiding me throughout all the process.

Second to my supervisors, Dr. Laurice Pouvreau for choosing me to be part of her project, and

Dr. Fred Van de Velde, for all the support and guidance.

To the technicians, Guido Staring, for teaching me a lot about the mass spectrometry software

and for spending a lot of his time with my work, and running my project samples, and a special

acknowledgment, to Jolan de Groot for helping me inside and outside of NIZO, for all the support,

and availability for helping me with anything in the 6 months I was in the Netherlands.

I am very grateful to the friends I made in these 6 months in the Netherlands that kept me sane

and happy, to my front desk colleague Larissa Margalho, and to my housemates Barbara

Tacconelli and Raquel Silvestre.

Also, to my close friends and colleagues that supported me throughout all the course from day 1

until this internship, and without whom I couldn’t finish this degree.

Lastly, to my parents and my whole family for always supporting me no matter what. Obrigada!

iii

Abstract

Enzymatic protein hydrolysis is a step used to allow the increase of protein content in certain food

formulations, that have each day more demand due to an increased preoccupation with a healthy

lifestyle, and food. Consumers and Suppliers are looking for the use of sustainable food products,

and so animal proteins are being replaced by vegetable proteins, and this applies also to

hydrolysates. It is also used to reduce allergenicity, increase solubility and create bioactivity.

Practical examples on vegetable proteins hydrolysis were produced in this project and also

compared to an animal protein (D) hydrolysis in same conditions. The reactions were all done at

same conditions: 50°C; pH=8 and mass E:S=1:100 using the enzyme Alcalase. Also, a reaction

without pH control was studied. The vegetable proteins studied were A, B and C.

During hydrolysis, aggregation of the intact proteins is inducing a lowering of the yield, as result

of protein-protein interactions. Industry is lacking understanding of this phenomenon. For the

reactions where the pH was controlled, the agglomeration can be ordered in the following

increasing order (g wet pellet/g intact protein): D (0 g/g), C isolate (2.6-4.6 g/g), B isolate (3-4.2

g/g), A isolate (3.9-4.6 g/g), C Commercial (4-5.4 g/g), B extract (4-5.6 g/g), A extract (6.6-9.2

g/g).

In terms of Degree of Hydrolysis, the results can also be ordered in a decreasing order: D (16%),

A isolate (14%), C Commercial (13%), B isolate (11%), C isolate (10%), A extract (9%), B extract

(4%).

Also, SDS-PAGE and LCMS analysis were done to try to identify the peptides pattern of the

hydrolysates. The average molecular weight (Da) of the hydrolysates peptides was also

compared: A extract (791 Da); B extract (770 Da); B isolate (708 Da); C isolate (660 Da); D

Commercial (640 Da); C Commercial (621 Da); A Isolate (616 Da).

Keywords: vegetable, proteins, hydrolysis, agglomeration, degree of hydrolysis, hydrolysates

iv

Resumo

A hidrólise enzimática de proteínas é um passo usado para permitir o aumento do teor de

proteína em certas formulações de alimentos, que têm cada dia mais procura devido a um

aumento da preocupação com um estilo de vida e alimentação saudável. Consumidores e

fornecedores procuram o uso de produtos alimentares sustentáveis, e por isso as proteínas

animais estão a ser substituídas por proteínas vegetais, facto que também se se aplica aos

hidrolisados. A hidrólise enzimática de proteínas também é utilizada como forma de reduzir a

alergenicidade e criar bioatividade.

Neste projeto foram produzidos exemplos práticos de hidrólise de proteínas vegetais e também

comparados à hidrólise de proteína animal, D, nas mesmas condições. As reações foram todas

realizadas nas mesmas condições: 50 ° C; pH = 8 e E:S mássico = 1: 100 utilizando a enzima

Alcalase. Além disso, as mesmas reações sem controlo de pH foram estudada. As proteínas

vegetais estudadas foram A, B e C.

Durante a hidrólise, a agregação das proteínas intactas provoca uma diminuição do rendimento,

como resultado de interações proteína-proteína, fenómeno que não é compreendido pela

indústria. Para as reações em que o pH foi controlado, a aglomeração pode ser ordenada pela

seguinte ordem crescente (g de aglomerado molhado/g proteína intacta): D (0 g / g), isolado C

(2,6-4,6 g / g), isolado B (3-4,2 g / g), Um isolado (3,9-4,6 g / g), C comercial (4-5,4 g / g), extrato

B (4-5,6 g / g), extrato A (6,6-9,2 g / g).

Em termos de DH, os resultados também podem ser ordenados em ordem decrescente: D

comercial (16%), isolado A (14%), C comercial (13%), isolado B (11%), isolado C (10%), extrato

A (9%), extrato B (4%).

Além disso, a análise de SDS-PAGE e LCMS foi feita para tentar identificar o padrão peptídico

dos hidrolisados.

Palavras-chave: vegetal, proteínas, hidrólise, aglomeração, grau de hidrólise, hidrolisados

v

Table of Contents

Chapter 1| Introduction ................................................................................................................ xiii

1.1 Context and Aim of the project ....................................................................................... 1

1.2 Literature Overview ......................................................................................................... 1

1.2.1 Enzymatic Hydrolysis .................................................................................................... 2

1.2.2 Degree of Hydrolysis ..................................................................................................... 4

1.2.3 Kinetics of the Protein Hydrolysis ................................................................................. 5

1.2.5 Protein – Protein Interactions during hydrolysis ........................................................... 6

1.2.5.1 Aggregation ............................................................................................................ 6

1.2.5.2 Water Holding on aggregates - Gel Formation ...................................................... 7

1.2.5.3 Glycation and Glycosylation ................................................................................... 8

1.2.6 Resume of some similar studies on enzymatic hydrolysis ............................................ 8

1.2.7 Vegetable Proteins ...................................................................................................... 10

Chapter 2 | Methods .................................................................................................................... 13

2.1 Hydrolysis Reaction parameters and procedure ............................................................... 13

2.1.1 Protein Substrates ....................................................................................................... 13

2.1.2. Substrate Concentration ............................................................................................ 13

2.1.3. Enzyme Activity and Enzyme to Substrate ratio ........................................................ 13

2.1.4 pH and Temperature ................................................................................................... 14

2.2 Determination of Yield ....................................................................................................... 14

2.3 Determination of Degree of Hydrolysis (DH) ..................................................................... 15

2.3.1. pH-stat Method ........................................................................................................... 15

2.3.2 OPA Method ................................................................................................................ 15

2.4 Determination of Peptide Patterns ..................................................................................... 16

2.4.1. SDS-PAGE electrophoresis ...................................................................................... 16

2.4.2. LC-MS: Liquid Chromatography and Mass Spectrometry ......................................... 17

2.4.3. Software Agilent Mass Hunter Qualitative Analysis ................................................... 17

Chapter 3 |Results and Discussion ............................................................................................. 19

3.1. Agglomeration – General View ..................................................................................... 19

3.1.1. Effect of pH control .............................................................................................. 19

3.1.2. Effect of albumins ................................................................................................ 21

vi

3.3. Degree of Hydrolysis – General View ........................................................................... 22

3.3.1. Effect of pH control .............................................................................................. 22

3.3.2. Effect of albumins ................................................................................................ 23

3.3.3. Albumins participation in the hydrolysis .............................................................. 23

3.4. Detailed Analysis of Each protein ................................................................................. 25

3.4.1 Agglomeration ............................................................................................................. 25

3.4.1.1. Protein A .......................................................................................................... 25

3.4.1.2. Protein B .......................................................................................................... 32

3.4.1.3. Protein C .......................................................................................................... 37

3.4.1.4. Protein D .......................................................................................................... 43

3.4.2. Degree of Hydrolysis ........................................................................................... 45

3.4.2.1. Protein A .......................................................................................................... 45

3.4.2.2. B Protein .......................................................................................................... 48

3.4.2.3. C Protein .......................................................................................................... 51

3.4.2.4. D Protein .......................................................................................................... 54

3.4.3. Hydrolysates Protein Pattern ............................................................................... 54

3.4.3.1. Protein A .......................................................................................................... 55

3.4.3.2. Protein B .......................................................................................................... 58

3.4.3.3. Protein C .......................................................................................................... 60

3.4.3.4. Protein D .......................................................................................................... 63

3.4.4. Hydrolysates Peptide Pattern .............................................................................. 64

Peptide Average Molecular Weight .................................................................................. 64

MS identification by protein sequence .............................................................................. 64

3.4.4.1. Protein A .......................................................................................................... 65

3.4.4.2. Protein B .......................................................................................................... 67

3.4.4.3. Protein C .......................................................................................................... 68

3.4.4.4. Protein D .......................................................................................................... 70

Chapter 4 |Conclusion and Future Work ..................................................................................... 73

Bibliography .................................................................................................................................... ii

Appendix ........................................................................................................................................ 5

vii

Figure Index

Figure 1 – Hydrolysis Reaction at different pH [4] ......................................................................... 3

Figure 2- Hydrolysis of proteins with Alcalase under standard hydrolysis conditions. DH is

determined from pH stat. S=8%; A/S= 0.012 AU/g; pH=8; T= 50°C ............................................. 9

Figure 3 – Regression to obtain the molar extinction coefficient in order to calculate the increase

of NH2 during hydrolysis obtaining by measuring the absorbance of L-leucine standards at

different concentrations ............................................................................................................... 15

Figure 4- Average g wet pellet/g intact protein for the vegetable isolate and extract proteins ... 20

Figure 5- g wet pellet/g intact protein for the D protein trials with and without pH control .......... 20

Figure 6- g dry pellet/g intact protein for the A Extract Protein trials ........................................... 21

Figure 7- Degree of hydrolysis of animal protein D with and without pH control with time ......... 22

Figure 8- Average Degree of hydrolysis of vegetable proteins (A, B and C) with and without pH

control with time........................................................................................................................... 22

Figure 9 – Average concentration of 𝑁𝐻2 increase for vegetable proteins Isolates and Extracts

(B and A) ..................................................................................................................................... 23

Figure 10 - Comparison between the degree of hydrolysis of the isolate and the extract of protein

A when adjusting the extract reaction to only account with the globulins as protein content ..... 24

Figure 11- Comparison between the degree of hydrolysis of the isolate and the extract of protein

A when adjusting the extract reaction to only account with the globulins as protein content ..... 24

Figure 12 - g wet pellet / g protein during the hydrolysis of A isolate with and without pH control

..................................................................................................................................................... 25

Figure 13 -g dry pellet/ g protein present in protein A isolate at the different E:S ratios ............ 26

Figure 14- Mass Spectrometry results of A isolate agglomerates after hydrolysis with pH control

at times 0, 0.5, 2 and 6 ................................................................................................................ 27

Figure 15- SDS-PAGE of the agglomerates of protein A Isolate trough time. On the right: mass

compounds found by the Agilent Mass Hunter software matched with the SDS-PAGE bands .. 27

Figure 16 - g wet pellet / g protein during the hydrolysis of A extract with and without pH control

and E:S=1:100 and g dry pellet/ g protein present in A extract hydrolysis’ agglomerates with

E:S=1:100 .................................................................................................................................... 29

Figure 17- Liquid Chromatography results of protein A extract agglomerates after hydrolysis with

pH control at times 0, 0.5, 2 and 6 run on protein gradient ......................................................... 30

Figure 18 - SDS-PAGE electrophoresis from A Extract agglomerates formed in the trial with pH

control Here are represented the pellets formed during hydrolysis at times 0, 0.5, 1, 2, 4 and 6

hours. ........................................................................................................................................... 31

Figure 19 – g pellet/ g protein of B isolate formed during hydrolysis with and without pH control

with an E:S ratio of 1:100 ............................................................................................................ 32

Figure 20 – Liquid Chromatography results of B isolate agglomerates after hydrolysis with pH

control at times 0.5, 2 and 6 run on protein gradient................................................................... 33

Figure 21- SDS-PAGE electrophoresis of the B isolate supernatant hydrolysates time 0 h (A) and

agglomerates from times 0.5, 1, 2, 4 and 6 h (B) for the reaction with pH control ...................... 34

viii

Figure 22 - g pellet/ g protein of B extract formed during hydrolysis with and without pH control

with an E:S ratio of 1:100 ............................................................................................................ 35

Figure 23 - SDS-PAGE electrophoresis of the B Extract pellets for the trial with pH control at times

0, 0.5, 1, 2, 4 and 6 h .................................................................................................................. 35

Figure 24- Mass Spectrometry results of the B extract agglomerates after hydrolysis with pH

control at times 0, 0.5, 2 and 6 run on protein gradient .............................................................. 36

Figure 25- g wet agglomerate pellet/ g protein obtained during the hydrolysis of C Isolate ....... 37

Figure 26- g wet agglomerate pellet/ g protein obtained during the hydrolysis of a C Commercial

..................................................................................................................................................... 37

Figure 27- Agglomerates of the C isolate after 0.5, 1, 2, 4 and 6 hours of hydrolysis reaction .. 37

Figure 28 – Mass Spectrometry results of the C isolate agglomerates after hydrolysis with pH

control at times 0.5, 2 and 6 ........................................................................................................ 38

Figure 29- Comparison of the proteins found in the agglomerates with the intact proteins using

the SDS-PAGE of the supernatants and the agglomerates obtained during the hydrolysis of C

isolate .......................................................................................................................................... 39

Figure 30- Mass Spectrometry results of the C Commercial agglomerates after hydrolysis with pH

control at times 0.5, 2, 4 and 6 .................................................................................................... 41

Figure 31 – SDS-PAGE of the C Commercial supernatants for times 0, 0.5, 1, 2 and 6h (A) and

agglomerates for times 0, 0.5, 1, 2 and 6 h (B) ........................................................................... 42

Figure 32 – Comparison of SDS-PAGE of C Commercial (A) and C isolate (B) agglomerates with

the identified proteins .................................................................................................................. 43

Figure 33- Mass Spectrometry results of the D Commercial agglomerates after hydrolysis with pH

control at times 0.5, 2, 4 and 6 .................................................................................................... 44

Figure 34 – D protein agglomerates obtained at each time of hydrolysis reaction without pH control

..................................................................................................................................................... 44

Figure 35- Comparison of the results in Degree of hydrolysis obtained using the OPA and the pH-

stat methods ................................................................................................................................ 45

Figure 36 - Comparison of NH2 increase of the trials with and without pH control for the A isolate

..................................................................................................................................................... 46

Figure 37 – Degree of hydrolysis of A Isolate and A extract with pH control obtained by the pH

stat method with an E:S ratio of 1:100 ........................................................................................ 46

Figure 38- Comparison of the behaviour of the degree of hydrolysis for both extracts, A and B.

This results of %DH were calculated with the pH-stat method ................................................... 47

Figure 39 - Comparison of NH2 increase of the trials with and without pH control for the A extract

..................................................................................................................................................... 48

Figure 40 - Degree of Hydrolysis of B Extract and Isolate with pH control using an E:S ratio of

1:100 ............................................................................................................................................ 48

Figure 41- Degree of hydrolysis of B Isolate obtained using the pH-stat and the OPA methods 49

Figure 42 – NH2 increase of both trials of B extract, with and without pH control....................... 49

ix

Figure 43 – Comparison between the two methods used in this thesis to calculate the degree of

hydrolysis for the B extract .......................................................................................................... 50

Figure 44 – NH2 groups increase for the hydrolysis reaction of B extract during 6 hours of reaction

..................................................................................................................................................... 50

Figure 45 – Comparison of the degree of hydrolysis curves of the two C isolate proteins studied

in this thesis: the commercial and the self-extracted .................................................................. 51

Figure 46- Comparison between the two methods used in this thesis to calculate the degree of

hydrolysis for the C Commercial isolate ...................................................................................... 52

Figure 47 – Comparison of the NH2 groups increase between the two trials with and without pH

control with time for the C Commercial ....................................................................................... 53

Figure 48 - Comparison between the two methods used in this thesis to calculate the degree of

hydrolysis for the C isolate .......................................................................................................... 53

Figure 49- Comparison of the NH2 groups increase between the two trials with and without pH

control with time for the C isolate ................................................................................................ 54

Figure 50 - Comparison between the two methods used in this thesis to calculate the degree of

hydrolysis for D protein ................................................................................................................ 54

Figure 51 – A :SDS-PAGE of the Aisolate hydrolysates from t=0 to t=6h with (+) and without(-)

pH control .................................................................................................................................... 55

Figure 52 – SDS-PAGE of the B isolate (A) and extract (B) hydrolysates from t= 0h to t=6h with

and without pH control ................................................................................................................. 58

Figure 53 – SDS-electrophorese gels for Commercial (A) and Isolate (B) C hydrolysates with (1)

and without (2) pH control ........................................................................................................... 60

Figure 54 – Sequence Coverage Map of C1 G5, A3. The highlighted green area represents the

polypeptide found in C isolate at t=6h ......................................................................................... 61

Figure 55- Sequence coverage map of the C1 G3 A1b. The highlighted green peptide is the one

referenced in Table 16, compound with 12 771 Da. The green coloured letters represent all the

parts of this polypeptide found in the sample at t=6 h combining all compounds found. ............ 62

Figure 56- Sequence coverage map of the C1 G5 A3. The highlighted green peptide is the one

referenced in Table 16, compound with 10 552 Da. The green coloured letters represent all the

parts of this polypeptide found in the sample at t=6 h combining all compounds found. ............ 62

Figure 57- Sequence coverage map of the C2 α’. The highlighted green peptide is the one

referenced in Table 16, compound with 10 321 Da. The green coloured letters represent all the

parts of this polypeptide found in the sample at t=6 h combining all compounds found. ............ 62

Figure 58 - SDS-PAGE of the D hydrolysates from t= 0h to t=6h with and without pH control .. 63

Figure 59 – Mass Spectrometry results on A isolate hydrolysates after hydrolysis with pH control

at times 0, 0.5, 2 and 6 h. ............................................................................................................ 11

Figure 60 - Mass Spectrometry results on A extract hydrolysates after hydrolysis with pH control

at times 0, 0.5, 2 and 6 h. ............................................................................................................ 11

