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THE EFFECT OF DIFFERENT STORAGE CONDITIONS ON THE

QUALITY OF ORANGE JUICE

Mary lene Lagace

Department of Food Science and Agricultural Chemistry

McGill University, Montreal

October, 1 998

A thesis submitted to the Faculty of Graduate Studies and Research in partial Fulfilment

of the requirements of the degree of Master of Science

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ABSTRACT

Unpasteurized (condition A) and pasteurized (condition 8) orange juice samples

were stored frozen for eight months. In addition, pasteurized samples were also

aseptically packaged and stored at +1 OC in polyethylene bags (condition C). Nine quality

parameters were monitored during the eight months of storage: sedimentation of the pulp.

cloud measurement. aroma volatiles, ascorbic acid concentration. viscosity, density,

colour. sugar content (sucrose. glucose and fructose). organic acids (malic and citric), in

addition to sensory analysis. The optimum storage condition for freshly processed orange

juice was the unpasteurized frozen storage method (condition A). The juice retained most

of its chemical and physical properties and was rated by a sensory panel to have the

highest sensory score.

Des echantillons de jus d'orange non-pasteurises (condition A) et pasteurises

(condition B) sont congeles pour huit mois. De plus, des echantillons pasteurises sont

aussi emballCs aseptiquement et entreposes a +I OC dam des sacs de polyethyhe

(condition C). Neuf paramktres de qualite sont ttudies durant les huit rnois

d'entreposage: la sedimentation de la pulpe, la mesure de l'opacite, Ies arhes volatiles.

I'acide ascorbique. la viscosite, la densite. la couleur. le contenu en sucre (sucrose,

glucose and fructose). les acides organiques (malique et citrique), de mOme que

l'evaluation sensorielle. La condition d'entreposage optimale pour un jus d'orange frais

presse est la methode de congelation sans pasteurization (condition A). Ce jus retient la

plupart de ces proprietes. chimiques et physiques. et obtient la note la plus haute lon de

l'evaluation sensorielle.

ACKNOWLEDGEMENTS

I would like to thank my supervisor Dr. V.A. Yaylayan for his advice and his supervision,

and my co-supervisor Dr. E. Farnworth, researcher at the Food Research and

Development Center at Saint-Hyacinthe, for his availability and his patience.

Dr. R. Couture and A. Lassonde Inc. company who permitted me to do a master's degree

while working hl l time.

Jean Ledoux and Diep Vu froin A. Lassonde company for their help with HPLC analyses

and viscosity measurements. Guylaine Dery and Carole Marchand in charge of sensory

evaluation.

Brian Stewart for his availability and his expertise in gas chromatography analysis.

And finally thank you to my family for their suppon, my boyfriend Francois Brion (for

his patience), and all my colleagues at FRDC, especially Pascal Daigle. Julie Barrette.

Denise Chabot. Franqois St-Germain. Christine Gendron. Claude Gagnon. etc.. who

helped me in many ways during my master's degree.