Figure 61- Mass Spectrometry results on B isolate hydrolysates after hydrolysis with pH control

at times 0, 0.5, 2 and 6 h. ............................................................................................................ 12

x

Figure 62 - Mass Spectrometry results on B extract hydrolysates after hydrolysis with pH control

at times 0, 0.5, 2 and 6 h. ............................................................................................................ 12

Figure 63 - Mass Spectrometry results on C isolate hydrolysates after hydrolysis with pH control

at times 0, 0.5, 2 and 6 h. ............................................................................................................ 13

Figure 64 - Mass Spectrometry results on C commercial hydrolysates after hydrolysis with pH

control at times 0, 0.5, 2 and 6 h. ................................................................................................ 13

Figure 65 - Mass Spectrometry results on D commercial hydrolysates after hydrolysis with pH

control at times 0, 0.5, 2 and 6 h. ................................................................................................ 14

Table Index

xi

Table 1 - Overview of studies that investigated the effect of system condition, i.e. temperature,

pH, enzyme to substrate ratio (E:S) and protein concentration, on the extent of protein hydrolysis

....................................................................................................................................................... 8

Table 2- Protein Substrate concentrations for each of the hydrolysis realized ........................... 13

Table 3- g wet pellet/g intact protein obtained in the different hydrolysis for each protein type . 19

Table 4- Percentage of sequence of each Albumin that was identified at t=6h in the A extract

supernatant .................................................................................................................................. 25

Table 5 – Common compounds identified by Mass Spectrometry analysis for the A isolate

agglomerates at both t= 0 h and t=6 h ........................................................................................ 29

Table 6 – Compounds common to t=0 h and t= 6 h hydrolysates of B isolate ........................... 34

Table 7- Compounds range of molecular weights found in B extract hydrolysates by t=0h and t=6h

..................................................................................................................................................... 36

Table 8 – Proteins found by the MS software in the agglomerates at the beginning of reaction, by

t=0.5 hours .................................................................................................................................. 39

Table 9 – Compound Mass found by the software at t=0.5 h of C commercial agglomerate ..... 41

Table 10 - Compounds found in the t=6h hydrolysate for A isolate and A extract hydrolysis with

pH control with a molecular weight between 19 and 20 kDa ...................................................... 56

Table 11 - Identification of the peptides with a mass of approximately 20 kDa at t=6h of the A

isolate .......................................................................................................................................... 56

Table 12 – Common Compounds found at t= 0 h and t=6 h hydrolysate for A isolate and A extract

hydrolysis with pH control with a molecular weight between 19 and 20 kDa .............................. 57

Table 13- Compounds identified for t=0 h of A extract ................................................................ 58

Table 14 – Compounds found by the MS software at t=0 and t=6 h for the B extract and isolate

..................................................................................................................................................... 59

Table 15- Compounds found by the MS software for the self-extracted and the commercial

isolates of C in the beginning and end of reaction ...................................................................... 61

Table 16 – Matched Sequences with the masses found at t=6h C Commercial hydrolysate ..... 61

Table 17 – Compounds found at t=0 supernatant of D protein, with respective correspondence

based on literature molecular weights ......................................................................................... 63

Table 18 - Average molecular weight of the peptides obtained by A proteins’ hydrolysis, after 6

hours ............................................................................................................................................ 65

Table 19 – Percentage of peptides identified by the MS software for time 6 h of the A proteins 65

Table 20 – Identification of the peptides at t=6h of A extract comparing with literature sequence

of amino acids ............................................................................................................................. 66

Table 21- Identification of the A isolate compounds ................................................................... 66

Table 22- Average molecular weight of the peptides obtained by B proteins’ hydrolysis, after 6

hours ............................................................................................................................................ 67

Table 23 - Percentage of peptides identified by the MS software for time 6 h of the B proteins 67

xii

Table 24 - Identification of the peptides at t=6h of B isolate comparing with literature sequence of

amino acids ................................................................................................................................. 68

Table 25 Identification of the peptides at t=6h of B extract comparing with literature sequence of

amino acids ................................................................................................................................. 68

Table 26-Average molecular weight of the peptides obtained by C proteins’ hydrolysis, by t=6h

..................................................................................................................................................... 69

Table 27-Percentage of peptides identified by the MS software for time 6 h with C proteins .... 69

Table 28- Identification of the peptides at t=6h of C commercial comparing with literature

sequence of amino acids ............................................................................................................. 69

Table 29- Identification of the peptides at t=6h of C isolate comparing with literature sequence of

amino acids ................................................................................................................................. 70

Table 30- Average molecular weight of the peptides obtained by D protein’s hydrolysis, after 6

hours ............................................................................................................................................ 70

Table 31- Percentage of D hydrolysates identified by the MS software for time 6 h .................. 70

Table 32- Average molecular weight of the peptides obtained by D protein’ hydrolysis, after 6

hours ............................................................................................................................................ 71

Table 33 - Compounds found for t=0h agglomerate of A isolate .................................................. 5

Table 34- Compounds with a molecular weight higher than 10 kDa found in the hydrolysate of A

isolate after 6 hours of reaction by the Qualitative Mass Hunter Software ................................... 5

Table 35 - Compounds found at t=0 sample agglomerate of the A extract .................................. 5

Table 36- Compounds found at t=0 sample agglomerate of the B isolate .................................... 6

Table 37 - Compounds found at t=6h agglomerate of the B isolate ............................................. 6

Table 38- Compounds found at t=0h agglomerate of the B extract .............................................. 7

Table 39 - Compounds found at t=6h sample agglomerate of the B extract ................................ 8

Table 40 - Literature Molecular Weights of A proteins .................................................................. 9

Table 41- Literature Molecular Weights of B proteins ................................................................... 9

Table 42- Literature Molecular Weights of C proteins ................................................................. 10

Table 43- Literature Molecular Weights of D proteins ................................................................. 10

xiii

Acronyms

DH – degree of hydrolysis

OPA -O-phtaldialdehyde

pI – Isoelectric point

𝐴𝑏𝑠 – Absorbance at 340 nm

d- dilution factor

𝜀 – molar extinction coefficient

t – time (h)

PSCAAS -Protein Digestibility Corrected Amino Acid Score

E:S - enzyme to substrate ratio

aa- amino acids

MS- Mass Spectrometer

LCMS – Liquid Chromatography and Mass Spectrometry

DTT - DL-Dithiothreitol

SDS – Sodium dodecyl sulphate

SDS-PAGE - Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

xiv

List of variables

Equation Variable Description

1

𝐷𝐻 Degree of Hydrolysis (%)

𝑉 Volume of titrant - NaOH (mL)

𝑁 Normality of the titrant - NaOH (meq/mL)

𝑚 mass of protein (g)

ℎ𝑡𝑜𝑡 number of peptide bonds per gram of protein

∝𝑁𝐻2 mean degree of dissociation of amino groups

4

𝜗 reaction rate

ϑmax maximum rate achieved by the system

[S] Substrate concentration

Km Michaelis constant

6

𝜗1 reaction rate of substrate degradation

𝑉𝑠 Maximum velocity of substrate degradation

𝐾𝑠 Constant of the reaction of substrate degradation

𝑆 Substrate Concentration

7

𝐾𝑃 Constant of the reaction of intermediate peptides (P) degradation

𝑉𝑃 Maximum velocity of intermediate peptides (P) degradation

𝑃 Intermediate peptides Concentration

𝜗2 reaction rate of intermediate peptides (P) degradation

9

𝑘2 Constant of the reaction of product (P) formation

𝑘3 Constant of the reaction of enzyme inactivation

𝐾𝑀 Michaelis constant

𝑒0 Initial concentration of enzyme

𝑟 reaction rate of hydrolysis reaction

12

𝐴𝑏𝑠 Absorbance (nM)

𝑑 length of the cell (cm)

𝜀 Molar extinction coefficient

[𝑁𝐻2]𝑡 Concentration of 𝑁𝐻2 for a specific time (mmol/l)

13

𝐶 Concentration of protein (g/l)

𝑁 Number of peptide bonds in the protein

𝑀 Molecular weight of the protein (g/mol)

[𝑁𝐻2]0 Concentration of 𝑁𝐻2 at time zero of the hydrolysis reaction

(mmol/l)

xv

1

Chapter 1| Introduction

1.1 Context and Aim of the project

In industry, protein enzymatic hydrolysis is a step used to allow the increase of protein content in

certain food formulations. This is possible since the solubility of the hydrolysates is higher than

the one of the intact proteins, because hydrolysates have smaller size. However, hydrolysis is an

expensive step and any lowering of the yield will reflect in the process profit. The major

phenomenon inducing this lowering of the yield is aggregation, as result of protein-protein

interactions during the hydrolysis reaction. Industry is lacking understanding of this phenomenon

and accepts the reduction in yield. A better understanding of the phenomenon will allow a better

design of the process. This work is part of a project of research in plant protein, in the company

NIZO.

The aim of this project is to study aggregation, define differences in yield, degree of hydrolysis,

and peptide patterns depending on the starting material of protein, creating practical examples of

protein hydrolysis. Small changes in the starting material (like aggregation and glycation) are

having large influence on not only the yield during hydrolysis but also in the application of the

hydrolysates in formulations. It is very interesting for industry (dairy or plant protein suppliers) to

determine the optimal starting material or to know what possible differences occur between

starting from a protein powder or a liquid stream before spray-drying. Different materials are

envisaged: commercially available dairy and plant protein powder and plant protein solutions

during the extraction process.

1.2 Literature Overview

Proteins are essential to the structure and functionality of all living organisms. They are

constituted by chains of amino acids linked by peptide bonds and folded in complex structures.

In order to supply the carbon, nitrogen, minerals and other growth factors needed to support cell

metabolic processes, proteins must be broken down into smaller peptides and free amino acids.

This process is made naturally by the stomach and intestine during digestion but the enzymatic

hydrolysis of dietary proteins as a preparative step to their ingestion has been developing.

2

The first commercially available protein hydrolysates for food appeared quite early during World

War II in two different contexts: first, casein hydrolysates for use in dietetic feeding and second,

pepsin modified soy proteins to replace the lack of egg whites for industrial use. [1] Nowadays,

food products with increased protein concentrations are widely used with different finalities, as

protein supplements in sport nutrition, weight-control or geriatric products and also to treat special

digestion or absorption clinical conditions, that for various reasons cannot get adequate nutrition

from ordinary solid food. [2]

For the industry, the value of hydrolysed proteins is based on the fact that these are more soluble

than the intact proteins’ solutions allowing the increase of protein content on formulations,

increasing nutritional value. The increased solubility is due to the smaller size of peptides,

together with the increase of charged groups. Protein hydrolysates can be used not only to

improve nutritional quality of the food but also the functional properties such as solubility,

emulsifying, foaming or gelling properties.[1, page 6]

Protein hydrolysates are prepared by degrading the intact protein molecules to smaller peptides

and amino acids. This may be performed in various ways. The more common processes are

carried out by acid, alkali or enzymes called proteases. Acid hydrolysis is a very harsh process,

carried out at high temperatures. This process attacks all peptide bonds in the protein substrate,

destroying some of the individual amino acids. Contrarily, enzymatic hydrolysis, since is carried

out under mild conditions, pH (6- 8) and temperature (40-60°C) [2], maintains the nutritional

quality of the amino acids, and that’s why it’s the best way to produce hydrolysates for the food

industry.

Proteins are not completely digested to single amino acids but to peptides. This peptides have

bio functional properties different from the amino acids itself: they have of course the nutritional

properties of the amino acids; they have bioactive properties as mineral binding, anti-microbial,

anti-hypertensive, opioid-like, immuno-modulating antithrombotic activities: and techno-functional

properties like the increasing of solubility of proteins.

1.2.1 Enzymatic Hydrolysis The proteolytic modification of food proteins is an ancient technology used by the man to improve

flavour and storage stability of the protein resources. Different societies have been developing

proteolytic processes to obtain different foods. These range from the dairy French cheeses to the

vegetable Japanese soy sauces. The proteins in these foods are degraded by the proteolytic

enzymes secreted by the microorganisms which take part in the fermentation process, from the

intact raw material or that can be added. This process is no different from what happens in an

enzymatic hydrolysis, in the normal digestive system, in vitro, or in a big scale reactor. [1]

3

To use proteins in food applications, depending on the application, the proteins must have certain

functional properties such as solubility, emulsifying, foaming or gelling properties. Hydrolysis of

proteins is known to be a mean to improve these functional properties.[3]

Since the 1940’s, protein hydrolysates have been used to treat patients who, for various reasons,

cannot digest intact proteins. Later these days, the preoccupation with food and nutritional value

of food has been creating an even bigger interest in obtaining high protein content foods. [4] As

it was said before, protein hydrolysates are more soluble than the intact proteins and that’s why

they are interesting for this high protein foods that are so demanded this days: because they allow

an increase of the proteinic value of food.

Figure 1 illustrates the enzymatic hydrolysis reaction.

Figure 1 – Hydrolysis Reaction at different pH [4]

The hydrolysis reaction results in one carboxylic group (-COOH) along with one amino group (-

NH2), at the C-terminus and N-terminus of the two peptides produced, respectively. At neutral or

basic pH, the neo-formed carboxylic groups release their proton, which may therefore be titrated

by a basic solution (NaOH typically). However, most of the co-produced amino groups also

consume a proton to form ammonium groups at neutral pH, hence counteracting the majority of

protons released by the carboxylic groups.

To stop the reaction the enzyme has to be inactivated. This can be performed by changing the

pH, heating the hydrolysate to denature the enzyme, or by the addition of an enzyme inhibitor.

4

1.2.2 Degree of Hydrolysis

The degree of hydrolysis (DH), defined as the percentage of hydrolysed peptide bonds of the total

number of peptide bonds, is often used as an indicator to express the extent of hydrolysis. There

are several methods to determine this, like, pH-stat; trinitrobenzenesulphonic acid (TNBS) and o-

phtaldialdeyde (OPA). The most common one is the pH stat method [5][6][7], where a basic

titration is made. In this method, to convert the volumes of basic titrant into degrees of hydrolysis

(DH in %) of proteins, which represent the percentages of hydrolysed peptide bonds, it is

necessary to take the mean degree of dissociation of amino groups at the pH considered, ∝𝑁𝐻2,

into account according to the following equation [8]

𝐷𝐻 = 100 ×

𝑉 × 𝑁

𝑚 × ℎ𝑡𝑜𝑡 ×∝𝑁𝐻2 (1)

where V is the volume of titrant (mL), N its normality (meq/mL), m is the protein mass (g), and htot

is the number of peptide bonds per gram of proteins.

The mean degree of dissociation can be estimated from the pKa of the amino groups trough,

∝NH2=

10(pH−pKA(NH2))

1 + 10(pH−pKA(NH2)) (2)

The pKa value was first determined by Adler-Nissen [8] by comparing the base consumption with

the analysis of the free α-amino groups released during hydrolysis. To determine the pKa at 50

°C Adler-Nissen made five experiments with a C protein concentrate at different pH values (6.5,

7.0, 7.5, 8.0 and 9.5) and two experiments with casein at pH 7.0 and 7.5, all using Alcalase 0.6L

and a solution of 50 g protein/l. [9]

The author followed the progress of hydrolysis with reference to the addition of the base

necessary to keep pH constant and an analysis of the free α-amino groups using the

trinitrobenzene sulphonic acid (TNBS) method (Adler-Nissen, 1979). Then developed a method

for determining pKa based upon a comparison between two experiments made at different pH

values. Adler-Nissen concluded that the differences were insignificant, since from a theoretical

point of view the pKa values might be expected to be practically identical, and thus he decided to

average them out, and proposed a value of pKa of 7.1 at 50 °C, in which he included both

substrates used (C protein, mean pKa 7.15 and casein, mean pKa 6.85). Most researchers in this

field have since used these results (Antila, 1988; Gonzalez-Tello et al. 1994; Margot et al. 1994;

Camacho et al. 1998; Dzwolak & Ziajka, 1999). Other authors (Margot et al. 1994) have tried to

relate base consumption with other factors pertaining to the conditions of the hydrolytic reaction,

such as soluble nitrogen, i.e. nitrogen that remains dissolved in an aqueous trichloroacetic acid

solution under normalized conditions. [2, page 252]

5

After that, the department of Chemical Engineering of Granada University [9, page 262],

developed another method that propose a practical method to determine the relationship between

pK and pH for any given protein/protease system. They proposed the expression:

pK = 3.8­0.45 pH (3)

The other methods, OPA and TNBS, are based both based on a reaction with the primary amino

groups, which number increases with the DH. The advantage of the pH-stat method over OPA

and TNBS methods is that in the first the DH can be followed in real time during hydrolysis.

1.2.3 Kinetics of the Protein Hydrolysis

The activity of the enzymes defines the rate at which the substrate is converted, and it is

determined based on the formation of product or degradation of substrate.

The enzymatic protein hydrolysis is can be followed by the DH curve as seen above. To

characterize and compare DH curves, kinetics parameters are used, such as ϑmax and Km.

According to Michaelis-Menten, the kinetics are given by,

ϑ =

ϑmax × [S]

Km + [S] (4)

and in fact, the overall hydrolysis has been fitted to this equation. It has been demonstrated that,

for the hydrolysis of soy protein with Alcalase at high substrate concentrations, the kinetics are

determined by substrate saturation throughout the reaction. This suggests a zero order kinetic

scheme rather than first order kinetics. However, the underlying assumptions of Michaelis-Menten

equation don’t agree with the system. This equation describes the conversion of just one subtract

and doesn’t take into account the fact that the intermediate products are also possible substrates

for the enzyme. Because of this, this model is not correct.

Adler-Nissen, [1], proved that the shape of the hydrolysis curve can be explained as a result of

substrate competition between the original substrate and the peptides which are continuously

formed during hydrolysis. This principle can be illustrated by this simple kinetic model, S is the

substrate, P are the intermediate peptides formed, R are the final peptides and amino acids:

𝑺 𝝑𝟏→ 𝑷

𝝑𝟐→ 𝑹 (5)

𝜗1 =

𝑉𝑠

1 +𝐾𝑠𝑆× (1 +

𝑃𝐾𝑃)

(6)

6

𝜗2 =

𝑉𝑃

1 +𝐾𝑃𝑆× (1 +

𝑃𝐾𝑆)

(7)

and the overall kinetics is

𝝑=𝝑𝟏+𝝑𝟐

(8)

The optimization of the reactors depends on the kinetic of the process. In the case of protein

hydrolysis, the large number of peptide bonds which are cleaved sets a limitation to the possibility

of estimating the basic kinetic parameters. To circumvent this problem, Márquez and Vásquez

[10], presented a study where they used a simple empirical rate equation to express the hydrolysis

curves. From this equation the objective was to deduce the kinetic parameters, so that the

reactors could be optimized. In this study they used hemoglobin as substrate and Alcalase as

enzyme. In their work, the kinetics were controlled trough the DH and then, using the curves given

and what is known about the reaction development, they created a model to determine the kinetic

constants of the hydrolysis performed. With this, they achieved an expression,

𝑟 = 𝑘2𝑒0𝑒𝑥𝑝 [

−𝑘3𝐾𝑀𝑘2

(𝐷𝐻)] (9)

Assuming,

𝑬 + 𝑺 𝒌𝟏↔ 𝑬𝑺

𝒌𝟐→𝑬 + 𝑷 (10)

𝑬 + 𝑬𝑺 𝒌𝟑→ 𝑬𝒂 + 𝑬𝒊 + 𝑷 (11)

where E is the enzyme, S is the Substrate and P are the final peptides and amino acids. The

model is valid for different conditions of substrate and enzyme concentrations.

1.2.5 Protein – Protein Interactions during hydrolysis

1.2.5.1 Aggregation

The conformation adopted by a protein under a particular set of environmental conditions is a

delicate balance between forces that promote and hinder unfolding, as for example the

hydrophobic interactions.[5] During hydrolysis, the structure of proteins is altered leading to the

exposure of the hydrophobic groups, which may result in aggregate formation. [3] Globular

7

proteins retain native conformation within a particular temperature range. Whey proteins, for

example, are sensitive to unfolding at temperatures above 60 °C. The potential for interaction

among the individual whey protein fractions during thermal treatments also needs to be taken into

consideration. The individual fractions that constitute the whey proteins differ in their thermal

stability. While heating native α-la on its own does not produce agglomerates at temperatures

≤75°C, free cysteine residues in β-lg and BSA lead to disulphide interchange reactions with other

β-lg/BSA molecules and with α-la. Heat treatment of whey protein substrates has been shown to

induce proteolysis as a result of protein unfolding and disulphide bond reduction. For example,

native β-lg is resistant to hydrolysis by pepsin, however, heat treatment of β-lg at 82 °C results in

peptic hydrolysis. The exposure of hydrophobic residues can lead to greater reactivity, and heat-

denatured whey proteins can be readily solubilized during hydrolysis. It is noteworthy that not all

thermal treatments result in an increase in hydrolytic susceptibility. Whey and Soy aggregation

has been studied before. [11],[12],[5].

Nagai an Inouye [11] proposed a mechanism for the aggregation of Soy proteins. In the beginning

of the hydrolysis, the hydrophilic surface areas of the proteins are degraded, not resulting in

aggregation. After that, the hydrophobic core is hydrolysed and so the tertiary structure of the

protein is decomposed, and this is followed by aggregation due to the hydrophobic interactions.

Whey protein conformational changes arising from heat treatment may quickly progress to a point

where aggregation takes place.

In enzymatic hydrolysis there are two factors that can induce aggregation: on the one hand the

formation of peptides that have a stronger tendency to aggregate compared to the parental

protein, and on the other hand a decrease in pH towards the pI of the protein, which may also

promote aggregate formation.[11]

1.2.5.2 Water Holding on aggregates - Gel Formation

Water holding is defined as the ability of a matrix of molecules to entrap water in such way that

exudation is prevented. Protein capacity to gel (to form a spatial network) results in water

entrapment within the gel, and therefore in a water holding ability. For example, soy proteins are

known for their ability to retain water or to improve water holding of products, but the origin of this

property is still largely unknown. [13]

Unfolded molecules associate to form aggregates of irreversibly denatured molecules, which may

lead to extended aggregation, precipitation and gelatine. The formation of the network or

aggregate/precipitate arises from a combination of covalent and non-covalent bonds. The main

type of covalent bonds are the disulphide bonds of cysteine residues. [14] These bonds stabilise

the structure of many proteins and are stronger than non-covalent interactions: hydrogen bonds,

electrostatic bonds and hydrophobic interactions. Electrostatic bonds may be stronger than the

other non-covalent bonds, but their existence is determined by the pH and the salt concentration

(ionic bond). Unfolded proteins may be more susceptible to protein−protein interactions via

calcium bridging in addition to hydrophobic bonding as well as disulphide interchange reactions.

8

1.2.5.3 Glycation and Glycosylation

Enzymatic hydrolysis is sensitive to multiple conditions, that can promote modifications in the

proteins, and between them.

In an industrial point of view, processing can promote this modifications through both chemical

and enzymatic reactions. Maillard reaction is the most common chemical reaction that can occur.

This modifications are sometimes applied on purpose to improve the techno-functional properties

of proteins, like emulsion and foam stability [15], [16]. In this cases, when the addition of sugars

is done by enzymatic reactions and to improve functionality, it is called glycosylation. In other

cases, this reaction is a non-desired side effect of production and storage of food products, and

called glycation.[17],[18] The first step of the Maillard reaction is called glycation. During glycation

the end of a carbohydrate reacts with a free amino group. Due to this attachment of the

carbohydrates the primary structure of the proteins is changed. When the reaction occurs in a

protein or peptide, the amino groups can be the side chain of a lysine residue and at the N-

terminal amino acid of the protein. The glycation of the side chain of arginine was also reported

in β-lactoglobulin. Later on, the Maillard reaction secondary reactions occur, which can lead to

changes in the secondary, tertiary or quaternary structures due to protein cross-linking. Glycation

can occur in nature, in animal or vegetable proteins, and most microbial proteins are actually

highly glycated.

1.2.6 Resume of some similar studies on the DH of

enzymatic hydrolysis

Within one enzyme­substrate combination, the extent of hydrolysis is still largely affected by

system conditions. In 2018, Deng resumed on her work [19], an overview of studies done before

about the effect of the conditions of reaction on hydrolysis, Table 1, where we can see the impact

of those conditions changes on the DH extent.

Table 1 - Overview of studies that investigated the effect of system condition, i.e. temperature, pH, enzyme to substrate ratio (E:S) and protein concentration, on the extent of protein hydrolysis

This fact makes the comparison between the hydrolysis of different works quite difficult to perform

because the system conditions are always different at some extent. For perfect comparison, the

9

studies to which one could be compared had to be performed in the exact same conditions as the

other.

There are multiple studies focused on the hydrolysis of whey proteins, as Table 1 also shows.

The authors Gonzalez-Tello, Camacho, Jurado, Paez, and Guadix, in [20], performed multiple

hydrolysis on whey protein. In this study, the hydrolysis are accomplished using 2 proteases of

bacterial origin, being one of them Alcalase, and one of animal origin. The results are that greater

DH extents are achieved using bacterial origin proteases, like Alcalase.

Their aim was to model the kinetics of the hydrolysis and propose a mechanism of reaction and

for that different conditions of reaction are studied in terms of the effect they have on the DH

extent. The two conditions changed are initial substrate concentration and initial enzyme

concentration. It is verified a decrease in DH when of the increase of initial substrate

concentration. This effect suggests that the substrate may play a part in deactivating the enzyme

and lends weight to the idea that the concentration of hydrolysed bonds is not the controlling

factor in the rate of hydrolysis.

In 2012, in [21], the authors try to identify which factor is the main reason for the decrease of DH

at increased substrate concentrations. For that purpose, whey protein isolate (1−30% w/v) was

hydrolysed by Alcalase and Neutrase at constant enzyme to substrate ratio. With increasing

concentration, both the hydrolysis rate and the final DH decreased. The presence of NaCl

decreased the rate of hydrolysis for low concentration, resulting in similar rates of hydrolysis for

all substrate concentrations. The conductivity increase (by increasing the protein concentration,

or by addition of NaCl) has significant effects on the hydrolysis kinetics, but the reason for this is

not yet well understood. In this study, casein, rapseed and haemoglobin degree of hydrolysis’

extents are also reviewed in this terms. The values of decrease presented are up to 8.5%, 13%

and 0.1-0.8% (w/v), respectively.

In Adler-Nissen studies, [22], also some DH extents vs. time can be retrieved. In this study

different proteins were studied, like maize, Soy, wheat, cotton seed and gelatine.

Figure 2- Hydrolysis of proteins with Alcalase under standard hydrolysis conditions. DH is determined from pH stat. S=8%; A/S= 0.012 AU/g; pH=8; T= 50°C

10

1.2.7 Vegetable Proteins

The worldwide demand for proteins is increasing and consequently there is a need for new

sources of food proteins. [23] Food crops, and therefore, vegetable proteins, are the most

abundant source of protein in the world, and also less expensive than animal proteins. The

development of foods with high protein content from vegetable proteins is gaining a lot of attention

in order to combat malnutrition problems in developing countries. [6],[24]

Vegetable proteins consumption is increasing over the years also because of the problems with

the animal proteins, like: food safety problems related to diseases such as bovine spongiform

encephalopathy; the use of animal hormones and the environmental issues. Also, a strong

demand for wholesome and religious (halal) food. [25]

As an example of economic disparity, under industrial conditions, the energy consumption per

kilogram of animal protein is 8 to 10 times higher than for vegetable protein.[23]

The sources of vegetable proteins that are now under study or already at the market are:

• Legume grains as peas (Pisum sativum), soybeans (Glycine max), lupins (Lupinus spp),

chickpeas (Cicer arietinum) or peanuts (Arachis hypogaea);

• Cereals as wheat (Triticum spp), maize (Zea mays), rice (Oryza sativa), barley (Hordeum

vulgare), rye (Secale cereale), oat (Avena sativa) or sorghum (Sorghum spp);

• Oilseeds as sunflower (Helianthus annuus), rapeseed (Brassica napus), sesame

(Sesamum indicum), cottonseed (Gossypium spp), or safflower (Carthamus tinctorius);

• Root vegetables as potato (Solanum tuberosum), cassava (Manihot esculenta) or

sweetpotato (Ipomoea batatas).;

• Leaves from alfalfa (Medicago sativa), cassava, amaranth (Amaranthus spp) or aquatic

plants.;

• Fruits as grape seed (Vitis vinifera), tomato seed (Solanum lycopersicum) or papaya

kernel (Carica papaya). [20]

Legumes are recognized as the best source of vegetable protein. [26] The partial replacement

of animal foods with legumes has been shown to improve nutritional status due to lower

cholesterol level in plant foods (Guillon and Champ, 1996). In addition, plant food diets increase

the level of fibre intake which reduces the risk of bowel diseases, including cancer of the colon

and also reduction in incidence of osteoporosis (Sirtori and Lovati, 2001).[27] In the developed

countries, plant proteins are now either regarded as versatile functional ingredients or as

biologically active components more than as essential nutrients (Marcello and Guius, 1997).

The nutritional value or quality of structurally different proteins depends on the amino acid

composition, ratios of essential amino acids, susceptibility to hydrolysis during digestion, source,

and the effects of processing.[23] Together this gives the nutritional value given as a PDCAAS

11

value. In [23], the analysis of protein nutrition shows that plant proteins have an amino acid

composition that is generally nutritionally less favorable than animal proteins. However, each

class of vegetable protein has a wide range of amino acids. Of course, in natural evolution of plant

proteins the nutritional factor was not the main consideration. New developments in plant breeding

and molecular biology should catalyse revolutionary changes in the biosynthesis of more

nutritious proteins.

Among plants, legume seeds such as Soy bean (Glycine max), beans (Phaseolus spp.), peas

(Pisum sativum), lupins (Lupinus spp.) and lentils (Lens culinaris) represent rich sources of

proteins, carbohydrates, several water-soluble vitamins, and minerals. [28],[25]

12

13

Chapter 2| Methods

2.1 Hydrolysis Reaction parameters and procedure

2.1.1 Protein Substrates

The substrates used in this project all come either from commercial isolates or vegetable flours.

The isolates or the extracts can be produced from the flour, by what we call extraction process.

A, B and C are vegetable proteins while D is an animal protein. A and B were extracted from the

flours to obtain the extract and the isolate. The difference between the two is the presence of

albumins. In the extract only, the starch is removed and, in the isolate, also the albumins are

removed. The protein here called protein A, in nothing has to do with the actual Protein A. Here

A is just a letter used to codify the protein.

2.1.2. Substrate Concentration

The substrate concentration varied from hydrolysis to hydrolysis since the self-extracted proteins

solutions obtained were used directly to perform the hydrolysis reaction. The concentration of the

substrate is not a factor of influence of the results here desired to achieve according to the fact

that the DH calculation formula used has this parameter accounted in the calculus. Table 2.1

shows the substrate concentration used for each reaction. After the extractions the protein

concentrations were determined using FT-IR. This method can be consulted in literature [29].

Table 2- Protein Substrate concentrations for each of the hydrolysis realized

Protein Substrate Concentration (%w/w)

A Extract 3

A Isolate 7

B Isolate 9.5

B Extract 3.5

C Isolate 7.5

Commercial C 7.5

Commercial D 7.5

2.1.3. Enzyme Activity and Enzyme to Substrate ratio

Proteases are an important class of enzymes, which constitute more than 65% of the total

industrial enzyme market. They can hydrolyse proteins to short peptides or free amino acids and

14

catalyse peptide synthesis in organic solvents or in solvents with low water content. Proteases

have a large variety of applications in food industries. These include beer chill proofing, meat

tenderization cheese manufacture, flavour development in fermentation, baking, and health

products.

Alcalase is an industrial, and food grade enzyme with bacterial origin produced by a selected

strain of Bacillus licheniformis and it is mainly constituted by subtilisin Carlsberg. Alcalase was

developed by Novo Nordisk (Bagsvaerd, Denmark) to the detergent industry. Although it has been

used extensively in various biotechnological applications, the specificity of this enzyme has not

been comprehensively characterized. In [17], Alcalase was shown to have a broad specificity and

to cleave peptides bonds on the carboxyl side of Glu, Met, Leu, Tyr, Lys and Gln.

The enzyme used in all the hydrolysis was Alcalase 2.4L R from Novozymes. Different

researchers have proven that this is one of the most effective enzymes to prepare protein

hydrolysates. [3] Alcalase activity is of 2.4 Anson units/gram (AU/g) [6]. The enzyme-substrate

ratio used for all the hydrolysis reaction was of 1:100. Only for special results treatment, as the

case of aggregation observation the ratio was changed to half, 1:200. to slow down the reaction.

2.1.4 pH and Temperature

Two different experiences were realized for the various protein substrates. For all of them, a trial

with pH control and one without pH control was carried out. The pH of the starting solutions of

both trials was adjusted to pH 8, since the optimal pH for the maximum enzyme activity is between

7 and 9 [6]. For the same reason the temperature chosen to realize the hydrolysis was of 50°C,

being the optimal between 30°C and 65°C. For the trial with pH control the pH was kept at 8 using

an automatic burette (pH-stat device).

2.2 Determination of Aggregation

In order to control the aggregation of the hydrolysis reaction the pellets weight was monitored.

The protein matter that doesn’t participate in the hydrolysis reaction is the one that agglomerates

and then precipitates, i.e., the pellet collected after centrifugation. Therefore, using this parameter

to control the yield of reaction becomes the clearest option. The pellets were weighted as wet

pellets, meaning that they contain some of the solution that gets trapped inside. For better

understanding of the water contained in the pellets freeze drying was applied in some occasions.

15

2.3 Determination of Degree of Hydrolysis (DH)

To characterize hydrolysis, there are five descriptors normally used: Degree of hydrolysis (DH);

Proportion of intact protein; Molecular weight distribution of the peptides; Identification of the

peptides; Quantification of the peptides. [30]

The DH represents the percentage of hydrolysed peptide bonds during the hydrolysis reaction,

as stated in Literature Overview. In this study, two methods were used to calculate this parameter,

the pH-stat and the OPA methods. The first method can only be used for the hydrolysis done with

pH control, since in the non-controlled reaction there is no addition of basis. The second one was

used to measure both controlled and not controlled pH reactions.

2.3.1. pH-stat Method

This method is described in Chapter 1.2.2.

2.3.2 OPA Method

The OPA method has the purpose to measure the increase of concentration of 𝑁𝐻2 in solution.

For that, it is necessary to create a series of standard samples with different concentrations (L-

leucine in this particular work) so that it is possible to make a regression that relates concentration

of 𝑁𝐻2 with the absorbance. For both the standard curve points and the samples the absorbance

was measured at 340nm of the (2.75ml) OPA solution in the cuvette. Then 30 µl of

standard/sample were added to the cuvette and the same measurement was done.

Figure 3 – Regression to obtain the molar extinction coefficient in order to calculate the increase of NH2 during hydrolysis obtaining by measuring the absorbance of L-leucine standards at different concentrations

This regression slope corresponds to the molar extinction coefficient at 340 nm. For each

measurement using the OPA method it was necessary to create a new calibration curve to

determine the molar extinction coefficient. However, its value of course was always in the range

of 6.4 ±0.1 𝑚𝑀−1𝑐𝑚−1.

16

After obtaining this coefficient, ε, it is possible to calculate the concentration of 𝑁𝐻2 in the samples

with the absorbance read in the UV spectrometer.

[𝑁𝐻2]𝑡 =

𝐴𝑏𝑠 × 𝑑

𝜀× 100 (12)

And then, the DH value is given by the following expression

𝐷𝐻 = 100 ×

[𝑁𝐻2]𝑡 − [𝑁𝐻2]0𝐶/ (𝑀/𝑁)

(13)

The UV spectrometer used was Cary 4000. OPA stands for o-phtaldialdehyde, a compound that

in conjunction with reduced sulfhydryl groups reacts with primary amines to form fluorescent

moieties. To perform this method, it is necessary to prepare the following solutions. 0.1M Borax:

20,12 g (water free) disodium tetraborate in 800 ml of distilled water and adjust to 1L. 10%: SDS,

50g sodium dodecylsulfate and adjust to 500ml with distilled water. OPA reagent: 40mg o-

Phthalaldehyde in 1ml methanol, 25ml of 0.1M Borax solution, 220 mg DTT and 5 ml 10% SDS.