TABLE OF CONTENTS

ABSTRACT ii

iii

ACmO WLEDGEMENTS iv

TABLE OF CONTENTS v

LIST OF TABLES ilr

LIST OF FIGURES xiii

GENERAL INTRODUCTION I

LITERA TURE REVIEW 3

2.1 Principal parts o f an orange 3

2.2 Pasteurization I

2.2.1 Purpose of pasteurization 4

2.2.2 Pasteurization Time-Temperature 5

2.2.3 ChemicaI changes during pasteurization 5

2.3 Storage 6

2.3.1 Frozen juice 6

2.3.2 Aseptically packaged juice 7

2.3.3 Storage studies of orange juice 8

2.3.4 Indicators of chemical degradation 9

2.4 Principal compositional analyses performed during storage studies 9

2.42 Ascorbic acid IS

2.4.3 Sugars and organic acids 18

2-43. I Sugars I 8

2-4-32 Organic acids 19

2.4.4 Colour 20

2.4.5 Sedimentation of pulp, cloud. viscosity and density -- 7 7

2.1.5.1 Sedimentation of pulp 12

2.4.5.2 Cloud 23

2.4.5.3 Viscosity and density 23

2.4.6 Sensory analysis 24

CHAPTER 3

M4 T E W S AND METHODS 26

3.1 Experimental design 26

3 5 Sampling of otange juice at Mexico 28

3.3 Sample preparation for analytical analyses 29

3.5 Analytical methods 29

3 . 4 Sedimentation of the pulp 29

3.4.2 Cloud measurement 3 0

3.4.3 Volatiles 5 0

5-43.! Gas chromatography coupled with a mass spectrometer (GC-MS) 30

A. Internal standard mixture 3 0

B. Volatile standards 3 1

Primary stock solution 3 I

Secondary standard solutions 51

C. Sample preparation 34

D. GC-MS conditions 34

3.4.3.2 Gas chromatography with a headspace injector (Headspace-GC-FID) 35

A. Internal standard mixture 35

B. Primary stock solution 35

C. Sample preparation 36

D. Headspace-GC conditions 36

3-44 Viscosity and density measurements 36

3.4.5 Ascorbic acid 37

3.4.6 Colour measurement 38

3.47 Sugars and organic acids 38

3 N . 1 High performance liquid chromatography (HPLC) 38

A. Standard sugar soiutions 38

vii

B- Standard organic acid solutions 38

C. Standard calibration cume 39

D. Sample preparation 39

E. HPLC conditions 39

3.4.7.2 "Brix 40

3.4.7.3. Titratable acidity 40

3.4.8 Sensory evaluation 41

3.4.9 Statistical treatment 42

RESUL TS AND DISCUSSION 43

4.1 Effect of storage condition on individual parameters 43

4.1 . I Density 43

4.1.2 Cloud and sedimentation of the pulp 45

4.1.3 Sugars 47

4.1.3.1 Brix measurement 47

4. 1.3.2 HPLC measurements 48

4. I .-I Organic acids 52

4. I A. 1 Titratable acidity measurements 5 2

4.1 A.2 HPLC measurements 54

4.1.5 Ascorbic acid 57

4.1.6 Viscosity 60

4.1.7 Colour 63

4.1.8 Volatiles 67 - - - --

4. 1.9 Sensory evaluation 83

4.2 Summary of all the parameters 90

4 3 Correlation between chemical and physical changes and sensory evaluation 92

CHAPTER 5

CONCL LlSIOlV 94

REFERENCES 96

APPErnLXA

PROFIL DESCRIPTIF QUANTITA TIF* I05

viii

APPENDIX B

EXAlMPLES OFSTATISTlCAL CALCULATIONS 108

APPErnIX C

CONCENTRATION OF VOLA TILES IN ORANGE JUICE FOR

THE MONTHS 2-6 112

LIST OF TABLES

Table number Page

Volatile components which contribute to fresh orange flavour 10

Thermally degraded compounds derived from essential oil constituents 12

Degradation products in canned single strength orange juice after

12 weeks storage at 35 O C 15

Parameters measured 27

Volatile standards for the primary stock solution ( 100 mL) 32

Density value of orange juice over an eight month storage period 44

Equation of the regression curves and coefficients of regression (R')

of the density of orange juice over an eight month storage period 44

Sedimentation of the pulp (measured at 3 time intervals) of orange

juice over an eight month storage period 45

Cloud stability (measured at 3 time intervals) of orange juice over

an eight month storage period 16

%rix value of orange juice over an eight month storage period 47

Equation of the regression curves and coeficients of regression (R')

of the "Brix of orange juice over a eight month storage period 38

Sucrose concentration of orange juice over an eight month

storage period 39

Glucose concentration of orange juice over an eight month

storage period 49

Fructose concentration of orange juice over an eight month

storage period 50

Results obtained from the t-test for each sugar contained in orange juice-5 1

Table number

X

Page

Equation of the regression curves and coefficients of regression

(R') of the three sugars of orange juice over an eight month

storage period 52

Titratable acidity measurements of orange juice over an eight month

storage period 53

Equation of the regression curves and coefficients of regression (R') of the

titratable acidity measurement of orange juice over an eight month

storage period. 53

Citric acid concentration of orange juice over an eight month

storage period 54

Malic acid concentration of orange juice over an eight month

storage period 55

Results obtained from the t-test for each organic acid contained in orange

juice 56

Equation of the regression curves and coefficients of regression (R')

of the two organic acids of orange juice over an eight month

storage period 56

Ascorbic acid concentration of orange juice over an eight month

storage period 57

Equation of the regression curves and coefficients of regression (R')

of the ascorbic acid concentration of orange juice over an eight month

storage period 57

Viscosity measurement of orange juice over an eight month

storage period 61

Equation of the regression curves and coefficients of regression (R')

of the viscosity measurement of orange juice over an eight month

storage period 61

Table number

xi

Page

Colour measurement (L value) of orange juice over an eight month

storage period 64

Equation of the regression curves and coefficients of regression (R')

of the colour measurement (L value) of orange juice over an eight

month storage period 65

Volatile concentration of orange juice for the first month of storage 68

Methanol concentration of orange juice over an eight month

storage period 70

Equation of the regression curves and coefficients of regression (R')

of the methanol concentration of orange juice over an eight month

storage period 70

I -Hexan01 concentration of orange juice over a six month

storage period 72

Equation of the regression curves and coefficients of regression (R')

of the 1-hexanol concentration of orange juice over an eight month

storage period 73

P-Myrcene concentration of orange juice over a six month

storage period 75

Equation of the regression curves and coefficients of regression (R')

of the P-myrcene concentration of orange juice over an eight month

storage period 75

Limonene concentration of orange juice over a six month

storage period 78

Equation of the regression c w e s and coefficients of regression (R')

of the limonene concentration of orange juice over an eight month

storage period 78

a-Terpineol concentration of orange juice over a six month

storage period 8 1

xii

Table number Page

39. Equation of the regression curves and coefficients of regression (R')

of the a-terpineol concentration of orange juice over an eight month

storage period 8 1

40. Mean scores of sensory evaluation of orange juice over eight months

of storage 85

4 1. Summary of the changes during the period of storage 90

LJST OF FIGURES

Figure number Page

Cross section of a citrus fruit 3

Volatile quality indicators 13

Principal formation pathway of alpha-terpineol 14

Anaerobic and aerobic degradation of vitamin C (AA) in orange

juice: DKA, diketogulonic acid: HF. hydroxyfurfural 16

Experimental design of the three storage conditions 27

GC-MS chromatogram of the 36 volatile standards used in the analysis

of the orange juice samples 33

HPLC Chromatogram of the three sugars in orange juice processed by

condition A at the last month (8) of the storage period 50

HPLC chromatogram of the two organic acids in orange juice

processed by condition A at the last month ( 8 ) of the storage period 55

Ascorbic acid concentration of orange juice over an eight month

storage period 58

Viscosity of orange juice over an eight month storage period 61

Colour measurement (L value) of orange juice over an eight month

storage period 66

Methanol concentration of orange juice over an eight month

storage period 7 1

1-Hexanol concentration of orange juice over a six month

storage period 74

Beta-myrcene concenvation of orange juice over a six month

storage period 76

Limonene concentration of orange juice over a six month

storage period 79

Figure number

xiv

Page

Alpha-terpineol concentration of orange juice over a six month

storage period 82

Evolution of the attribute "orange taste" in orange juice over an eight

month storage period 86

Evolution of the attribute "fresh pressed" in orange juice over an eight

month storage period 87

Evolution of the attribute "homogeneity" in orange juice over an eight

month storage period 88

Evolution of the attribute "presence of pulp" in orange juice over an

eight month storage period 89

CHAPTER I

GENERAL INTRODUCTION

Orange juice is the most popular fruit juice but it is also the least stable (Shaw.

1996a) due to its delicate and complex flavor which is easily altered during heat treatment

and storage (Shaw er al., 1993; Shaw. 1996a). In Canada, the refrigerated juice market is

worth 190 millions dollars and more than 56 millions dollars only in Quebec (Auger.