Adjust to 50 ml with distilled water. It is also needed to prepare a dilution buffer: 25ml of 0.1M

Borax solution, 200 mg N, N-dimethyl-2-2mercaptoethyl-ammonium chloride (DMA) and 5ml 10%

SDS. Adjust to 50ml with distilled water and a L-Leucine solution (6 mM): 40 mg of L-Leucine in

a 50 ml solution with distilled water.

2.4 Determination of Peptide Patterns

2.4.1. SDS-PAGE electrophoresis

The protein profile of all samples was determined by sodium dodecyl sulphate-polyacrylamide gel

electrophoresis (SDS-PAGE) under reducing conditions. The samples were solubilised to 2 mg

protein/ml. Then mixtures of the solubilized sample and of Laemmli Sample buffer (Bio Rad) with

15 mg/mL of dithiothreitol (DTT) were prepared in 1:1 (v/v). This mixtures were then incubated for

5 min at 90°C followed by centrifugation at 15 000 g for 4 min in order to remove any insoluble

material. Solutions were allowed to cool and sample volumes corresponding to 5 µl were loaded

onto 4–20% polyacrylamide gel, 13.3 × 8.7 cm (W × L) Criterion™ TGX™ Precast Midi Protein

Gel, 26 wells from Bio Rad. Precision Plus Protein™ Standards (Bio Rad) with molecular weight

standards ranging from 10 to 250 kDa was used as molecular weight marker. Electrophoresis

was performed at 200 V for about 40 min. Following electrophoresis, the gels were stained with

Instant Blue™ Coomassie (Expedeon). The gels were destained by washing with distilled water

until a clear background was obtained.

17

2.4.2. LC-MS: Liquid Chromatography and Mass Spectrometry

Liquid Chromatography/Mass Spectrometry (LCMS) is fast becoming the preferred tool of liquid

chromatographers. It is a powerful analytical technique that combines the resolving power of liquid

chromatography with the detection specificity of mass spectrometry. Liquid chromatography (LC)

separates the sample components and then introduces them to the mass spectrometer (MS). The

MS creates and detects charged ions. The LC/MS data may be used to provide information about

the molecular weight, structure, identity and quantity of specific sample components.[31] The

protein samples were mixed with 3 volumes of a reducing buffer, containing Bis-Tris, urea,

trisodium citrate and DTT, pH 7.0. The mixture was left at room temperature for 60 min, after

which it was diluted further by the addition of 4 volumes of a urea solution adjusted to pH 2.5.

Prior to application to the column, samples were filtered through 0.22 µm filters.

2.4.3. Software Agilent Mass Hunter Qualitative Analysis

Mass Hunter uses dedicated software modules for instrument-specific tasks including

identification, characterization, quantitation, target analyte screening and confirmation, unknown

identification, characterization of biomolecules, nanoparticles, elemental species, and protein and

metabolite identification. [32]

During this project, this software was used to identify the biomolecules (amino acids, peptides

and proteins) in the hydrolysis samples in both supernatants and agglomerates. For this the MS

input of the samples was analysed using both modules to find molecules: “Find by protein

deconvolution” and “Find by molecular feature”. In this two, the program decodifies the signal

obtained in the MS to transform this to a mass value.

Then, it was also used to identify the origin of these molecules. For that, the sequence of amino

acids of each protein found in literature had to be inserted. The amino acids sequences and their

molecular weight considered was obtained from Uniprot/SwissProt Bioinformatics Resource Tool

[33],[34]. For this we use the “Define and match sequence” module. Here, the identification was

difficult because proteins suffer a lot of modifications and so the proteins used in this project can

differ from the ones found in literature. Also, glycation can affect the proteins like it was explained

in the introduction. Because of that, it was impossible to identify almost all of the proteins because

it would need a lot of time to first be able to identify the modifications.

18

19

Chapter 3 |Results and Discussion

3.1. Agglomeration – General View

The next table shows the concentration of wet agglomerates in each protein solution, with or without pH control over time. The only reaction that didn’t form pellets at any time point was the commercial D protein with pH control. Contrariwise when the reaction is done without pH control, there is formation of pellet as we can read in

Table 3.

Table 3- g wet pellet/g intact protein obtained in the different hydrolysis for each protein type

3.1.1. Effect of pH control

All the vegetable proteins have agglomerates formation with and without pH control. In this study

the variations in the substrates are the presence or not of albumins in the vegetable proteins, the

pH control, and the commercial protein isolates.

For an easier understanding of the impact of these factors, the vegetable proteins were divided

into two groups, the extracts and the isolates, obtained as explained before in Chapter 2.1.1. In

this section the vegetable proteins will be analysed as groups and not as particular proteins.

Figure 4 shows the average of the extracts from proteins A and B and the average of the isolates

from proteins A, B and C with and without pH control.

D Commercial A Isolate A Extract C Isolate C Commercial B Isolate B Extract

t(h) + - + - + - + - + - + - + -

0 0.00 0.00 2.09 2.09 1.14 1.14 0.00 0.00 5.35 5.60 0.00 0.00 0.68 0.68

0.5 0.00 2.45 4.55 4.87 8.69 8.97 8.69 4.23 4.26 8.26 4.24 3.67 0.99 2.25

1 0.00 1.89 3.92 4.81 9.20 8.67 4.10 3.62 4.08 5.12 3.92 3.76 4.04 5.19

2 0.00 1.61 4.19 4.52 8.37 7.90 3.35 3.64 3.96 4.62 3.49 3.37 5.51 4.92

4 0.00 1.62 4.27 4.09 7.63 7.08 2.66 3.28 4.31 3.82 3.28 2.99 5.22 4.75

6 0.00 1.85 3.87 3.62 6.63 6.11 4.62 4.37 4.09 3.64 3.05 2.69 5.57 4.79

20

Figure 4- Average g wet pellet/g intact protein for the vegetable isolate and extract proteins

In the isolates it is not possible to see a big difference between the reactions made with and

without pH control but in the case of the extracts, there is a noticeable difference between the

two. The pH-controlled reactions are actually producing more pellet than the reactions without pH

control, fact that was not expected.

During hydrolysis the pH decreases, and this means that the protein solutions will be closer to the

pI of proteins. Because of that, it could be expectable to affect aggregation for the reactions

without pH control, and therefore, a higher agglomeration, like it happens for the animal protein

hydrolysis (see Figure 5), and not the contrary.

Figure 5- g wet pellet/g intact protein for the D protein trials with and without pH control

In order to understand better the difference between the extracts’ pellets, new trials were done,

with the A protein as example. The formed pellets were freeze dried, so that it was possible to

say how much protein mass is present in each of the agglomerates. The results are presented in

Figure 6.

21

Figure 6- g dry pellet/g intact protein for the A Extract Protein trials

The results of the dry pellets show that there is not such a big impact of pH on the amount of

protein that agglomerates, neither in the isolate or the extract, since they don’t differ significantly

between the trials with and without pH control. Comparing this to the results of the wet pellets,

what this means is that the agglomerates formed in the pH-controlled trials hold more water in

their structure than the ones formed without pH control, in the extracts case.

At higher pH, as is the case for the pH-controlled reactions, the proteins will be more negatively

charged. The negative charges establish repulsive electrostatic forces between the linked

proteins creating empty spaces where there can be water molecules. As said before, the pH drops

during the hydrolysis reaction and this means that the protein molecules are less impacted by the

electrostatic forces, in the reactions that don’t have controlled pH. Because of that the molecules

will be closer to each other, not allowing water to go inside the agglomerates. That is why it is

observed that the wet pellets of the trials with pH control form heavier pellets, because in fact they

hold more water inside.

3.1.2. Effect of albumins

When it comes to the effect of albumins, in Figure 4 it is clear that the aggregation is affected by

it. The extracts always have a larger amount of agglomeration than the isolates. Looking to Figure

6 it is confirmed that there is a lot more protein precipitation in the extracts than in the isolates.

The difference between the isolates and the extracts are the albumins removed. The fact that

there is a lot more precipitating in the extracts than in the isolates can leads to think that the

albumins could be all precipitating and not taking part of the reaction, but that doesn’t happen as

it is proven in Chapter 3.3.3. So, the albumins presence is affecting agglomeration but by inhibiting

the reaction and not by just precipitating.

22

3.3. Degree of Hydrolysis – General View

The degree of hydrolysis represents the percentages of hydrolysed peptide bonds during the

hydrolysis reaction. In this study two methods were used to calculate this parameter. First the pH-

stat method, where the calculation is made using the amount of NaOH used to keep pH constant

and then the OPA method, where the calculation is made by measuring the quantity of NH2 groups

present in the samples by reaction with o-phtaldialdehyde. The first method can only be used for

the hydrolysis done with pH control, since in the other trial there is no addition of basis. The OPA

method was used for both controlled and not controlled pH reactions.

Before exploring each protein in specific, a general analysis is done. Further in this project every

protein can be seen in specific.

3.3.1. Effect of pH control

In contrary to the results for the agglomeration, in terms of DH, the pH control seems to be of

importance for both animal and vegetable proteins. For both protein types, the pH-controlled

hydrolysates have a higher increase of concentration of 𝑁𝐻2 (see Figure 7 and Figure 8). A higher

concentration of 𝑁𝐻2 means that there are more peptides being formed and therefore a higher

extent of DH.

Here the OPA method is being used to compare DH because only for the pH-controlled trials, the

pH-stat method can be calculated. So, in order to able to compare the effect of pH control, the

increase of concentration of 𝑁𝐻2 is used.

Figure 7- Degree of hydrolysis of animal protein D with and without pH control with time

Figure 8- Average Degree of hydrolysis of vegetable proteins (A, B and C) with and without pH control with time

23

3.3.2. Effect of albumins

In this study, both extracts and isolates were studied. To do this only A and B where used since

this were the flours available at NIZO. The C protein was also studied but only as an isolate. The

difference between the extract and the isolate is the presence of albumins in the extract. When

hydrolysed this were the results obtained by the OPA method, for the increase of 𝑁𝐻2

concentration - Figure 9.

Figure 9 – Average concentration of 𝑁𝐻2 increase for vegetable proteins Isolates and Extracts (B and A)

It is clear that the isolates hydrolyse much more than the extracts, complementing what was seen

for the agglomeration, where the extracts agglomerate more than the isolates.

3.3.3. Albumins participation in the hydrolysis

After noticing that the extracts have such smaller DH than the isolates and higher agglomeration,

the question was if albumins were in fact inhibiting the enzymatic reaction or if they were just not

participating in the reaction.

In order to understand this, the protein content was reduced to only the globulins content in the

calculation of DH. This test was made only for the pH-controlled trials. To do this, it was necessary

to know the albumin to globulin ratios. To calculate this ratio, it is necessary to identify the amount

of globulins and albumins existing in the extracts. This identification was realized by Jolan de

Groot, for the case of protein A and Marieëtte Nelissen, for the case of protein B, at NIZO. In this

project only, the ratios were calculated. The proteins were run in HPLC, and in time, samples

were collected and then run in SDS gel with the purpose of identifying the proteins by molecular

weight.

24

After the identification it was possible to estimate the ratio of albumins and globulins by the peak

areas found by the HPLC. For both proteins A and B, the ones where the isolates and the extracts

were tested, an albumin ratio was obtained using the HPLC. 1

After knowing how much albumin is in each extract, the DH can be recalculated to consider only

globulins as protein matter, and therefore found out if their hydrolysis is being inhibited by the

albumins or not. The calculation of the DH only with the globulins as protein matter is calculated

with the expression (10) since 𝑚 is the mass of protein.

%𝐷𝐻 =

𝑉 × 𝑁

∝𝑁𝐻2× (1 − 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛) × 𝑚 × ℎ𝑡𝑜𝑡 (14)

The results of the curves of DH for A and B are presented in Figure 10 and Figure 11, respectively.

As we can see, removing the albumins fraction from the protein content is not enough to

compensate the difference in DH found between isolates and extracts, meaning that the albumin

fraction is inhibiting the reaction and not just being resistant to hydrolysis.

To verify if albumins are in fact being part of reaction, through the Qualitative Analysis Mass

Hunter software the hydrolysates of the extracts were identified. After inserting some of the protein

A albumins sequences in the software, for t=6h, the sequences were matched with peptides

found. The percentage of the albumins sequence that was identified is described in Table 4.

1 This calculation is not in the thesis because it is confidential.

Figure 10 - Comparison between the degree of hydrolysis of the isolate and the extract of protein A when adjusting the extract

reaction to only account with the globulins as protein content

Figure 11- Comparison between the degree of hydrolysis of the isolate and the extract of protein A when adjusting the extract reaction to only

account with the globulins as protein content

25

Table 4- Percentage of sequence of each Albumin that was identified at t=6h in the A extract supernatant

Protein chain % of sequence found

Albumin 1 A a 16.98 %

Albumin 1 B a 7.55 %

Albumin 1 C a 16.98 %

Albumin 2 2.60 %

With this, the participation of albumins in the hydrolysis reaction is proven, as well as that the

albumins are inhibitors of the hydrolysis reaction.

3.4. Detailed Analysis of Each protein

3.4.1 Agglomeration

3.4.1.1. Protein A

A Isolate

Figure 12 describes the agglomeration observed with time during the hydrolysis reaction. Most

agglomerates are formed in the beginning of the reaction, before 30 minutes. After that, the

concentration of agglomerates is almost constant, decreasing slightly.

Figure 12 - g wet pellet / g protein during the hydrolysis of A isolate with and without pH control

26

Since it was noticed that the aggregation started right in the beginning of the reaction, the E:S

ratio was reduced in order to try to deaccelerate the reaction. Figure 13 describes the dry

agglomerates formed with and without pH control at the two different E:S ratios.

Figure 13 -g dry pellet/ g protein present in protein A isolate at the different E:S ratios

When the reaction was done with an E:S ratio reduced to half of the initial value, the agglomerates

still increased concentration before t= 0.5 h and then kept constant. The only difference is that

with the lower E:S ratio (E:S=1:200) there are much more agglomerates formed than with higher

ratio (E:S = 1:100). This is simple to explain since a lower E:S ratio means that the reaction is

slower and therefore there will be more time for the proteins to interact with each other and to

form agglomerates.

Since the agglomerates seem to remain with constant concentration in solution, after the initial

formation, it could be expectable that they had reached some stability conformation and that they

would remain with the same composition during hydrolysis. To verify this, LC-MS and SDS-PAGE

techniques were applied to the agglomerates.

In the LCMS chromatogram it is known, from previous experience, that peptides are found more

in the beginning of the chromatogram (smaller retention times) and the intact proteins more

towards the end of the chromatogram (higher retention times).

Looking into the LCMS results (Figure 14) it is visible that the composition of the agglomerates is

changing with time towards a smaller concentration of intact proteins (found between 30 to 50

minutes) and higher concentration of peptides (found between 8 and 30 minutes). This decrease

of the intact proteins is more evident at t= 6h. It is important to say that the last peak found in this

chromatograms after 58 minutes does not correspond to the intact proteins. This is a not identified

peak.

27

Figure 14- Mass Spectrometry results of A isolate agglomerates after hydrolysis with pH control at times 0, 0.5, 2 and 6

The results from the SDS-PAGE of this agglomerates (Figure 15), show that the proteins present

by t=0h remain present in the agglomerates until the end of reaction.

The SDS-PAGE only shows proteins with a molecular weight superior to 10 kDa, which means

that this technique will only illustrate the polypeptides chains from the intact proteins, or other big

peptides that result from an initial hydrolysis present in the samples.

Figure 15- SDS-PAGE of the agglomerates of protein A Isolate trough time. On the right: mass compounds

found by the Agilent Mass Hunter software matched with the SDS-PAGE bands

28

However, it would be expectable a decrease in concentration, by t=6h, of the intact proteins as it

is shown in the LCMS, and this is not seen in the SDS-PAGE. There is no noticeable decrease

despite all samples were prepared with the same dilution.

Using the software Agilent Mass Hunter Qualitative Analysis this agglomerates were analysed in

order to try to identify the proteins present in the agglomerates. The procedure of the software

can be read in Chapter 2.

By t=0h, it was possible to find a lot of masses but that are divided mainly in 4 different molecular

weights: 12 kDa; 19 kDa; 35 kDa and 48 kDa. The table with all the compounds identified can be

consulted at Appendix A Table 34. The different compounds with similar molecular weight have

origin in the same protein sequences but that have suffered modifications. That is why this

compounds were aggregated in groups of similar masses in order to do an easier identification

by comparison with the SDS-PAGE.

The SDS-PAGE reveals a similar pattern: 4 dominant stripes close to the mass groups range

found in the software (see Figure 15

Figure 15). Comparing to literature, these proteins found could be A2 for the 48 kDa proteins, A1

α for the proteins with 35 kDa and A1 β for the 19 kDa. Only the 12 kDa protein seems to not

correspond to any protein referenced in literature. Protein masses found in literature can be

consulted in Table 40, Appendix B.

Since the SDS-PAGE indicates the presence of the same proteins from the beginning to the end

of the hydrolysis reaction, the masses of the compounds found in the agglomerates by t=6 h were

also identified with the software Agilent Mass Hunter. Table 5 shows the intact proteins found by

t=6h that are also present in t=0h, confirming that these, identified before as the basic and acid

polypeptides of A1, stay present in the agglomerates unhydrolysed. Only A2 protein was not found

29

by the software and is detected by the SDS-PAGE. The list of all the compounds found at this

time can be consulted in Table 34 Appendix A.

Table 5 – Common compounds identified by Mass Spectrometry analysis for the A isolate agglomerates at both t= 0 h and t=6 h

Compound Mass Probable protein

1 35131 A1 α

2 19829 A1 β

The software allows to identify compounds using the sequence of the proteins. When this was

applied to this compounds there was no identification. This means that the sequences of the

proteins found in literature don’t correspond to the ones used in this project as already seen

before. With this said it is possible to conclude that these can be modified compared to the ones

from literature or they can be different varieties. Because of that, the software couldn’t match the

compounds with any of the proteins, but it is still possible to understand that there is one variety

of A1 α and β at the agglomerates by the end of the reaction.

A Extract

In Protein A extract it is possible to see a similar behaviour to Protein A isolate. The weights of

the wet and dry pellets are represented in Figure 16. For this protein, the agglomerates are again

formed right in the beginning of the reaction and then the concentration in solution stays almost

constant. As proven before, the loss of protein matter is not influenced by pH control, what

happens is that the agglomerates with pH control hold more water inside than the non-controlled

ones. This fact is explained in Chapter 3.1.1, Figure 6.

Figure 16 - g wet pellet / g protein during the hydrolysis of A extract with and without pH control and E:S=1:100 and g dry pellet/ g protein present in A extract hydrolysis’ agglomerates with E:S=1:100

30

To check on their composition both LC-MS and SDS-PAGE were realized in the agglomerates’

pellets, Figure 17.

Figure 17- Liquid Chromatography results of protein A extract agglomerates after hydrolysis with pH control at times 0, 0.5, 2 and 6

From the LCMS results of the controlled trial, it is possible to see that there is not a big difference

in the pellets composition from t=0 to t=2 h but there is a significative change in composition of

the agglomerates after 6h. Again, by this time there are less molecules in high retention times,

which correspond to the intact proteins. However, there is no significative increase in the

molecules at lower retention times (first 20 minutes), which relates to no increase of the peptides.

The small molecules that exist get attached to the agglomerates already at t=0.5h and their

concentration in the agglomerates seems to keep constant in the LCMS results.

Like the protein A isolate, agglomerates change with time and some of the intact proteins present

in them are hydrolysed. In this case, there is a bigger concentration of the polypeptides still

present in the agglomerates at the end of reaction (t=6h) than in protein A isolate, which is

comprehensible since the extract has lower DH than the isolate. Because of that, it is normal that

by the end of reaction there are more intact proteins in the extract than the isolate and so, that

also translates to the agglomerates.

In the SDS-PAGE electrophoresis, Figure 18, the decrease in concentration of the intact proteins

is also visible after t=4h. Again, the proteins present don’t change in time, only their concentration

does. What this means is that through all times, all proteins are found but in different

concentrations, none of the proteins is completely hydrolysed.

In order to identify the intact proteins here present, the software Agilent Mass Hunter was used

to identify the masses found at t=0h. The masses of the agglomerates above 50 kDa, that are

31

showed in the SDS-PAGE were not found by the MS software. All the masses found with the

software Agilent Mass Hunter at the agglomerates by t=0h can be consulted in the Appendix A,

Table 33. Again, out of all the compounds found, we can find four main molecular weights, equal

to protein A isolate, which makes total sense because they are the same protein with the only

difference that the albumins were separated, so 12 kDa; 19 kDa; 35 kDa and 48 kDa.

Figure 18 shows the SDS-PAGE bands matched with the masses identified by the software, and

also one mass band visible that comparing to literature might be one albumin, but that the

software didn’t find.

Figure 18 - SDS-PAGE electrophoresis from A Extract agglomerates formed in the trial with pH control

Here are represented the pellets formed during hydrolysis at times 0, 0.5, 1, 2, 4 and 6 hours.

By the end of reaction, mostly the A1 proteins are present in the agglomerates meaning that this

are more difficult to hydrolyse. The A1, both polypeptides, acid and basic, are still present by the

end of reaction. No doubt is also that the A1 β is more concentrated than A1 α. This fact is

presumable when compared to other proteins with a similar structure in soy protein glycinin. The

resistance of the basic chains of glycinin to hydrolysis has been reported before.[3] In this study,

glycinin and β-conglycinin are studied separately. The hypothesis made by this author is that the

acid polypeptide is shielding the basic one quite well, and thereby preventing its degradation. This

occurs until the point that the basic polypeptide becomes exposed to the solvent, and

consequently to the enzyme. This results were obtained with non-heated glycinin.

A is a very complex protein with a lot of different varieties of intact proteins, which makes it hard

to study, and identify. In both forms of the A protein, isolate and extract, the agglomerates are

constituted by the intact polypeptides from the beginning to the end of reaction, so none of the

intact polypeptides are completely hydrolysed. The SDS and MS software analysis identified A2,

A1s α and β in the agglomerates and so all proteins seem to agglomerate, not just a specific one.

However, by the end of reaction the concentration of A2 and albumins (in the extract case)

decreases, while A1s are still very present, for both forms. In the case of A isolate, the

agglomerates by t=6h are mostly composed by smaller peptides that resulted from hydrolysis, but

32

for the A extract there are still a lot of the intact polypeptides. This seems logical since the A

extract hydrolyses less than the A isolate, approximately 14% and 10%, respectively. Therefore,

there will be less intact proteins and more small peptides in the A isolate than the A extract, and

that translates also for the agglomerates.

3.4.1.2. Protein B

B Isolate

Figure 19 describes the agglomeration observed with time during the hydrolysis reaction of B

isolate. Like protein A, the B isolate agglomerates are formed in the beginning of reaction, before

t=0.5h. The behaviour after that, is also similar to A. The concentration of agglomerates is almost

constant until the end of reaction.

Figure 19 – g pellet/ g protein of B isolate formed during hydrolysis with and without pH control with an E:S ratio of 1:100

To check the proteins present in the agglomerates, LCMS and SDS-PAGE techniques were

realized.

In the LCMS chromatogram, Figure 20, it is possible to see compounds with higher retention

times – associated to intact proteins- increasing from t=0.5h to t=2h and then decreasing again

from t=2h to t= 6 h. This intact proteins existing at t=0.5 h seem to exist in the same order of

concentration of the ones on t=6 h but the peaks’ shapes are different, which indicates that

perhaps there are some differences in the proteins existing in each time. By t=6h, the quantity of

small peptides attached to the agglomerate is very high compared to the other times, which is

completely expected since it represents the end of reaction.

33

Figure 20 – Liquid Chromatography results of B isolate agglomerates after hydrolysis with pH control at times 0.5, 2 and 6

This change on the intact polypeptides/proteins is visible in the SDS page, Figure 21. As it was

said before, this technique will not show the results for the peptides, but it is just used to analyse

the intact proteins present in the agglomerates. Figure 21 shows that the protein pattern of the

agglomerates doesn’t change much with time in terms of the proteins present in the agglomerates

but only in terms of these proteins’ concentration. There is a decrease of the concentration of the

heavier proteins after 4h of reaction, and that is probably what causes the difference in the peak

format, at t=6h, in the LCMS. So, the heavier proteins are partially hydrolysed, but the other

proteins, a big quantity still stays intact in the agglomerates.

The great difference of the agglomerates by the end of reaction from the ones of the beginning is

the quantity of smaller peptides that are attached to the agglomerates. Those we can see in the

LC chromatogram and also by identification on the MS software.

34

Figure 21- SDS-PAGE electrophoresis of the B isolate supernatant hydrolysates time 0 h (A) and

agglomerates from times 0.5, 1, 2, 4 and 6 h (B) for the reaction with pH control

When comparing the bands of the intact proteins sample (image A) in the SDS-PAGE to the

literature masses, that can be consulted in Appendix B Table 41, the identifications made in Figure

21 seem to be the most reasonable.

Over 10 kDa, the MS software, for t=6 h, found masses of approximately, 12, 14, 16, 18 and 20.

Six compounds of 20 kDa were found in the B isolate and these are probably the various B1s β

that we can see in the SDS-PAGE that stay in the agglomerate until the end of reaction. The list

of all compounds found at t=0h and t=6h is in Appendix A, Table 36 and Table 37.

Table 6 displays the compounds found both in t=0h and t=6h, meaning that they are intact

proteins, that in this case were identified as being, B1 β. So, B1 β are the polypeptides that stay

in the agglomerate, meaning they don’t hydrolyse. The rest of the proteins seem to hydrolyse to

not be present in the agglomerates by the end reaction, or at least to have a really low

concentration. The same phenomenon has been detected before in soy proteins, like it can be

read in the discussion of the A agglomerates.

Table 6 – Compounds common to t=0 h and t= 6 h hydrolysates of B isolate

Compound Mass (Da)

1 20 166

2 20 150

3 20 124

35

B Extract

In the B extract the agglomerates keep being formed until later than the ones from the B isolate -

Figure 22. The agglomerates increase concentration in solution until about 1 h for the trial without

pH control and 2 hours for the one with pH control.

Figure 22 - g pellet/ g protein of B extract formed during hydrolysis with and without pH control with an E:S

ratio of 1:100

The increase of concentration is also visible in the SDS-PAGE. Figure 23 shows the agglomerates

from the trial with pH control. After 2 hours is achieved the most concentrated solution, as the

previous figure indicated. In comparison to the other proteins studied so far, there is no change

in terms of the proteins present in this agglomerates, i.e., none of the proteins present in the first

agglomerates formed disappear completely after some time, they can only get more or less

concentrated. To check on peptides that can get attached and to confirm the information given by

the SDS-PAGE, LCMS was used.

Figure 23 - SDS-PAGE electrophoresis of the B Extract pellets for the trial with pH control at times 0, 0.5,

1, 2, 4 and 6 h

36

The LCMS results, Figure 24, agree with the SDS-PAGE and the pellets mass control. The

agglomerates in this case are pretty stable and they almost don’t change composition. The protein

content gets higher until 2 hours and then it maintains constant. There is also a small increase of

the molecules with smaller retention times meaning that there are some peptides getting attached

to this agglomerates during hydrolysis, but in much smaller quantity than what was seen for B

isolate.

Figure 24- Mass Spectrometry results of the B extract agglomerates after hydrolysis with pH control at times 0, 0.5, 2 and 6

For the B extract, a lot of intact protein masses were found. Since there were a lot of compounds,

again, a gradient was created considering only differences in the thousands Da, meaning that

compounds with masses like the ones presented here were found. For example, since here 48

kDa is present than it means that at least one protein with mass approximately 48 kDa was found.

The list of all compounds found at t=0h and t=6h is in Appendix A, Table 38 and Table 39.

Table 7- Compounds range of molecular weights (Da) found in B extract hydrolysates by t=0h and t=6h

Compound t=0h Compound t=6h

1 48 1 35

2 41 2 34

3 40 3 30

4 34 4 28

5 32 5 22

6 27 6 21

7 24 7 20

8 21 8 15

9 20 9 14

10 12 10 12

11 10

37

Looking to Table 7, the major difference between the two times is that from t=0 h to t=6 h proteins

around 48 kDa are not present anymore. So, like we saw in the SDS-PAGE, this proteins are

hydrolysed while all the others stay present in the agglomerates. The 48 kDa protein is most

probably B2.

3.4.1.3. Protein C

Figure 25 and Figure 26 represent the variation of the wet agglomerates with time, of C isolate

that didn’t suffer any industrial treatment (identified as C isolate) and of a C Commercial isolate

(identified as C Commercial), respectively.

By time 0.5 h, a high concentration of agglomerates seems to be formed in the trials with pH

control for both C proteins studied. Looking at the samples, this agglomerates revealed to be gels,

as it is visible in

Figure 27. So, actually this high concentration result is due to the fact that the gel holds a lot of

water inside, making the agglomerate heavier and not to the actual protein content.

Figure 27- Agglomerates of the C isolate after 0.5, 1, 2, 4 and 6 hours of hydrolysis reaction

Figure 25- g wet agglomerate pellet/ g protein obtained during the hydrolysis of C Isolate

Figure 26- g wet agglomerate pellet/ g protein obtained during the hydrolysis of a C Commercial

38

Gels can be formed by heat treatment, and so this gel was formed in the heat inactivation step.

Normally hydrolysis is assumed to not contribute for the gel formation since it increases the

number of charged groups and decreases molecular weight, which hamper gelation.[3] On the

other hand, the hydrolysis exposes the hydrophobic groups that can form aggregates. In both

cases, with and without pH control there is aggregate formation, but only the trials with pH control

form a gel because, naturally, aggregates from the pH-controlled trials are charged more

negatively and that is why a space filling network is created, a network that can hold water, and

therefore form a gel. In the non-controlled pH trial, where the solution will be at lower pH, the

aggregates will be less charged and therefore will take less space and not hold as much water

when precipitating, and consequently not forming a gel. The fact that the gel formation only occurs

at this time is explained by the said before, gel formation is favoured by big molecular weights,

and after t=0.5 h, the molecular weight is probably not high enough to continue to form gels.

Excluding this big difference, at t=0.5 h, due to the gel formation, the agglomerates of C behave

in a very similar way as the previous proteins for the rest of the time. After t=0.5h, the

agglomerates’ concentration stays almost constant.

Figure 28 describes the C isolate chromatograms results of mass spectrometry, and Figure 30

the same for the C Commercial. This chromatograms show that for the isolate, there are some

peaks with high retention time, which probably correspond to proteins, increasing and that there

are not a lot of peptides attaching to this agglomerates, since there is no increase of masses in

the lower retention times. There is one particular peak that increases a lot by t=6h.

Figure 28 – Mass Spectrometry results of the C isolate agglomerates after hydrolysis with pH control at times 0.5, 2 and 6

39

For the C isolate, when looking at the SDS-PAGE, Figure 29, it is possible to see the intact

proteins decreasing concentration after 4h, the ones close to 50 and 37 kDa. Since the LCMS

pointed to an increase of protein content with time, this seems to oppose to it. However, when

identified by the software Agilent Mass Hunter, the peaks in the mass spectrometer for t=6h

revealed to belong to two compounds: one with 19kDa and other with 20 kDa. With this said, it

means that the increase of protein content that is visible in the chromatograms is due to the

aggregation of this proteins of 19-20 kDa, and that is the highest peak at t=47 min, and not the

other two proteins.

Figure 29- Comparison of the proteins found in the agglomerates with the intact proteins using the SDS-PAGE of the supernatants and the agglomerates obtained during the hydrolysis of C isolate

This two proteins that are visible decreasing in the SDS-PAGE, are hydrolysed and not even

found by the MS software at t= 6 h, so they either don’t exist by the end of reaction or are in really

small quantity. In conclusion, the proteins with masses around 19 and 20 kDa are the proteins

taking a bigger part in the aggregation, at least after t=4h.

To try to identify the proteins seen above, as being the most responsible for the agglomeration in

the C isolate, the Agilent Mass Hunter Software was used to find the masses present in the

agglomerates. For the C isolate the masses founded by the software are in Table 8.

Table 8 – Proteins found by the MS software in the agglomerates at the beginning of reaction, by t=0.5 hours

Compound Mass (kDa) Probable protein

1 32 551 C1 α

2 31 938

3 21 973

C2 β 4 21 147

5 21 103

6 19 297

40

As it is visible in Table 8, the software only finds two major different types of protein, one around

32 kDa and the other around 19-21 kDa. This clearly corresponds to the two lower bands on the

SDS-PAGE. The band at 50 kDa that is visible in the SDS-PAGE (see Figure 29) was not

identified by the software.

This compounds were found in t=0.5 h of reaction which means that to some extent some

hydrolysis has occurred and so it is possible that this agglomerate has peptides in it, not only

intact proteins. To check If this compounds are in fact intact proteins, this was compared to the

intact solution of the C isolate, figure A.

The conclusion of Figure 29 is that the proteins found in the agglomerates are the same found in

the intact protein solution. That confirms that the compounds present in the agglomerate are intact

proteins, and therefore, are C1 acid and basic chains and C2 β, by comparison to literature,

Appendix B Table 42. By the end of reaction, 6 hours, only the C1 basic chains seem to resist in

the agglomerates.

Here it is quite clearly that the basic polypeptide is degraded after the acid polypeptide of C1 is,

looking at the differences from the hydrolysates from times 2 to 6 hours, in Figure 30 A. Again,

this C1 protein has the same structure as glycinin, and so it is quite expectable that the basic

chain is the last one to be degraded, because it is shielded by the acid polypeptide, like it was

already explained in page 29.

For the C Commercial, the amount of proteins in the agglomerates (represented by the peaks at

higher retention times) decreases with time meaning that the proteins are re-solubilizing and then

from t=0.5 h on, it is possible to see peptides composing the agglomerates, but in small quantity.

41

Figure 30- Mass Spectrometry results of the C Commercial agglomerates after hydrolysis with pH control at times 0.5, 2, 4 and 6

The SDS-PAGE of the commercial C ( Figure 31 B) showed that after t=0.5 h of reaction there

are no more compounds heavier than 10 kDa in the agglomerates.

After using the Agilent Mass Hunter software, it was found that the agglomerates are formed by

large peptides instead of proteins. The masses found in this agglomerates range between 113

and almost 6 000 Da, in both t=0.5 h and t=6 h. So, that is why we don’t see bands in the SDS-

PAGE, and there is a blue stain in the end of the wells. Proteins from C Commercial are quickly

broken into big peptides, like we can see by Figure 31 - because we only see intact proteins at

time zero - and then those peptides are the ones that form the agglomerates. However, by t=0.5h

there is still a big compound found, with a molecular weight that can be from an intact protein.

The software identified this mass as described in Table 9.

Table 9 – Compound Mass found by the software at t=0.5 h of C commercial agglomerate

Compound Mass

1 20719

To understand if this is an intact protein or a very big peptide, the SDS-PAGE of the agglomerates

was compared to the intact proteins’ one, Figure 31. For sure it is possible to understand that this

compound is not an intact protein since it doesn’t exist in the intact proteins’ solution. So, this

42

compound is a big peptide formed after hydrolysis of one of the intact proteins that we can see in

Figure 31 A.

When identified with the Agilent Mass Hunter software, using the literature sequence (that was

consulted at [34]), the software identified this peptide as C2 α from the amino acids positions 219

to 400 (this positions exclude the signal peptide amino acids).

In Figure 31 A, it is possible to see the same peptide in solution (t=0.5h and 1h) and in the

aggregate (t= 0.5h). This component was already identified as being C2 α, and so it is known that

this peptide is the largest one existing also in solution until 1 hour of reaction. After that, the

peptide is finally broken down.