1997). Since World War 11. the emergence of new technologies has permitted processing

industries to make orange based products market favorites. Canned, dry crystals, frozen

concentrate, chilled ready-to-serve, "not from concentrate" and fresh squeezed are

possible products for orange juice developed through the years (Brown er al., 1993). The

consumers are now considering orange juice "not from concentrate" as a premium quality

product. The composition of orange juice can depend on many factors such as growing

condition. various treatments and practices. maturity. rootstock. variety and climate.

These factors are associated with the orange fruit whereas each of them have intrinsic

properties (Velduis. 1971). Different cultivars of sweet oranges such as Harnlin. Mam,

Parson. Brown. Pera. Pineapple. Sharnouti and Valencia are available. The juice made

from freshly squeezed Valencia oranges has an excellent quality and is considered by

many as a standard by which all other juices must be compared with (Nagy, 1 996).

The shelf life of citrus juices and related beverages are primarily determined by

microbial growth and by chemical changes. Many factors have an impact on shelf life

such as processing conditions (pasteurization time and temperature), oxygen content,

properties of and type of container, storage temperature and type of product (concentrate.

single-strength, chilled. unpasteurized) (Shaw el al., 1993).

The most important factor in determining the shelf life of aseptically packaged

orange juice, concentrate and fresh squeezed juice is the storage temperature (Graumlich

et a/., 1986; Fellen, 1988). The juice can be stored aseptically (stainless steel tank or

polyethylene bag) or in frozen blocks. The pasteurized product stored aseptically above

freezing temperatures is more economicd than storage of frozen product. However

aseptically stored product can lose some of its desirable flavour top-notes and develop

non-desirable flavours (off-flavour) if it is stored for a long period (Shaw et al., 1993).

Many studies have been done on the storage of concentrated juice since the 1940's

(Curl, 1947) by measuring parameters that have been shown to be related to orange juice

quality, whereas the "not from concentrate" juice has not been studied much. This fact

will probably change in the next few years considering the popularity of this type of

product. since its quality depends in part or. the mode of storage used after pressing. This

study is the most complete research done on the applicable storage condition of the juice

"not from concentrate". A variety of instrumental and sensory evaluation techniques were

used to measure changes in the juice composition and properties. The micriobiological

aspect was not a part of this study because with low temperature and pasteurization, no

change was expected.

The objective of this project was to identify the optimum storage

conditions (time, temperature and type of container) for freshly

squeezed orange juice samples (pasteurized or not pasteurized).

CHAPTER 2

LITERATURE RE MEW

2.1 Principal parts of an orange

SEGMENT MEMBRANE

Figure 1 . Cross section of a citrus fruit (Ting and Rouseff. 1986).

The flavedo (or epicarp) is the colored portion of the peel. This part contains the

carotenoids which give the colour of the citrus fruits. Under the flavedo, is the albedo or

mesocarp. This fraction is a thick. white and spongy layer. A large part of the pectic

substance is found in the albedo. The juice vesicules are found in 9 to 13 segments

separated by segment membranes (Ting and Rouseff, 1986). During the pressing, the

fruit is cut in two parts and squeezed to obtain a mix of serum juice and pulp. Thus

components arc found from all the parts of the fruit.

2.2 Pasteurization

The juice is subject to quality degradation especially at ambient temperature due

to microbiological, enzymatic activities and chemical reactions (Chen er of., 1993). All

these changes reduce the shelf life of the orange juice because of the effects on the

nutritional quality, colour and flavour.

2.2.1 Purpose of pasteurization

Pasteurization of orange juice is required for two reasons: inactivation of pectic

enzymes and destruction of microbial population capable of causing spoilage (Carter

198 1 : Graumlich er a/., 1986). The pectic enryrne, pecdnmethylesterase (PME), naturally

present in orange juice. catalyses chemical reactions with the pectin molecule (Carter

198 1). This reaction produces a loss of cloud (clarification) in juices or gelation of

concentrates (Graumlich et (11.. 1986). Maintaining cloud is important to eye appeal and

retention of certain flavour compounds associated with the cloud matrix (Sadler et a/.,

1992). Once inactived. the PME cannot regenerate or increase in concentration (Carter.

198 1). Pasteurization is used to achieve commercial stability of cloud of various citrus

products not just orange juice (Graumlich el al.. 1986).

Shorter time-temperature pasteurization is required for microbial than for PME

inactivation (Carter, 1981). Heat pasteurization procedures established for enzyme

inactivation are therefore sufficient for microbial inactivation (Chen et al., 1993), but due

to non-aseptic transfer of the juice a second pasteurization is required for complete

microbial destruction.

2.2.2 Pasteurization Time-Tern perature

The time-temperature relation is the most important factor when pasteurization of

orange juice is carried out. At lower temperatures the time required is longer. and shorter

for higher temperatures. Most of the time two pasteurization steps are done. The first is

performed when the juice is extracted. The pectin enzymes are inactived by immediate

heat treatment at 90 C for 7 seconds. This treatment is often used in the production of

Florida orange concentrate according to Carter ( 198 1). The second pasteurization is done

prior to packaging for commercial purposes on reconstitued juices. which don't have

residual enzyme activity. A temperature ot about 74 O C for 16 seconds is usually

recommended (Carter 198 1 ).

Many time-temperature pasteurization procedures exist: the combination chosen

depends on the product.

2.23 Chemical changes during pasteurization

Even when the heat treatment has been used to increase the shelf life of orange

juice. certain chemical processes can continue that will adversely affect the flavor of the

product due to the formation of off-flavour compounds during storage. The most

significant changes due to pasteurization are increases in the following known oxidation

products of the major oil component d-Iirnonene: alpha-terpineol, 4-terpineol. carve01

and pans-mentha-2.8-dien- 1-01 (Schreier er a!.. 1977) The most effective way to

minimize the degradation of flavour has been to store the juice at refrigerated temperanue

(Shaw el ai.. 1993).

2.3 Storage

The major factor affecting the quality of orange juice concentrate is the

temperature of storage and the composition of the storage container (Bokhari er al., 1995).