Figure 31 – SDS-PAGE of the C Commercial supernatants for times 0, 0.5, 1, 2 and 6h (A) and agglomerates for times 0, 0.5, 1, 2 and 6 h (B)

Finally, with respect to the agglomeration, comparing both C proteins it is possible to say that C

Commercial agglomerates in a very different way from C isolate, since the agglomerates from C

isolate are constituted by big peptides and the C isolate agglomerates are constituted by proteins

and a small amount of peptides attached. This proteins on C isolate agglomerates hydrolyse

during reaction but, after 6 hours of reaction, there is still intact basic C1 protein in solution. Figure

32 compares the two agglomerates and shows the match of the identification done with the

software with the SDS bands. Despite the differences in constitution, the amount of agglomerates

between the two C proteins is very similar, so in terms of yield of raw material, the result it is quite

similar.

43

Figure 32 – Comparison of SDS-PAGE of C Commercial (A) and C isolate (B) agglomerates with the identified proteins

In the C isolate the basic chains from C1 stay intact trough the 6 h, and the reason was explained

above, but this doesn’t happen with the C Commercial. From the same reference study where the

basic chain presence in the glycinin agglomerate was detected [3], it is known that when heated,

the quaternary structure of C1 is changed in such way that the acidic chain of C1 does not protect

the basic one sufficiently anymore, and that results in better and easier access of the enzyme to

this polypeptide. In this project C1 is not isolated like glycinin was in [3], we have not only C1 but

all C proteins, but still the same denaturation must happen when C is heated during the process

to obtain the commercial powder. C Commercial protein powder was processed with heat, and

so C1 quaternary structure is changed and that is why the basic polypeptide from C1 is easily

hydrolysed in contrary to the isolate.

3.4.1.4. Protein D

The impact of pH on protein D has been studied before on Chapter 3.1.1. There, is described that

there is only formation of agglomerates for the non-controlled reaction. In this section the

identification of the compounds on these agglomerates will be presented.

Figure 33 shows the MS chromatogram obtained for the agglomerates trought time for protein D

hydrolysis. The proteins that exist by t=0.5 h (that belong to bigger retention times) are clearly

reducing with time, and a lot of peptides, with lower rentention times, are getting attached to this

agglomerates since the beginning (see first chromatogram, time 0.5 h). So it is possible to say

that during hydrolysis the proteins agglomerated are hydrolysed and their concontration in the

agglomerates decreases a lot.

44

Figure 33- Mass Spectrometry results of the D Commercial agglomerates after hydrolysis with pH control at times 0.5, 2, 4 and 6

The SDS-PAGE shows this decreasing concentration of D1 and D2 present in this agglomerates.

Also, it is visible an increase of a big protein, around 70 kDa on the agglomerates.

Figure 34 – D protein agglomerates obtained at each time of hydrolysis reaction without pH control

When checked with the MS software many masses were found. When they were identified it was

found that most of the masses belong to D2, proteins or its peptides and just a small percentage

has origin in D1. Only 5 out of 34 compounds found here were identified as part of D1.

45

3.4.2. Degree of Hydrolysis

3.4.2.1. Protein A

A Isolate

Figure 35 shows the DH obtained using both OPA and pH-stat methods. The results obtained are

similar, so we can assume that they are correct, and conclude that the A isolate solution after 6

hours reaches a DH of about 14%.

Figure 35- Comparison of the results in Degree of hydrolysis obtained using the OPA and the pH-stat

methods

46

The trials with and without pH control were also compared, in Figure 36. Here the value of 𝑁𝐻2

groups increase, in relation to t=0 h, is used to compare the trials. This value is obtained using

the OPA method, explained in Chapter 2.3.3. For the A isolate, the results follow the expected,

the trial with pH control has in general a higher increase of 𝑁𝐻2 groups, in the same amount of

time, than the trial without pH control. The difference is quite considerable since by t=6h of

reaction the 𝑁𝐻2 groups increase obtained without pH control is still lower than the increase

obtained with pH control, by t=2 h.

Figure 36 - Comparison of NH2 increase of the trials with and without pH control for the A isolate

A Extract

The difference between the extract and the isolate is immediately noticeable. In A isolate, the

hydrolysis follows the same rate as other proteins studied before. On the other hand, the rate of

hydrolysis of the A extract shows to be almost linear in time, as represented in Figure 37.

Figure 37 – Degree of hydrolysis of A Isolate and A extract with pH control obtained by the pH stat method

with an E:S ratio of 1:100

The kinetics of the A extract hydrolysis are different from all the other proteins studied in this

thesis, even from the other extract, the B extract, as it is possible to see in Figure 38. Because of

this fact, it is understandable that this change in kinetics is not due to the present of albumins but

47

that this is due to a specific characteristic of the A extract proteins, and since this is not verified

by the A isolate, a specific characteristic of the A albumins.

Figure 38- Comparison of the behaviour of the degree of hydrolysis for both extracts, A and B. This results

of %DH were calculated with the pH-stat method

The downward in the DH’ curves observed in the majority of the proteins is explained by the

presence of product inhibitors at higher DH, meaning that some of the products formed during

hydrolysis, the peptides, inhibit the reaction. [1]

In the case of the A extract, this downward is not observed which means that there aren’t product

inhibitors in this case. Since it was proven that the reaction is happening, by the analysis done to

measure DH, like the OPA measurement (Figure 39) for example, then the peptides are being

produced but they are not inhibiting the reaction. This means they are not competing with the

original substrate proteins. The fact is that, this peptides can, like all others, still be broken by the

enzyme and so they should compete with the intact proteins as substrate for the enzyme and

deaccelerate the reaction. The reason why this happens needs more research at molecular level,

to understand why this molecules are not inhibiting the reaction.

To compare the impact of the pH on this hydrolysis, the NH2 increase was measured using OPA

method and those results are present in Figure 39. The measuring for the A extract without pH

control produced hydrolysates was irregular, like Figure 39 can show. As it is visible this method

didn’t apply well to this protein type. A decrease of NH2 groups is visible at t=0.5h, and t=2h which

doesn’t make any sense since the reaction is occurring, and proteins are being broken down.

Some type of mechanism is occurring preventing OPA to detect the NH2 groups present.

The extract proteins are very complex solutions since they contain soluble and insoluble proteins

and so it is always very complicated to work with them.

48

Figure 39 - Comparison of NH2 increase of the trials with and without pH control for the A extract

3.4.2.2. B Protein

The DH of B is in general lower than the A, only around 10% for the isolate protein and even lower

for the extract. In opposition to A, B extract and Isolate DH’ curves are similar and in agreement

with the kinetics seen before for protein hydrolysis by other studies in this work and from literature-

Figure 40.

Figure 40 - Degree of Hydrolysis of B Extract and Isolate with pH control using an E:S ratio of 1:100

49

B Isolate

Both methods applied to the trial with pH control, gave similar results in what is the DH of the

protein by each of the times, making trustful the fact that this DH is close to reality.

Figure 41- Degree of hydrolysis of B Isolate obtained using the pH-stat and the OPA methods

In the B isolate, the pH control doesn’t have a big impact not only in the aggregation but also in

the DH, as it can be seen in Figure 42. The NH2 increase measure revealed that the hydrolysis

doesn’t vary a lot from the two different conditions, and even that by t=2 h, the result is exactly

the same. However, the pH-controlled hydrolysis extends further than the non-controlled.

Figure 42 – NH2 increase of both trials of B extract, with and without pH control

B Extract

50

Once again, just like with the A extract, the B extract behaved much less according to the

expected and was very difficult to analyse. First, there is a quite considerable difference between

the two methods used to do the calculation of the DH, Figure 43.

Figure 43 – Comparison between the two methods used in this thesis to calculate the degree of hydrolysis for the B extract

Second, in Figure 44, a decreasing of NH2 concentration groups for the first hour is observed,

which makes no sense since hydrolysis has already started, and of course the number of NH2

groups could never decrease, only increase, or in case of no reaction it could stay the same. Like

it was explained for the A, the OPA method revealed to be quite sensitive when it comes to the

extracts without pH control. With this said, the difference between the pH controlled and not

controlled reaction is not certain. Anyway, the obtained results are shown in Figure 44.

Figure 44 – NH2 groups increase for the hydrolysis reaction of B extract during 6 hours of reaction

The increase of concentration of the NH2 groups measured is very small for both reactions. The

OPA measure method is quite sensitive, any modification on the hydrolysates after the reaction

can imply problems in the readings, since we are talking about absorbances. In this case, since

the DH is so low, and therefore the differences in the increase of concentration of the NH2 groups

51

are smaller makes the method even more sensitive since the differences between absorbances

are so small. Because of that, and the fact that the pH stat method has been more studied, than

the pH stat method is most probably more precise than the PA method.

3.4.2.3. C Protein

Both C proteins studied follow the same kinetics as the proteins found in the literature. Comparing

the two, C Commercial has a higher DH than C isolate. Both proteins are C isolates, the difference

between the two is the fact that the C Commercial isolate suffered processing, and with that, some

heat treatments, that the isolate didn’t. Also the starting material of C commercial is a powder, in

contrary to C isolate (self-produced) that is a liquid. Heat treatments provoke heat denaturation

of the proteins, and that means that the proteins are unfolded and so more exposed to the

enzyme, so it is natural that the process of hydrolysis is faster

Figure 45 – Comparison of the degree of hydrolysis curves of the two C isolate proteins studied in this

thesis: the commercial and the self-extracted

C Commercial Isolate

The difference between the two methods used to determine the DH for the C Commercial isolate

was very small (Figure 46), giving a quite good approximation of what is the real DH of this protein

after 6 hours, 12%.

52

Figure 46- Comparison between the two methods used in this thesis to calculate the degree of hydrolysis

for the C Commercial isolate

When comparing the impact of pH on this protein, it was found that there isn’t a big difference,

Figure 47. However, it still is verified that the trial with pH control produces more hydrolysis

products.

If proteins are denaturated, then they are unfolded, and so the electrostatic forces, that are bigger

or smaller depending on the pH, don’t have as big impact as on the folded molecules. Therefore,

also the pH doesn’t have a big impact on the reaction. As seen before, the electrostatic forces

are the strongest non-covalent forces, and so they are the most impacting on the proteins’

interaction.

The difference seen between the two trials, for this protein, is reflecting only the fact that the

enzyme is deviated from its optimal pH, and not the protein-protein interactions from the intact

proteins, as the other proteins are, since this are denaturated.

53

Figure 47 – Comparison of the NH2 groups increase between the two trials with and without pH control with time for the C Commercial

C Isolate (Self-produced)

The comparison between the two methods for the C isolate, is not as good as the C Commercial

isolate. In this case, there is a 2% deviation between the two. Nevertheless, the DH should situate

somewhere between 10 and 12%.

Figure 48 - Comparison between the two methods used in this thesis to calculate the degree of hydrolysis for the C isolate

Also, the impact of pH in this protein was quite considerable. Figure 49 shows that the increase

of 𝑁𝐻2 groups for the trial without pH control was very small compared to the trial with pH control,

and so it is concluded that the pH has a really big impact in this protein, in terms of DH.

54

Figure 49- Comparison of the NH2 groups increase between the two trials with and without pH control with time for the C isolate

3.4.2.4. D Protein

For D protein the difference between the two methods to determine the DH was also about 2%,

like the C isolate. D protein has a high DH compared to all other proteins, going up to 14-16%

after 6 hours.

Figure 50 - Comparison between the two methods used in this thesis to calculate the degree of hydrolysis for D protein

The impact of pH on the DH of D protein has been seen before on Chapter 3.3.1.

3.4.3. Hydrolysates Protein Pattern

55

In this chapter, trough the SDS-PAGE gels and the LCMS analysis, is identified the hydrolysation

pattern of each protein/polypeptide for all A, B, C and D proteins. Most specifically, which proteins

hydrolyse better, and which proteins stay unhydrolyzed until the end of reaction. The previous

chapter shows the SDS-PAGEs and LCMS results of the pellets and here the supernatants of the

hydrolysis reaction are analysed. The chromatograms obtained in the LCMS for all proteins are

presented in Appendix C.

3.4.3.1. Protein A

Figure 51 shows the hydrolysates obtained for both A proteins, with and without pH control, for t=

0h to t=6h, in an SDS-gel electrophoresis.

Figure 51 – A :SDS-PAGE of the Aisolate hydrolysates from t=0 to t=6h with (+) and without(-) pH control

B :SDS-PAGE of the Aextract hydrolysates from t=0 to t=6h with (+) and without(-) pH control

Looking into this figure, the first big noticeable difference is that the protein close to 50 kDa stays

in solution for the A isolate but not for the A extract. This protein, comparing to the literature,

should be A2 - see Appendix B Table 40. In Appendix B Table 40 are presented the literature

molecular weights of A proteins.

The second noticeable fact in the SDS-gel is a band with the molecular weight of approximately

20 kDa that stays in all the hydrolysates in a big concentration trough time for both A isolate and

A extract. From literature, this band could correspond to the β fractions of A1.

To try to identify these molecules, the samples were analysed in the software Agilent Mass

Hunter, after running them in the LC-MS. For the A isolate and the A extract, these were the

masses found by t=6h, Table 10.

56

Table 10 - Compounds found in the t=6h hydrolysate for A isolate and A extract hydrolysis with pH control

with a molecular weight between 19 and 20 kDa

Compound A Isolate A Extract

Mass (Da)

1 19 892.65 19 892.90

2 19 876.29 19 876.60

3 19 829.11 19 829.08

4 19 846.08

The masses found for both proteins are quite similar, only for A isolate one more compound with

a slightly different molecular weight was found. In fact, all these compounds have quite close

molecular weights and they probably belong to the same proteins, but each one of them has one

or more different modifications that change the molecular weight.

Looking for the masses from literature, none of these corresponds exactly to the molecular

weights described. This can mean either that the A1 β existing in this A protein is altered and

stays intact in solution or that this aren’t intact proteins, but large peptides from bigger intact

proteins as, for example, A3. This alterations can be attached molecules, like sugars for example

(glycation) or even a modification in the sequence of amino acids of the protein. There is no

certain that the protein A variety used in this project is the same as the one reported on the

literature and so the amino acid sequence can be different.

For the A isolate results, without introducing any modifications to the software, the masses found

before were identified as Table 11 describes.

Table 11 - Identification of the peptides with a mass of approximately 20 kDa at t=6h of the A isolate

Compound Mass (Da) Identified sequence (part of

the aa sequence)

1 19 892.65 A3 (173-345)

2 19 876.29 A3 (99-266)

3 19 829.11 A4 (55-229)

4 19 846.08 A3 (105-273)

However, this results can be untrue to reality. The software makes an approximation to the

masses by cutting the sequences and comparing that mass to the mass the software founded in

the sample by mass spectrometry. Since A3 and A4 are such big proteins it is obvious that is

much easier to find a peptide between the A3 sequence, for example, that matches the masses

founded than to match this with an intact protein without knowing the modification it went through.

This said, the approach to check the veracity of the software identification was to see if any of

these compounds found are or aren’t present in t=0h. If this same compounds found at t= 6h are

57

present in t=0h than it means that those compounds were not hydrolysed by Alcalase, and that

this compounds stay unhydrolysed in solution.

Since this is common to both A extract and isolate, both were analysed. A lot of compounds were

found for t=0 h with molecular weights between 19 and 20 kDa, but here are only present the

ones that seem to be similar in at least both solutions. The list of all the hydrolysates found by the

end of reactions is not available in the Appendix because each reaction results in something like

900 to 2500 compounds, like will be described in the next chapter 3.4.4. and so the lists are too

long to attach in this thesis. This lists were kept in excel files for future consultation.

The compounds that are present at t= 0 and 6 h are represented in Table 12.

Table 12 – Common Compounds found at t= 0 h and t=6 h hydrolysate for A isolate and A extract hydrolysis with pH control with a molecular weight between 19 and 20 kDa

A Extract t=0 h A Isolate t=0 h A Extract t=6 h A Isolate t=6 h

Average Mass (kDa)

19829.31 19829.13 19 829.08 19 829.11

19844.87 19846.54 19 846.08

19876.45 19 876.60 19 876.29

19892.57 19 892.90 19 892.65

The A extract and isolate are obtained from the same raw material, so they should have the same

globular proteins present in their composition. In reality, not all the masses were found in both

samples, but we can consider that the ones found in the A isolate will also exist in A extract and

vice-versa, because they come from the same flour.

So, there are some compounds that are present in both t= 0 h and in the hydrolysate at the end

of reaction, t=6h, meaning that this compounds are not hydrolysed and therefore the most

probable is that this are intact proteins. Since the modifications on the proteins were not studied

in this thesis, the match will be made by approximation, and so the closest fit for this 19 to 20 kDa

proteins is the A1 β, but still there is a deviation of at least 352 Da. This deviation can easily

correspond to a couple of sugar molecules, for example glucose, fructose, galactose that have

molecular weights of approximately 180 g/mol.

With this said what is possible to affirm is that this proteins common to t=0h and t=6h, are proteins

that stay intact in solution during all hydrolysis and that are different from the proteins referenced

in literature.

The identification by protein sequence of t=0 h for all compounds found, with a tolerance of 1 Da,

resulted in the identification of two polypeptides from A1: A1 A2 β and A1 K β for the protein

extract, so this two A1 β polypeptides are hydrolysed because they are not found in t=6h. For the

A isolate, with the same tolerance none of the compounds was identified, but both of the A protein

extract and the A protein isolate are originated from the same flour, so the intact proteins should

58

be the same. The extraction process doesn’t have such severe conditions that could explain

modifications of the proteins.

Table 13- Compounds identified for t=0 h of A extract

Sequence Mass

Compound 66 A, A1 A2, β 20182,31

Compound 67 A, A1 K, β 20135,10

None of the compounds present in Table 12 found by the end of reaction, t=6h, was identified by

the MS software.

3.4.3.2. Protein B

Figure 52 shows the hydrolysates obtained from both B proteins, the isolate and the extract, with

and without pH control, for t=0h to t=6h, in an SDS-gel electrophoresis.

Figure 52 – SDS-PAGE of the B isolate (A) and extract (B) hydrolysates from t= 0h to t=6h with and without pH control

Looking at the t=0h SDS-PAGE for both B proteins, the isolate and the extract, a lot of bands of

intact proteins (t=0h) appear in the gel, but we can highlight mostly two to three bands around 20

kDa; one band below the 37 kDa standard mark; one below the 50 kDa and one between 75 and

50 kDa. Only the proteins with higher molecular weights, below 50 kDa, seem to be totally

hydrolysed. A2, is found in literature with a molecular weight of 48 kDa, so we can say that the

band below 50 kDa is probably A2 and that this protein is totally hydrolysed.