The permeability of the container to oxygen is an important factor for the shelf life of

stored pasteurized orange juice (Marshall et al., 1986), because oxidization is the primary

reason for flavour and vitamin C degradation during storage. Glass bottles are the

containers that provide the longest shelf life (Shaw. 1996b) compared to other types of

packaging. like plastic and polyethylene. If they are sealed correctly. no oxygen can pass

through the glass and no absorptioil of aroma by the packaging occurs.

The juice "not from concentrate" can be stored frozen in blocks. and thawed when

the final product is required, or stored aseptically for the needed period of time above

freezing temperature (Shaw et al.. 1993; Shaw. 1996b).

2.3.1 Frozen juice

Freezing large blocks of single-strength juice requires more energy than

aseptically stored products and therefore the fieezing method involves more cost (Shaw er

al., 1993). Many reactions are stopped at fieezing temperatures and others are slowed

down. Freeze preservation is known to keep degradative processes to a minimum during

long periods of storage (Olson, 1968). The quality of natural juice preserved by fieezing

without heat treatment and consumed immediately after thawing, is the closest to that of

freshly squeezed orange juice (Merin and Shorner, 1984). Certain studies on storage of

juice concentrate have used as a reference control juice stored at -18 OC (Tatum er al.,

1975; Marcy e l ul., 1984; Marcy et al., 1989). However, even at this temperature,

reactions between the components are not prevented. For example, Kefford et al. (1959)

and Kanner et al. (1982) reported small losses of ascorbic acid during storage of

concentrate at -18 OC. Bokhari ef a!. (1995) found little change for vitamin C and acidity

for a concentrate stored at -15 OC. Therefore, changes can still occur in frozen orange

juice. However. the effect of freezing on juice "not fiom concentrate" has not been

studied thoroughly and the application of the results obtained for juice fiom concentrate,

probably are not applicable.

According to Merin and Shomer (1984) the rate of separation of insoluble

particles from the serum provides an indication of physical changes which occur in juice

during frozen storage. The primary freezing rate is an important factor. If the juice is

quickly frozen. the separation of thawed juice after a relatively long storage is delayed.

The tendency for separation is greater after a slow freezing rate because large ice crystals

formed cause compression of insoluble particules causing aggregation. Aggregation

appears in thawed juices for all type of treatments after a long period of storage in the

frozen state. Particular attention must be taken when the juice is frozen to minimize the

effect of crystallization of water especially for not concentrated juice.

2.3.2 Aseptically packaged juice

Low density polyethylene (LDPE) is the most commonly used material for the

inner layer of a multilayer package. Aseptic packaging produces a sterile product. On the

other hand. certain factors affect the shelf life of product packaged aseptically such as

oxygen which can permeate along the seams of the package and react with orange juice

components (Varsel, 1980). Another problem is the absorption by LDPE of nonpolar

volatiles particularily terpene hydrocarbons (such as d-lirnonene) and aldehydes. This

phenomenon is called "scalping" and it decreases the level of flavour components (Shaw

et a!., 1993). Another major factor affecting the shelf life of the product is storage and

distribution temperature. It is essential to maintain the juice at refrigerated temperatures

during storage, transportation and retail marketing in order to keep the quality high (Shaw

et a/., 1993).

2.3.3 Storage studies of orange juice

Many studies have been carried out on the effects of temperature of storage on

orange juice concentrate. The studied parameters are always related to these three

important criteria of quality: the flavour, the colour and the texture.

Kanner ef a!., (1982) studied the stability of orange juice concentrate. Their

samples were stored in metal cans at -18, 5 , 12, 17. 25 and 36 "c. Nonenzymatic

browning, ascorbic acid degradation. furfural accumulation and sensory quality were

measured over a period of 18 months. In 1984. Marcy er ai. stored in metal cans orange

juice concentrate at -12.2, -6.6, -1.1 and 4.4 OC and analyzed O~rix . % acidity, ascorbic

acid, furfural. serum viscosity. apparent viscosity. browning, Hunter colour values and

taste panel scores at monthly intervals for one year. Later, a study on processed

pasteurized orange juice was done by Kaanane el al. (1988). Total solid. pH and acidity.

form01 index. total sugar. ascorbic acid content and furfural production were investigated

on samples stored at 4. 22.5, 35 and 45 OC respectively in glass bottles for 14 weeks.

Marcy el ai. (1989) undertook another study on aseptically packaged orange juice

concentrate and orange drink concentrate. The juice was stored in laminated bags at 4.

15, 22 and 30 O C for 6 months. Ascorbic acid, nonenzymatic browning and sensory

quality were measured monthly. More recently, Bokhari er al. (1995) examined the

quality of packaged and stored Kinnow (mandarin or tangerine oranges) juice concentrate.

Samples were placed at three temperatures: -15,4 and 32 "C in high density polyethylene

bags and low density polyethylene bags. The target parameters were @Brix, acidity,

ascorbic acid. pectinesterase activity, organoleptic evaluation, total bacterial plate count

and yeast.

2.3.4 Indicators of chemical degradation

Some chemical compounds are useful tools to evaluate the quality of a product.

Lee and Nagy (1996) have drawn a list of seven chemical markers for the citrus industry:

ascorbic acid. dehydroascorbic acid. hydroxymethylfurfura1, furfural, 2.5-dimethyl-4-

hydroxy-3(2H)-hone, 2,3-dihydro-3.5-dihydroxy-6-methyl-4H-py-4-one. 4-vinyl

guaiacol and alpha-terpineol. These compounds can give some indications of temperature

and storage abuse.

2.4 Principal compusilionai analyses performed during storage studies

Freshly pressed orange juice has a full. fruity flavour quality that has not been

completely duplicated in any orange juice product. The overall total quality of a juice

depends on many criteria. Various analyses are done in order to have as much

infomation as possible on the changes in composition of the juice as a hc t ion of the

storage temperature. Flavour degradation is considered as the most important factor in

quality loss of citrus juice products.

2.4.1 Volatiles

A volatile fluid is a liquid with the tendency to become vapour at low temperature

and pressure values. Volatiles contribute to the flavour, and the flavour degradation is

considered as the most important factor in quality loss of citrus products. The volatiles

have been studied to understand the degradation process due to storage temperature. The

scarce information available on the contribution of volatile compounds to the sensory

impression of a complex aroma is vague and partially contradictory (Ziegler, 1970).