59

Below 37 kDa it is possible to find bands from the beginning until the end of reaction, but it seems

that the bands found in this range, from t=0.5h on, are not the same as the one in the intact

protein. The strong band found at t=0h disappears and a new band close to this molecular weight,

but slightly lighter is formed. This can be for example, a peptide prevenient from the A2. So, this

one is the one that stays in time and doesn’t hydrolysate. This happens for both isolate and

extract, with and without pH control.

Just like it happened when analysing the agglomerates, the MS output for the A extract t=0h,

resulted in a lot of compounds. Once again, this compounds despite being a lot, all fall into the

same mass ranges. Because of this it was possible to group this proteins/polypeptides in ranges,

just like it was done for the agglomerates. Table 14, resumes this groups of proteins found for

each time and initial protein solution.

Table 14 – Compounds found by the MS software at t=0 and t=6 h for the B extract and isolate

Extract t=0 h Isolate t=0h Extract t=6h Isolate t=6h

48 48

35 34 35

31 31

20 20 20 20

14 14 16

12 12 11 10

When comparing this masses to the literature, the only mass around 30 kDa is B1 B4 α

polypeptide, which mass is 32 kDa. This should be the protein band more outstanding below the

37 kDa standard mark, so probably the 31 kDa found by the MS at t=0h.

The masses close to 20 kDa, are most probably the polypeptides of B1 β. Despite the big variety

of peptides found in that area of the gel we can see that this stay in solution, and also the results

from the software Agilent Mass Hunter Qualitative analysis point the same results. In B protein,

the B1 α in general has a molecular weight of approximately 17 kDa, corresponding to the lowest

dark band that is visible in the SDS-PAGE at t=0, in both proteins. That band disappears with

time, meaning that this B1 α are being hydrolysed. So, once again B1 β is the last one to be

hydrolysed, since it is in a globular structure where B1 α is shielding it, and in this case, it stays

in solution until the end. This structure issue is explained in more detail in chapter 3.4.1.3. page

29.

It is important to refer that literature for B protein is not as extensive as for the other 3 proteins in

this project. Definitely this is a protein that still needs a lot more work of research. Only a few

intact proteins of protein B are documented and the SDS reveals a big variety of masses. This

turns the identification job very difficult.

60

3.4.3.3. Protein C

C Isolate

Figure 53 shows the hydrolysates obtained from both C proteins, the commercial and the self-

extracted isolates, with and without pH control, for t=0 to t=6 h, in an SDS-gel electrophoresis.

For the two types of C, the difference in the hydrolysates is pretty clear only looking to the SDS-

PAGEs. C Commercial (image A) hydrolyses much faster than the self-extracted isolate (image

B), and after t=0.5 h there is no intact proteins anymore. This has probably to do with the fact that

C Commercial proteins were heated and so their quaternary structure is modified. Because of

that the proteins became easier for the enzyme to reach, and therefore are hydrolysed faster.

Figure 53 – SDS-electrophorese gels for Commercial (A) and Isolate (B) C hydrolysates with (1) and without (2) pH control

As we can see in the Appendix B Table 42, C is mainly constitued by β-C2 and C1. In the SDS-

PAGE, has it is described in the methods, the samples are denaturated and so the proteins are

broken into polypeptides. At t=0h, we can say that it is possible to identify in both C, commercial

and isolate, a band below 75 kDa; a band below 50 kDa; a band below 37 kDa and a band below

20 kDa. Comparing to literature this must be β-C2 α; β-C2 β, A1/A2/A4 C1 α chains and C1 β

chains, respectively.

In the isolate protein trial with pH control (image B1), it is possible to see the basic chain of C1

being more resistant to hydrolysis, and only after 6 hours this proteins are not present anymore.

Another fact that is visible in this figures, is a polypeptide around 15-16 kDa. This one is present

in both Commercial and Self Extracted Isolates and stays present until the end of reaction.

The MS output resulted in the following spectrum of masses. Here it is found only one compound

at t=6 of the C isolate with a molecular weight of 21 kDa. This could look like a C1 β since the

61

mass is similar to the one from literature but since the same mass doesn’t exist by time zero, it is

only possible that it is a peptide formed in an intial phase of hydrolysis of other protein. Looking

to t=6h of the C protein isolate (image B1) it is possible to see a light band with superior weight

to the intact proteins that were identified as B1s β confirming this.

Table 15- Compounds found by the MS software for the self-extracted and the commercial isolates of C in the beginning and end of reaction

C Isolate C Commercial

t=0 t=6 t=0 t=6

37 21 33 12

36

32 10

35

31 10

20

30

29

27

20

When matched with the literature sequences, this compound was identified as C1 G5, A3 chain,

which means one of the acid chains of C1, from positions 73 to 259, figure 54.

Figure 54 – Sequence Coverage Map of C1 G5, A3. The highlighted green area represents the polypeptide found in C isolate at t=6h

For the C Commercial, the peptides found by t=6h were also identified by the MS software. The

results are in Table 16.

Table 16 – Matched Sequences with the masses found at t=6h C Commercial hydrolysate

Sequence Name Mass Amino acid position

C, C1, G3, A1b 12771 101-210

C, C1, G5, A3 10552 228-317

C, C2 α’ 10321 344-434

The highlighted parts on the amino acid sequences on Figure 55, Figure 56 and Figure 57

represent the compounds on Table 16.

62

Figure 55- Sequence coverage map of the C1 G3 A1b. The highlighted green peptide is the one referenced in Table 16, compound with 12 771 Da. The green coloured letters represent all the parts of this polypeptide

found in the sample at t=6 h combining all compounds found.

Figure 56- Sequence coverage map of the C1 G5 A3. The highlighted green peptide is the one referenced in Table 16, compound with 10 552 Da. The green coloured letters represent all the parts of this polypeptide found in the sample at t=6 h combining all compounds found.

Figure 57- Sequence coverage map of the C2 α’. The highlighted green peptide is the one referenced in Table 16, compound with 10 321 Da. The green coloured letters represent all the parts of this polypeptide found in the sample at t=6 h combining all compounds found.

The bigger peptides found by the end of reaction of C were possible to identify unlike what

happened before for the B and the A proteins.

63

3.4.3.4. Protein D

Figure 58 shows the hydrolysates obtained from D protein, with and without pH control, for t=0 to

t=6 h, in an SDS-gel electrophoresis. It is clear that D proteins hydrolyse much faster that all other

proteins. After only 0.5 h of reaction there is no intact protein remaining and the polypeptides

formed are really small.

Figure 58 - SDS-PAGE of the D hydrolysates from t= 0h to t=6h with and without pH control

From the supplier, this protein powder must contain only D1 and D2 and in fact this are the

proteins detected in the SDS-PAGE gel. There are some other molecules with higher molecular

weights also appearing in the SDS-PAGE but in much lower concentration.

The MS software output gives confirmation of the presence of D1 and D2 by t=0 but it gives also

some other compounds with a close molecular weight from D2. Table 17 shows the masses found

by the software. Comparing to literature, that can be found in Appendix B Table 43, the masses

found are similar. Still there are 3 other masses that differ a little from D2 mass. However, this 3

are probably D1 proteins with some post translation modifications associated to them that might

occurred during the processing of this commercial D protein.

Table 17 – Compounds found at t=0 supernatant of D protein, with respective correspondence based on

literature molecular weights

Compound Mass (Da)

1 14186 D1

2 18281 D2

3 18367

4 18368

5 18388

64

3.4.4. Hydrolysates Peptide Pattern

Peptide Average Molecular Weight

In order to try to compare the peptides formed by each protein, an average molecular weight was

calculated, for the end of the hydrolysis reactions, this is for the t=6h hydrolysates. To do this the

hydrolysis supernatants were run in the LCMS (see Appendix C) and after with the MS Software

Qualitative Mass Hunter, a compound identification was done on the hydrolysate’s samples. After

obtaining the masses of the peptides, the calculation to obtain an average mass was the one

represented by equation

Average MW =

∑ Molecular Weighti × Peak Heightini=1

∑ Peak Heightini=1

(15)

As it was reviewed in Chapter 3.4.3, by the end of the hydrolysis reaction there are still some

intact proteins chains in solution. Because of that two average molecular weights were calculated:

the first one complies all the compounds of found, and the second one only the compounds below

10 kDa. This separation was done since the compounds with a molecular weight above 10 kDa

correspond to intact proteins that weren’t hydrolysed, or at least large polypeptides, while the

ones below 10 kDa correspond to the actual peptides formed in the hydrolysis. This will help to

understand what is the real average size of the product peptides without compromising the

average by the large protein remaining in solution.

MS identification by protein sequence

The compounds found by the Qualitative Analysis Mass Hunter software, were then matched to

the respective amino acids’ sequences, found in literature[33], by using the function “Match

Sequence” in the same software. The software identifies proteins by approximating the molecular

weights of the peptides found, with possible peptides formed during hydrolysis taking the literature

amino acids sequences [33] as reference. So, basically, the software breaks the amino acids’

sequences from literature [33] to try to find peptides with the same molecular weight as the ones

found by spectrometry. Then, it gives a list of possible matches and decides on the closest match.

The fact that the software works on approximation and that we are dealing with a lot of variety of

proteins makes this software very sensitive. A simple modification on the proteins from the project

can alter this identification. Anyway, this analysis was done, and without introducing any

modifications and only using the proteins varieties found in literature [33], this were the results

obtained.

65

3.4.4.1. Protein A

In Table 18, despite having still some intact proteins in solution, the peptides produced by

hydrolysis of the A isolate (616 Da) are, in average, smaller than the peptides produced by the A

extract hydrolysis (791 Da). This is an expected result since the DH of the isolate is higher than

the one of the extract. Also, when looking to all compounds existing in solution the difference

between the two averages is even bigger, 1671 kDa of the A extract to 658 kDa of the A isolate,

because this average complies all the proteins and polypeptides that aren’t hydrolysed yet.

Despite having heavier peptides resulting from hydrolysis in the A isolate, there is a much bigger

quantity of compounds above 10 kDa in the A extract that give this difference in the average

molecular weight.

Table 18 - Average molecular weight of the peptides obtained by A proteins’ hydrolysis, after 6 hours

A isolate A Extract

Number of compounds found by the MS software 2 740 997

MW of the smallest compound (Da) 102 119

MW of the largest compound (Da) 19 893 29 843

Average MW of all compounds (Da) 658 1 671

Average MW of all peptides below 10 kDa (Da) 616 791

MS identification by protein sequence

In the case of protein A, the great majority of the compounds was identified as Table 19 shows.

Between the two, a lot more peptides were found in the case of the isolate than the hydrolysate,

which agrees with the fact that the DH is higher in the isolate, and so produces more peptides.

Table 19 – Percentage of peptides identified by the MS software for time 6 h of the A proteins

A Extract A Isolate

# different identified compounds 971 2600

# total different compounds 997 2740

% identified 97% 95%

Table 20 and the tables of the same genre displayed in the next page for all proteins A, B, C and

D, are a resume of the identification results. The first column gives us the percentage of sequence

from each intact protein from A, B, C or D, that was found when combining all the peptides. The

second column gives us the number of different compounds found, remembering that this is a

qualitative analysis and not quantitative.

66

Table 20 – Identification of the peptides at t=6h of A extract comparing with literature sequence of amino

acids

Original intact protein sequence

% of sequence found

# different peptides

A1 A, α 96% 210

A1 A, β 94% 120

A1 A2, α 90% 16

A1 A2, β 0% 0

A1 B, α 76% 42

A1 B, β 81% 58

A1 J, α 96% 100

A1 J, β 70% 30

A1 K, α 22% 8

A1 K, β 27% 15

A2 96% 190

A2 14 kDa 78% 22

A3 92% 112

A4 86% 48

For A extract, just A1 A2 β was not found in the peptides but also it is not found in the pellets.

This can only mean that the variety of pea used in this project doesn’t have this protein in it. All

the rest of the proteins were hydrolysed and a big percentage of them is found in the hydrolysates

peptides by t=6h.

Table 21- Identification of the A isolate compounds

Original intact protein sequence

% of sequence found # different peptides

A1 A, α 99 % 598

A1 A, β 100 % 346

A1 A2, α 53 % 56

A1 A2, β 5 % 4

A1 B, α 98 % 134

A1 B, β 98 % 210

A1 J, α 97 % 243

A1 J, β 70 % 65

A1 K, α 55 % 20

A1 K, β 74 % 36

A2 98 % 462

A2 14 kDa 89 % 49

A3 97 % 277

A4 95 % 100

67

In the case of the isolate, the results say that 4 different peptides that are originally from A1 A2 β

exist. This can be true, but more probably, since the value is so low, we can consider that the

identification is fallacious for the reason already explain in the beginning of this subchapter.

3.4.4.2. Protein B

The B results show that, again, the peptides produced from the isolate, with average molecular

weight of 708 Da, are smaller than the ones from the extract, that have an average molecular

weight of 770 Da. Still, the difference is not that big between the two, but the number of peptides

found is. As it is visible in Table 22, the number of peptides is one order of magnitude different.

When it comes to all compounds found, it is visible that the average molecular weight of the B

extract, 758 Da, is higher that the B isolate, 857 Da, meaning that there are more intact proteins

found in the B extract that the B isolate, which is normal since it doesn’t hydrolyse as much.

Table 22- Average molecular weight of the peptides obtained by B proteins’ hydrolysis, after 6 hours

B isolate B Extract

Number of compounds found by the MS software 2402 109

MW of the smallest compound (Da) 113 117

MW of the largest compound (Da) 20 465 20 464

Average MW of all compounds (Da) 758 857

Average MW of all peptides below 10 kDa (Da) 708 770

MS identification by protein sequence

For B protein, like A protein, the great majority of the compounds was identified, as Table 23

describes.

Table 23 - Percentage of peptides identified by the MS software for time 6 h of the B proteins

B Extract B Isolate

# identified compounds 458 2234

# total compounds 487 2402

% identified 94% 93%

In the results from Table 24 and Table 25 some varieties of the proteins described in the literature

seem to not exist in the one used in this project since the identification is 0% for both extract and

isolate, like B1 B2 α; B1 B6 α; B1 B7 β.

In these two tables it is also visible that B2 protein gives the most different types of peptides,

which is normal since this is a protein with a long amino acids’ sequence. Comparing the two we

can see that the isolate produces a higher variety of peptides than the extract, which agrees with

the fact that the DH of the isolate is higher and that the average molecular weight of the peptides

is lower (Table 22).

68

Table 24 - Identification of the peptides at t=6h of B isolate comparing with literature sequence of amino

acids

Original intact protein sequence

% of sequence found # different peptides

B1 B2 α 0 % 0

B1 B2 β 100 % 489

B1 B4 α 92 % 349

B1 B4 β 100 % 491

B1 B6 α 0 % 0

B1 B6 β 40 % 16

B1 B7 α 19 % 5

B1 B7 β 0 % 0

B2 99 % 884

Table 25- Identification of the peptides at t=6h of B extract comparing with literature sequence of amino

acids

Original intact protein sequence

% of sequence found # different peptides

B1 B2 α 0% 0

B1 B2 β 97% 93

B1 B4 α 98% 78

B1 B4 β 100% 77

B1 B6 α 0% 0

B1 B6 β 87% 7

B1 B7 α 19% 3

B1 B7 β 0% 0

B2 99% 200

3.4.4.3. Protein C

Comparing the two isolates (table 26), the self-produced C isolate and the commercial C, the

biggest difference is that the commercial C produces the double of peptides of the self-produced

C isolate, which was expectable since in the previous chapter, 3.4.3 we could see that the

commercial C hydrolyses much faster. Also, the fact that the molecular weight of the largest

protein of C commercial is much lower than the one from the largest protein of C isolate evidences

this difference in DH. In terms of the sizes the average of the commercial C is also lower (621

kDa) than the self-produced isolate (660 kDa).

69

Table 26-Average molecular weight of the peptides obtained by C proteins’ hydrolysis, by t=6h

C isolate C Commercial

Number of compounds found by the MS software 1243 2926

MW of the smallest compound (Da) 102 100

MW of the largest compound (Da) 21 104 12 771

Average MW of all compounds (Da) 703 627

Average MW of all peptides below 10 kDa (Da) 660 621

The identification numbers are also quite disparate (table 27), fact that is related with identified

peptides percentage. This two facts can only mean that this two C proteins are very different

varieties, or that the commercial produced C, suffered such harsh conditions that the proteins are

very different from their natural state.

Table 27-Percentage of peptides identified by the MS software for time 6 h with C proteins

C Commercial C Isolate

# identified compounds 606 1188

# total compounds 2930 1243

% identified 21% 96%

The numbers of the percentages of sequences found (Table 28 and 29) show again a big disparity

between the two confirming that these are very different varieties of each proteins. The clearest

example is the C1 G1, A1a that we can consider inexistent in C isolate, and that in the C

commercial 50% of the sequence was found.

Table 28- Identification of the peptides at t=6h of C commercial comparing with literature sequence of amino acids

Original intact protein sequence

% of sequence found # different peptides

C1 G1, A1a 51% 40

C1 G1, B2 31% 32

C1 G2, A2 22% 34

C1 G2, B1a 7% 10

C1 G3, A1b 16% 22

C1 G3, B1b 18% 13

C1 G4, A5 27% 16

C1 G4, A4 28% 80

C1 G4, B3 44% 42

C1 G5, A3 22% 22

C1 G5, B4 17% 16

C2, α 41% 105

C2, α’ 37% 62

C2, β 41% 112

70

Table 29- Identification of the peptides at t=6h of C isolate comparing with literature sequence of amino acids

Original intact protein sequence

% of sequence found # different peptides

C1 G1, A1a 2 % 1

C1 G1, B2 99 % 283

C1 G2, A2 95 % 200

C1 G2, B1a 70 % 58

C1 G3, A1b 71 % 64

C1 G3, B1b 30 % 15

C1 G4, A5 83 % 76

C1 G4, A4 82 % 36

C1 G4, B3 83 % 76

C1 G5, A3 63 % 49

C1 G5, B4 51 % 25

C2, α 90 % 180

C2, α’ 64 % 73

C2, β 67 % 52

3.4.4.4. Protein D

The average molecular weight of the animal protein D is in the same range as the vegetable

proteins.

Table 30- Average molecular weight of the peptides obtained by D protein’s hydrolysis, after 6 hours

Commercial D

Number of compounds found by the MS software 2 591

MW of the smallest compound (Da) 109

MW of the largest compound (Da) 14 474

Average MW of all compounds (Da) 691

Average MW of all peptides below 10 kDa (Da) 640

We can see that a lot of peptides were found, 2591, and in Table 31, that 71% of this compounds

were identified. It is fair to say that this is the identification using the amino acids sequences

without any modifications, that can occur. For this protein, a some proposes for modifications are

already studied, [33] suggest some, in contrary to the others but they were not tested, since this

was used only as a method of comparison for the vegetable proteins.

Table 31- Percentage of D hydrolysates identified by the MS software for time 6 h

Commercial D

# identified compounds 1832

# total compounds 2591

% identified 71%

71

Anyway, for the two intact proteins were found peptides that completed all the amino acid

sequence, Table 32, showing that the proteins D1 and D2 in this protein powder are the same as

referenced in literature, and aren’t modified.

Table 32- Average molecular weight of the peptides obtained by D protein’ hydrolysis, after 6 hours

Original intact protein sequence

% of sequence found

# different peptides

D1 100% 720 D2 100% 1112 unidentified 759

total # 2591

72

73

Chapter 4 |Conclusion and Future Work

This work focused on the study of the hydrolysis of plant and animal proteins. It is known that

aggregation is the principal cause of material lost in industry and so the first objective of the work

was to observe this phenomena.

During hydrolysis the pH decreases, and this means that the protein solutions will be closer to the

pI. Because of that fact, it could be expectable that the aggregation would be increased in the

reactions without pH control. However, the study of agglomeration showed that the pH control

only impacts agglomeration significantly in animal protein, D, since it is verified that there is no

agglomeration when the pH is maintained throughout the reaction. In vegetable proteins, A, B and

C, the difference between agglomeration when controlling or not the pH of the protein solution is

not significant.

Two different types of vegetable proteins were studied, using proteins A and B: extracts and

isolates. The difference between them is that extracts contain both globulins and albumins, while

isolates only contain globulins. When compared with each other, extracts always have more

agglomeration than the isolates, meaning that the albumins promote a bigger interaction between

the proteins, either between them only or between albumins and globulins. In chapter 3.3.3. it is

confirmed that the albumins inhibit hydrolysis reaction and not only agglomerate. In terms of the

agglomerates time of formation, the general appreciation for all proteins is that they are mainly

formed right in the beginning of the reaction, varying slightly in concentration the rest of the time.

In terms of agglomeration, in order to compare the proteins, the results obtained in the thesis,

when the pH was controlled, can be ordered in the following decreasing order: D (0 g/g), C isolate

(2.6-4.6 g/g), B isolate (3-4.2 g/g), A isolate (3.9-4.6 g/g), C Commercial (4-5.4 g/g), B extract (4-

5.6 g/g), A extract (6.6-9.2 g/g). This results represent the grams of wet pellet per grams of dry

intact protein in the beginning of the reaction. Some proteins’ pellets were dried but unfortunately

not all of them were, because the project didn’t start with that method and it was not possible to

repeat all hydrolysis and dry all the pellets. Because of that, the values are above 1, because they

describe not only the grams of protein but also the grams of water held by the pellets. The most

correct way of comparing the actual protein mass that agglomerates would be comparing the dry

pellets, but since it was not possible the comparison was made like this. For protein A, the pellets

of both isolate and extract were dried and so a more precise comparison can be done. The dry

extract agglomerates of protein A were approximately (0.8 g dry pellet /g intact protein) while the

isolates resumed in approximately (0.3 g dry pellet /g intact protein). So, despite the fact that the

wet pellets take in consideration not only protein content but also water holding, we can still see

that overall the isolates form less agglomerates than the extracts, and that all of the vegetable

proteins form more pellet than the animal one, that doesn’t agglomerate.

74

When it comes to the DH, the pH control has a significant impact for both animal and vegetable

proteins. For both protein types, the pH-controlled hydrolysates have a higher increase of

concentration of 𝑁𝐻2 which means that there are more peptides being formed and therefore a

higher extent of DH. Comparing all proteins, in the trials with pH control, the results can also be

ordered in a decreasing order: D Commercial (16%), A isolate (14%), C Commercial (13%), B

isolate (11%), C isolate (10%), A extract (9%), B extract (4%).

Observing the two orders of DH and agglomeration, clearly D protein performs best in hydrolysis,

either in yield (less agglomeration) or extent of DH. Also, the isolates outperform the extracts in

both DH and yield. When comparing the C isolates, C Commercial (powder) and the C self-

produced isolate (liquid), the C Commercial powder allows a higher DH extent than the C isolate

liquid solution, but in terms of the yield (aggregation), the commercial powder is a worst choice

since it promotes more agglomeration, and therefore more loss of material (C commercial:4-5.4

g/g; C isolate:2.6-4.6 g/g). The choice between the two, has to consider which factor is most

important in the process. This needs to be evaluated, economically and in terms of the peptides

obtained from each type of protein during the same amount of time. In this work, there can already

be seen some identification of the peptides present, but they are not quantified. With this said, a

study of the pretended properties, like the solubility or foaming, and an economic study should be

done to both the C powder and the C liquid proteins in order to evaluate if the pretended

hydrolysates obtained from the liquid achieve the same properties pretended in the same amount

of time and using the same amount of raw material.

For future work, a different method should be applied to calculate the extent of the hydrolysis in

the extracts, since the OPA method revealed to be quite sensitive and unprecise.

Since C protein isolate solution was stable frozen the same was assumed for A and B isolates.

During the project, an abnormal quantity of precipitate was observed in protein A even before the

hydrolysis. After a study that involved testing the heating step to reaction temperature, enzyme

heat inactivation and freezing step, it was found that protein A cannot be frozen before hydrolysis.

This step, done in the lab for convenience of time, promoted an unnecessary precipitation of

protein, and therefore loss of raw material. For A hydrolysis purpose, it is not recommended to

freeze the solution at any time, at least in the conventional way. Freezing with liquid nitrogen was

not tested and can be a solution.

An unexpected observation after all the different hydrolysis performed in this work was the fact

that A extract hydrolysis followed different kinetics from all the others tested, and the ones

reported in literature. This fact was confirmed by multiple extractions and hydrolysis, which

resulted in the same kinetics. The downward in the DH curves observed in all the others is not

observed in A extract which leads to thinking that there aren’t product inhibitors in this case. It is

a fact that the reaction is happening and therefore the peptides are being produced but they are

not inhibiting the reaction, which means they are not competing with the original substrate

proteins. The reason why this happens was not determined by the end of this project and definitely

75

needs more research at molecular level, to understand why this peptides are not inhibiting the

reaction, or what is happening to them.

When it comes to the identification of the hydrolysates, as we can see in the tables in Chapter

3.4.4, the majority of the peptides were identified. For proteins A and B, the identification of the

peptides was always superior to 90%, as well as the C protein isolate. C commercial protein

isolate had the lowest identification of only 21% of the peptides, and the commercial D protein:

71% of the peptides. In the case of the C commercial, this lower identification can either mean

that the variety of protein, isn’t the same as the one consulted in the literature or that this is a very

modified protein. It is noticeable that all the peptides produced from the extracts and isolated self-

produced in the lab are better identified because they didn’t suffer any type of processing, and

therefore, only natural modifications can have occurred in this proteins. That is why the

percentage of peptides identified is so high in this cases. In each table can be consulted the

percentage of each protein identified by the end of reaction and accessing the program can be

verified each of the peptides formed. Due to the fact that the list of peptides for each hydrolysis is

so extensive only a resume and a range of molecular weights is presented, and this were not

attached in Appendix. Anyway, this work produced data bases of all the peptides formed during

hydrolysis that can be accessed using the Mass Spectrometry software. There, it is possible to

see every peptide formed and its original intact protein, when an identification was achieved.

The great barrier found in the identification of the molecules, in the hydrolysates, was the

undefined modifications of the proteins and also the great variety of proteins existing. Despite the

high values of identification of the peptides by t=6h, when compared to the initial intact proteins

there is practically zero identification. As it is visible in chapter 3.4.3. contrarily to the peptides,

the intact proteins identified are few. The truth is that the small percentage of peptides that aren’t

identified block the identification of the original intact protein. To do this, it is necessary to discover

which are the modifications that occurred to this unidentified parts. This procedure was tried but

the work revealed to be quite hard and, the software to be quite sensitive since it works with

approximations. A new software of statistics analysis was expected to help with this but

unfortunately it didn’t arrive in time, and so this was not possible to achieve. Also, this identification

work takes a lot of time, and this were results impossible to produce in such short time. A future

idea could be of using a technique to remove attached molecules, such as sugars, from the

peptides, without damaging them.

However, it was possible to calculate an average molecular weight of the hydrolysates obtained

after 6 h for each protein, since the LCMS identifies all the masses despite knowing or not to each

protein they belong to. The ordered results obtained were A Isolate (616 Da); C Commercial (621

Da); D Commercial (640 Da); C isolate (660 Da); B isolate (708Da); B extract (770 Da); A extract

(791 Da).

As a last observation, a similar pattern throughout all vegetable proteins was observed. A previous

study on soy protein proposed a thesis where the acid polypeptide of glycinin is shielding the

basic one quite well, and thereby preventing its degradation. [3] Because of that, α-glycinin is first

76

hydrolysed and just after that the enzyme has full access to the β-glycinin. When observing the

other vegetable proteins, A, B and C the same effect was detected for the respective A1, B1 and

C1 proteins. A1, B1 and C1 have the same structure of the glycinin, a globular α and β structure,

and so this is a quite understandable fact. A1, B1 and C1 β chains are always the last ones to be

hydrolysed and so they stay in solution and in the agglomerates longer.

ii

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5

Appendix

A. Agglomeration

Table 33 - Compounds found for t=0h agglomerate of A isolate

Compound Mass (Da) Peak Height

1 48887 8942

2 36584 19216

3 36584 9413

4 35213 16787

5 35187 19949

6 35162 8964

7 35130 5389

8 35130 44033

9 35023 15921

10 19892 18146

11 19876 31219

12 19846 28687

13 19829 122288

14 19694 65425

15 19559 38511

16 19559 83374

17 12404 57898

Table 34- Compounds with a molecular weight higher than 10 kDa found in the hydrolysate of A isolate after 6 hours of

reaction by the Qualitative Mass Hunter Software

Table 35 - Compounds found at t=0 sample agglomerate of the A extract

Compound Mass (Da) Peak Height

1 48958.42 21869

2 48889.16 29350

3 48855.4 27822

4 36584.16 27298

5 36496.27 24481

6 36424.83 14269

7 35217.58 16516

8 35187.64 46288

9 35159.88 17424

10 35130.84 136153

11 35109.47 7363

12 35023.71 59969

13 20802.9 18788

14 19909.49 18288

15 19893.08 32414

16 19876.98 46378

17 19860.62 17640

18 19846.29 45055

19 19829.35 127897

20 12404.65 62648

Compound Average Mass

1 11266.3

2 19829.1

3 19846.1

4 19876.3

5 19892.6

6

Table 36- Compounds found at t=0 sample agglomerate of the B isolate

Compound Mass (Da) Peak Height

1 30179.79 33203

2 30111.78 55922

3 20465.68 502751

4 20464.85 10793

5 20165.76 6426

6 20150.27 157373

7 20149.84 13472

8 20124.38 215119

9 20024.42 10263

10 20023.96 160726

11 19035.24 11121

12 18536.51 35780

13 18260.27 78432

14 17457.38 70919

15 17030.04 56922

16 16943.72 37515

17 16500.37 254278

18 16499.50 5631

19 16387.43 120802

20 15616.12 8974

21 15304.01 71720

22 14230.73 120521

23 14034.03 7553

24 13996.43 6156

25 12608.38 76622

26 12002.72 240736

27 12001.82 5681

28 11730.61 25940

29 10421.17 102228

30 9385.26 7146

31 8031.39 4592

32 6723.84 9879

33 5179.31 7591

34 5020.54 10211

Table 37 - Compounds found at t=6h agglomerate of the B isolate

Compound Mass (Da) Peak Height

1 30179.79 33203

2 30111.78 55922

3 20465.68 502751

4 20464.85 10793

5 20165.76 6426

6 20150.27 157373

7 20149.84 13472

8 20124.38 215119

9 20024.42 10263

10 20023.96 160726

11 19035.24 11121

12 18536.51 35780

13 18260.27 78432

14 17457.38 70919

15 17030.04 56922

16 16943.72 37515

17 16500.37 254278

18 16499.50 5631

19 16387.43 120802

20 15616.12 8974

21 15304.01 71720

22 14230.73 120521

23 14034.03 7553

24 13996.43 6156

25 12608.38 76622

26 12002.72 240736

27 12001.82 5681

28 11730.61 25940

29 10421.17 102228

30 9385.26 7146

31 8031.39 4592

32 6723.84 9879

33 5179.31 7591

34 5020.54 10211

7

Table 38- Compounds found at t=0h agglomerate of the B extract

Compounds Mass (Da) Peak Height

1 48782.67 13133

2 48651.66 8916

3 48609.45 16760

4 48582.29 31663

5 48579.28 13996

6 48563.91 20966

7 48556.01 13923

8 48511.83 18493

9 48451.29 29302

10 48409.71 15415

11 48383.27 37218

12 48363.67 27894

13 48348.73 7128

14 48282.50 4326

15 48255.40 7043

16 41185.25 24931

17 40355.34 23039

18 40338.19 61451

19 34188.85 13564

20 34142.49 50088

21 34121.60 23315

22 34102.16 15149

23 34073.57 84393

24 34053.37 29895

25 34028.40 64477

26 34007.16 33283

27 33959.36 92505

28 33939.41 22300

29 32009.89 36695

30 31990.46 76670

31 31961.68 439145

32 31946.89 59747

33 31940.75 55028

34 31926.54 3002

35 31922.14 18324

36 31875.52 87457

37 31847.58 66321

38 31818.88 46074

39 31761.15 12649

40 27459.87 31483

41 27188.45 92120

42 24307.62 22037

43 21620.22 47464

44 20214.54 31593

45 20167.45 67114

46 20150.11 139976

47 20136.48 67118

48 20119.49 70036

49 20100.88 33071

50 20088.15 16978

51 20071.45 182668

52 20024.82 99470

53 12442.80 56982

54 12372.05 469152

8

Table 39 - Compounds found at t=6h sample agglomerate of the B extract

Compounds Mass (Da) Peak Height

1 35799.70 28961

2 35766.54 32703

3 35761.77 50520

4 35675.40 52779

5 35675.37 134698

6 35674.63 11949

7 35674.44 11918

8 35666.81 38802

9 35417.47 15365

10 35213.42 21415

11 34127.31 27355

12 30226.77 15354

13 30180.40 141270

14 30159.77 31833

15 30111.38 223368

16 30091.49 31930

17 30042.31 42881

18 29781.86 58717

19 29712.87 46413

20 28381.38 94796

21 22286.51 116202

22 22269.88 214242

23 21858.34 31803

24 21855.45 31768

25 21836.58 144952

26 21826.44 35704

27 21800.12 22991

28 21749.81 101444

29 21262.90 37539

30 21089.51 28309

31 21071.07 22087

32 21054.19 152981

33 21041.63 360785

34 21021.20 7335

35 20926.12 67161

36 20925.23 79892

37 20854.19 573187

38 20854.18 21529

39 20836.76 19657

40 20771.24 59344

41 20712.17 16234

42 20589.53 27518

43 20466.36 36269

44 20465.49 856587

45 20450.18 119424

46 20287.38 372676

47 20277.26 20811

48 20245.28 53582

49 20235.80 43741

50 20219.26 148074

51 20214.24 287636

52 20182.47 85862

53 20166.72 449438

54 20150.26 25675

55 20150.18 1013154

56 20135.33 286737

57 20124.32 376503

58 20123.92 142983

59 20120.56 51369

60 20105.67 61322

61 20101.93 31762

62 20098.94 38291

63 20080.42 91569

64 20071.39 571548

65 20063.79 54945

66 20061.13 291683

67 20053.30 180405

68 20045.41 48444

69 20024.38 743323

70 20023.36 36878

71 19824.94 12573

72 19676.58 16513

73 19592.67 8272

74 15421.90 5915

75 15406.73 16328

76 15406.70 401319

77 15249.18 241735

78 15148.39 45648

79 14939.85 12910

80 14926.56 363983

81 14367.46 262312

82 14227.27 11057

83 12002.69 146939

84 10915.06 430795

85 10687.65 71680

9

B. Literature Proteins’ Molecular Weights

Table 40 - Literature Molecular Weights of A proteins

Table 41- Literature Molecular Weights of B proteins

10

Table 42- Literature Molecular Weights of C proteins

Table 43- Literature Molecular Weights of D proteins

11

C. Chromatograms obtained by the LCMS of the

hydrolysates

Protein A

Figure 59 – Mass Spectrometry results on A isolate hydrolysates after hydrolysis with pH control at times 0, 0.5, 2 and 6 h.

Figure 60 - Mass Spectrometry results on A extract hydrolysates after hydrolysis with pH control at times 0, 0.5, 2 and 6 h.

12

Protein B

Figure 61- Mass Spectrometry results on B isolate hydrolysates after hydrolysis with pH control at times 0,

0.5, 2 and 6 h.

Figure 62 - Mass Spectrometry results on B extract hydrolysates after hydrolysis with pH control at times 0, 0.5, 2 and 6 h.

13

Protein C

Figure 63 - Mass Spectrometry results on C isolate hydrolysates after hydrolysis with pH control at times 0, 0.5, 2 and 6 h.

Figure 64 - Mass Spectrometry results on C commercial hydrolysates after hydrolysis with pH control at

times 0, 0.5, 2 and 6 h.

14

Protein D

Figure 65 - Mass Spectrometry results on D commercial hydrolysates after hydrolysis with pH control at times 0, 0.5, 2 and 6 h.