Orange flavour is the result of a combination of volatile components in specific

proportions. The relative contribution of individual components to the overall juice

flavour is influenced by the threshold values and by possible synergy between

components. Also, volatile and non-volatile components present in orange juice may

interact with each other and influence its flavour (Amed er al., 1 978). There are several

classes of compounds that contribute to the distinct flavor of orange: terpenes, aldehydes.

esters and alcohols (Shaw. 199 1). Shaw (1 W6a) summarized volatile components known

to contribute to fresh orange flavour (Table 1).

Table 1.

1 996a).

Volatile components which contribute to Fresh orange flavour (Shaw.

Hydrocarbons d-limonene

I citronella1 I methyl butanoate I

myrcene a-pinene valencene

I sinensal I I

Aldehydes acetaldehyde

The hydrocarbons limonene, myrcene and pinene are the three major constituents

of peel oil (Shaw er al.. 1977). These components are easily absorbed and/or adsorbed by

the low density polyethylene (LDPE) layer used in packaging due to the affinity of the

LDPE for the non-polar components. Halek and Meyer (1989) found approximately 30%

total loss of d-limonene and alpha-pinene from the studied solution. Concerning the

myrcene. it seems to be the most affected with 40% loss due to sorption. The absorption

of d-limonene increases the permeation of oxygen through the packaging material. Sadler

and Braddock (1990) have shown that the permeability of the oxygen by the LDPE was

proportional to the absorbed mass of limonene. In addition, the temperature also has an

effect on the absorption. The absorption of volatiles and the permeability to oxygen were

substantially lower at 4 O C than at 25 O C (Pieper et al., 1992).

oc tanal nonanal decanal

Esters ethyl acetate

Alcohols ethanoI

ethyl butanoate ethyl 2-methylbutanoate

ethyl 2-hydroxyhexanoate

(E)-2-hexenol ' (2)-3 -hexen01

linalool

Two categories of volatile flavour compounds can be identified. Depending on

the polarity of the components, a volatile can be found in the oil-soluble constituent

present in peel oil and in juice oil, or in the water-soluble constituents present in the juice

(Nagy. 1996). The peel oil of citrus fruit is located in small, ductless glands present in the

flavedo, or the outer portion of the peel. During the extraction, a small amount of peel oil

ends up in the juice and provides the characteristic. pleasant citrus aroma of intact h i t

(Nagy, 1996). However, some of the flavour components. including the "top-notes", are

provided by volatiles present in the juice sac. including oil soluble components in juice oil

located within the juice sac (Shaw. 199 1). The water-soluble volatile components present

in the juice sac are considered to contribute most to the characteristic flavour of fresh

citrus fruit (Huet, 1969). However. the flavour of orange juice is a complex combination

of aqueous essence, essence oil and peel oil. The major component of orange and peel

oil. d-limonene, is generally considered as non-essential to orange flavour but it acts as a

precunor of alpha-terpineoi. which is know as an o ff-flavour compound (Dm, 1 980).

The changes occuring in orange juice after processing and storage can be divided

in two classes: ( I ) loss of original flavour and (2) development of off-flavours and off-

colours (Shaw er al.. 1993). Both of these events involve volatiles.

The storage is an important factor to consider. especially at ambient temperature.

The formation of compounds which impart off-flavour is more pronounced under these

conditions. A summary of off-flavour compounds derived from the thermal

decomposition of citrus oil constituents is presented in Table 2. The formation of off-

flavours during storage is often due to chemical changes o c c u ~ g during the initial heat

treatment of the juice (Shaw er al.. 1993).

Some volatile components are recognized as degradative indicators:

hydrox ymethy 1 fiufural commonly cal ied HMF, furfural, 4-viny lguaiacol. 2,5-dimethyl-4-

hydroxy-3(2H)-bone ( h e o l ) and alpha-terpineol (Lee and Nagy , 1996). 4-

Vinylguaiacol, h e 0 1 and alpha-terpineol were judged to be most responsible for

malodorous properties of time-temperature abused juice (Shaw et a[., 1993). Figwe 2

shows the structure of different chemical indicators (Lee and Nagy, 1996).

Table 2. Thermally degraded compounds derived from essential oil constituents

(Shaw et al., 1993).

Precursor I

d-limonene ' linaloo 1 '

I cis- 1.8-p-rnenthanediol* 1 I 1 .

1.8-cineole I Pungent. cam~horaceous a-terpineol

p-mentha- 1 (7),2-dien-8-01

Derived compounds a-terpineol a-terpineo 1

nerol

'Present naturally in fresh j~

Flavour response Stale, musty, piney Stale, musty, piney Sweet. rose fruity

geraniol cis4.8-p-rnenthanediol

*Also know as cis-terpineol.

Sweet, floral rose Sweet. carn~horaceous

p-cymene I Terpiney off-flavour

1.4-cineole p-mentha- l.5-dien-8-01

p-mentha- 1 (7).2-dien-8-01 cis-p-mentha-2.8-dien- 1-01 trans-p-mentha-2.8-die* 1 -

0 1 p-cymen-8-01

p-c ymene a,p-dimethy lstyrene

ice.

-- ------ Not characterized Not characterized Not characterized Not characterized

Nonspeci fied off- flavour Terpiney off-flavour Terpiney off-flavour

**Isomeric mixture of nerd and geranid.

Figure 2. Volatile quality indicators.

HMF Furfural

4-vinyl guaiacol

Furaneol

HMF is the primary breakdown product during the dehydratation of glucose or

fructose in an acid medium. In citrus products, the presence of HMF is suggested as the

first indication of change due to storage even though it is not responsible for the off-flavor

characteristic of stored citrus juice (Berry and Taturn. 1965). High concentration of HMF

in juice is considered to be due to excessive heat treatment during concentration or

pasteurization.

The second component. the furfurall, is produced by the aerobic and anaerobic

degradation of ascorbic acid (Bauemfeind and Pinken, 1970; Kaanane et al., 1988).

FurfUral and HMF may be related to the darkening of juice and are also useful indicators

of temperature abuse or storage time in diverse foods (Pompei er a[., 1986; Beeman.

1987). The furfiual and HMF content of tieshly processed citrus juice is essentially zero

but increases to significant amounts, during high-temperature storage (Nagy and Randall,

1973; Askar, 1984). F u r W does not contribute directly to the flavour changes but its

accumulation parallels to that of other compounds that alter flavour (Nagy and Smoot,

1977).

4-Vinyl guaiacol has been identified as the most detrimental off-flavour

compound in aged canned orange juice ( T a m et a/., 1975). It is formed by the

carboxylation of fermlic acid (Fiddler et al., 1967). The Cvinyl guaiacol imparts an old

h i t or rotten flavour to orange juice at a level of 0.075 ppm (Tatum et al.. 1975).

Like HMF, fbmneol is also a degradative product of sugar (Shaw et a!.. 1968).

Furaneol content gradually increased as a function of storage time and temperature (Lee

and Nagy, 1987). Futaneol gives a pineapple-like aroma to orange juice (Tatum ef al..

1975).

Concerning the alpha-terpineol, it is derived from the degradation of some

essential oil components of orange juice (Figure 3). I t s precursors are mainly linalool and

d-limonene, which are the major volatile organic constituents of orange juice.

Approximatively 2-3 ppm of alpha-terpineol is sensorially detectable as deterioration

(Diirr. 1980). Tatum et al. (1975) were more precise in indicating as little as 2.5 ppm of

alpha-terpineol added to freshly expressed juice caused a stale. musty or piney aroma to

orange juice. This compound is very important because of the large presence of limonene

in juice.

Figure 3. Principal formation pathway of alpha-terpineol.

Limonene

Hydration

Alp ha-terpineol

The volatiles are generally extracted fiom orange juice by distillation or by liquid-

liquid extraction with different solvents such as methyl chloride or diethyl ether. Gas-

chromatography (GC) is the method most often used for separation and quantification.

Mass spectroscopy (MS) and nuclear magnetic resonance (NMR) permit identification of

organic compounds. The most common GC system used for volatiles determination is

flame ionization detection. To measure very volatile components. a headspace injector

can be coupled with the GC-FID (flame ionization detector). Depending on the

composition of volatiles. either a polar or non-polar column can be used. Tatum el ul.

(1975) analyzed a canned single-strength orange juice and extracted the volatiles with

methylene chloride and used a gas chromatographic separation with a Carbowav column.

IR and MS spectroscopy was used for identification of the different peaks. Ten

degradation compounds were isolated from the canned juice stored at 35 O C for 12 weeks

(see Table 3).

Table 3. Degradation products in canned single strength orange juice after 12 weeks

storage at 35 "C (Tatum et al.. 1975).

FurfUral I

a-terpineo 1

2.4.2 Ascorbic acid

Degradation of ascorbic acid (vitamin C) in orange jl

cis- 1 .&p-menthanediol trans- 1 .8-D-menthanediol

I I

3-hydroxy-2-pyrone 1 4-vinylguaiacol

lice is well do'

2-hydroxyacetyl furan 2.5-dimethyl-4-hydroxy -3 (2H)-hanone

I

cumented,

probably because of its nutritional value. In 1980, Nagy studied the variability in the

vitamin C content of citrus fruits and their products as influenced by variety, cultural

practice, maturity, climate. fresh h i t handling, processing factors, packaging and storage

conditions. Storage time and temperature were found to be important factors in the loss

of vitamin C. Low temperature is imperative for the retention of vitamin C during storage

I

benzoic acid '1

5-hydroxymethy 1 Mimi b

(Nagy, 1980). The degradation of ascorbic acid can be effected by enzymatic or

nonenzymatic processes. Enzymatic degradation is controlled by pasteurization at high

temperature which disables the oxidative enzymes. The greatest loss of vitamin C in

processed product is due to aerobic and anaerobic reactions of a nonenzymatic nature

(Nagy, 1980). Both aerobic and anaerobic degradations occur in the same juice system

(Kennedy et d., 1992). Aerobic and anaerobic pathways for degradation of vitamin C in

aqueous medium (see Figure 4) have been proposed by Bauernfeind and Pinkert (1970).

Figure 1. Anaerobic and aerobic degradation of vitamin C (AA) in orange juice: DKA,

diketogulonic acid; HF, hydroxyfurfUml (Bauemfeind and Pinkert, 1970).

A A DHA OKA

HG-OH GHO

After oxygen is consumed (aerobic), vitamin C is degraded anaerobically but at

rates lower than the aerobic process (Nagy, 1980). However, according to Kennedy et ul.

(1992). the aerobic process generally predominates and the anaerobic process takes place

when the level of dissolved oxygen has reached equilibrium.

The processing of fruit to make juice products results in minimal loss of vitamin C

potency, but subsequent storage of the finished product at high temperatures results in

considerable losses. Shaw et al. (1993) summarized the factors correlating with

degradation rates of vitamin C reported by various studies. Solid content and storage

temperature, pH, presence of metal ions and fiee oxygen (headspace and dissolved in

juice) were considered important. Kennedy et al. (1992), studied the degradation of

ascorbic acid as a function of time and temperature ( -20, 4. 20. 37. 76 and 105 OC).

Samples stored at 20, 37, 76 and 105 OC showed dramatic decreases in vitamin C levels

after the tint few days of storage. This fact coincided with an initial drop of the dissolved

oxygen level. This correlation between dissolved oxygen and ascorbic acid stability was

not demonstrated in frozen samples (stored at -20 OC), but small loses of vitamin C were

observed after 23 days in the samples with the lowest losses of dissolved oxygen. In

1982, Kanner et al., demonstrated that the loss in ascorbic acid was limited at - 1 8, 5 and

12 'C but important at 25 and 36 OC. Generally all the authors (Kanner er 01.. 1982;

Marcy er a/., 1984; Kaanane et 01.. 1988; Marcy er a!., 1989. Bokhari et al., 1995) have

found a decrease in ascorbic acid at all temperatures (even at frozen temperatures); losses

increase with the temperature and storage time. Except Bokhari et af. (1995) who used a

titration with 0.1 N iodine. all the studies were perfomed with the AOAC method of

titration with 2.6-dichlorophenol indophenol to measure the ascorbic acid in solution

(Carter. 1981). Titration has been the most commonly used method. but new

developments in instrumental methodology provide the possibility of alternative methods

of analysis. Vitamin C can be quantified for example by fluorimetric procedures, the

dinitrophenylhydrazine method, or HPLC reverse-phase ion pair. These analyses have

been reviewed by Ting and Rouseff (1 986).

2.4.3 Sugars and organic acids

The popular flavour of orange juice is the result of a natural combination of

volatile compounds in a well-balanced system of sugars, acids and insoluble solids

(Shaw, 1986).

Sugars

The economically most important juice parameters. for the citrus grower or the

processor. are the total soluble solids ('Brix) and Brixacid ratio. Besides sugars, the

remainder of total soluble solids in the juice are organic acids. nitrogenous C O ~ Q O U ~ ~ S .

soluble pectic substances and other minor constituants (Ting and Rouseff. 1 986).

The main sugars found in orange juice are D-glucose. D-fructose and D-sucrose. D-

glucose and D-fructose are reducing sugars, while D-sucrose is non-reducing. The sugars

in orange juice are subject to change because of juice acidity. The ratio of sugar content

in processed juice can change during storage due to acid-catalysed hydrolysis of D-

sucrose to D-glucose and D-fructose (Chen et al.. 1993). When the orange is mature. total

reducing sugars ( D - ~ C ~ Q S ~ and D-glucose) are approximatively equal to D-sucrose. and

they are in almost equal proportion.

Sugar content of juice can be measured by several methods. Total sugar is often

measured as 'Brix. Sugars comprise 70-85 % of the total soluble solid material in orange

juice (Chen el al., 1993; Miller and Hendrix, 1996). O ~ r i x is a unit used to designate per

cent dissolved sugar (Carter. 1981). The official test for O ~ r i x is performed by a Brix

hydrometer. This instrument measures the specific gravity and it is calibrated to read

directly in degrees Brix, or percent of sucrose, at a standard temperature of 20 O C (Millers

and Hendrix, 1986). Unofficial method for reading of Brix content can be performed with

a rehctometer. Marcy et al. (1984) and Bokhari et al. (1995) used rehctometers in their

methods for determining of "Brix. Both methods are rapid and the results are ohen

expressed with acidity in the form of a ratio ('Bridacid). Brix and its acid ratio are

criteria used in determination of h i t maturity and juice quality (Carter. 198 1).

2.4.3.2 Organic acids

Organic acids in orange juice consist primarily of citric acid (85-95 Oh) and malic

acid. Smaller amounts of tartaric and succinic acids may be present (Chen et al.. 1993:

Miller and Hendrix. 1996). The two major organic acids in citrus juice (citric and malic)

can be separated and quantified using a reverse phase HPLC system (T ing and Rouse&

1986). However. the measure used to express acidity is often titratable acidity. A known

volume of juice is titrated to a phenolphthalin end point or pH of 8.1 using sodium

hydroxide. Results are expressed as g of citric acid / I OOmL (Carter. 198 1). This method

seems to be the most widely used for acidity (Marcy et al.. 1984: Kaanane er ol.. 1988:

Bokhari a ui.. 1995). All citric and malic acids in orange juice are not in their fiee form:

a part is in the salt form. The combination of acid and salt imparts great buffering

capacity to citrus juice (Chen et a!.. 1993). The brixacid ratio is first an index of legal

h i t maturity because acidity decreases when the soluble solids content increases (Chen

er a!.. 1993). This ratio plays an important part in acceptance (both by processors and

consumers) of the orange juice (Miller and Hendrix. 1996).

According to studies performed on stored orange juice concentrates. %rix and

acidity showed no significant change during the test period (Marcy et ai.. 1984; Kaanane

er al., 1988). Bokhari et al. (1995) found no significant change in OBrix but there was a

gradual decrease in the acidity throughout the entire storage period in all samples stored at

different temperatures. This fact caused a gradual increase in the "Brkacid ratio.

2.4.4 Colour

The two important factors affecting the appearance of the orange juice are opacity

and colour (Stewart, 1980). The orange colour of orange juice is due to carotenoids.

These C-40 compounds are primarily responsible for the red, orange and yellow pigments

found in citrus juices (Shaw et af.. 1993). The typical colour of orange juice is an

important factor for acceptance by the consumer. During storage, darkening of citrus

juices occurs. This phenomenon is called "browning". Browning reactions may be either

enzymatic or nonenzyrnatic (Paul. 1972). Browning increases with increasing storage

time and storage temperature. Enzymatic browning is usually not considered important

because of high temperatures used during pasteurization.

During storage studies of citrus juices, the nonenzymatic browning is often

measured. The reactions are very complex and many compounds found in orange juice

can undergo browning reactions. Nonenzymatic browning reactions frequently involve

reducing sugars or sugar-related compounds with amino acids in Maillard-type reactions

(Paul. 1972). Maillard was the first to describe the development of a brown color in

mixtures containing amino acids and reducing sugars. Varsel (1980) considered the

oxidation of ascorbic acid as the major factor in browning of citrus products. Besides

ascorbic acid. large amounts of other organic acids and their salts create favourable

conditions for degradation of sugars. amino acids, and phenolic compounds during

processing and upon storage via browning reactions (Lee and Nagy, 1988). The

formation of melanoidar pigments result fiom reaction of furfUral with amino acids or

fiom furfural polymerization. The interaction of nonemymatic browning and oxidation

reaction products with juice constituents is very complex and catalytic behavior of one

type of reaction on the other may reduce the predictability of the quality degradation that

can occur (Adams, 1989). Robertson and Sarnaniego (1986) found a highly significant

correlation between browning index, HMF and fumval formation. They suggested that

all three serve as chemical indicators of storage temperature abuse in lemon juices.

Handwerkand and Coleman (1988) studied the role of amino acids. sugars, ascorbic acid.

buffer and catalysts, sulfur containing amino acids and thiols in the browning reaction.

They concluded that reducing sugars, ascorbic acid and probably other carbonyl

compounds are reactants in browning reactions and are the precursors to compounds of

taste significance. Amines and amino acids present in juice catalyse the initial reaction

and also take part in later sequences of the reaction that occur.

The measurement of colour was initially performed visually by difference. Today.

instruments are used for colour determination. Meydav et a/. (1977) proposed a

procedure to measure browning in citrus products. Centrihgation. dilution and filtration

are followed by a transminance spectra at 420 nrn. This method gives a "browning

index". A simpler method which measures the colour rehctance is done with an

instrument such as the Hunter Color Difference. This intrument is based on the

tristimulus principle of X, Y and Z (Nickenon, 1946) related with LAB values where L

represents the lightness. A the redness and B the yellowness. For browning, L is the most

important value. This method is faster than that of Meydav et at. (1977).

The tristirnulis calorimeter was used by Kanner et al. (1982) and Marcy et a!.

(1989). The latter used absorbance at 420 nm to confirm the colour change. They found

changes in the tristimulus attribute L occured more rapidely for juice concentrate stored at

30 O C more than 2 months. The colour of juice stored at 4 O C was not significantly

different after 6 months of storage. Kanner et UL (1 982) found an increase in the attribute

L for juice concentrate stored at 25 OC for at least 200 days of storage. But the

concentrates stored at 5 and 12 O C for 18 and 12 months were stable. Marcy et ol. (1984)

used absobance at 420 run and observed no change in colour of orange concentrate kept at

-12 O C for 1 year. However, slight changes in samples stored at -6.6. -1.1 and 4 O C were

found. The rate of browning depends in large part on the temperature of storage. Low

temperature is recommended to delay the development of brown colour.

2.4.5 Sedimentation of pulp, cloud, viscosity and density

Sedimentation of pulp, cloud, viscosity and density S e c t the appearance and the

mouth feel of orange juice and are therefore important for acceptance by consumen.

2.4.5. I Sedimentation of pulp

In addition to contributing to apparence and mouth feel. pulp also contributes to

the perceived flavour because it preferentially absorbs nonpolar vola~iles. The presence

of pulp modifies not only the intensity but also the balance of the overall aroma (Itadfort

er al.. 1971). Most of the aroma volatiles are anached to the pulp panicles: therefore an

orange juice should contain a sufkient amount of pulp material (Diim. 1980). The

separated pulp may coalesce and float to the top or settle at the bottom of the bottle

leaving a clear or slightly hazy serum resulting in an unattractive non-homogeneous

appearance of the product (Rangarma and Raghuramaiah. 1970). The sedimentation of

pulp becomes parricularily imponant when the frozen juice is thawed. The separation of

insoluble particles (pulp) From the juice serum increases with increasing fiozen storage

time and is especially apparent after a slower primary fieezing rate.

Men a juice is frozen, depending on the primary freezing rate. the insoluble

panicles are compacted between ice crystals. This factor causes flocculation. the pulp

settles more rapidly and the separation is more rapid. Merin and Shomer (1984)

measured the height of separated serum Eom a suspension of thawed nanual Valencia

orange juice after 0, 30, 60 and 90 days. Measuremenu were made at 0.5. 1, 2 and 3

houn after thawing. Three different methods of freezing were used: liquid nitrogen

fieezing (10 rnin), blast fieezing (3 hr) and chamber fieezing (24 hr). The results showed

that the primary rate of fieedng is very important. Thc senling rate was advanced with

the frozen storage time and in accordance with the sequence of the primary fmdng rate.

The samples frozen by the liquid nitrogen had the lowest rate of sedimentation among the

three methods. Quick primary freezing delayed the separation of thawed juice over a

relatively long storage period.

2.4.5.2 Cloud

In o m g e juice. an opaque or cloudy appearance is considered a desirable

characteristic. The cloud is the opaque appearence of citrus juice due to colloidal

suspension of particles (Ting and Rouseff, 1986), and the pectin is the protective colloid

(Carter. 198 1). The partideb can be ce!lulose. protein or lipids ( h e and Olssofi, 1994).

The pectin substances found in juice are complex carbohydrates and consist of

galacturonic acid and its methyl ester in chains of undetermined length (Kertesz, 195 1).

Clarification results from the natural enzymatic effect of pectinmethylesterase (PME).

Cloud is primarily stabilized by pasteurization which causes the deactivation of PME.

Without pasteurization, a part of methyl ester linkages are split, forming pectinic acids or

possibly pectic acid. which. with the calcium naturally present in the juice. forms a

precipitate (insoluble calcium pectate) and causes clarification or gelation of concentrated

juice (Rangma and Raghurarnaiah, 1970). In additions, other cloud forming

components are entrapped in this precipitation process, resulting in total loss in single-

strengh juices (Irwe and Olsson. 1994). No studies of the effect of frozen storage seem to

have been reported on the stability of juice cloud. According to Carter ( 198 1 ), measuring

cloud in citms juice provides an indication of abuse and stability. This can be performed

with a calorimeter (at 650 run).

2.4.5.3 Viscosity and density

Viscosity is associated with pectin - the most important contributor to juice

viscosity. Serum viscosity affects the mouth feel and the body of the juice. It is usually

measured with a Brookfield viscosimeter (Ting and Rouseff. 1986). Marcy er ul. (1984)

found no signifiant change in serum viscosity during one year of storage.

The density measurements have not been reponed in literature.

2.4.6 Sensory analysis

Sensory analysis is a very complex domain. Many studies on organoleptic

evaluation have been performed on various materials using a wide variety of methods and

producing a variety of results. Sensory analysis is an important method for evaluating

consumer acceptance of a product. The panel can consist of untrained. semi-trained or

trained (expert) members. The perception of flavour depends on two classes of

compounds: (1) the non-volatile compounds that possess taste attributes and are sensed

by taste buds in the mouth. and (2) the volatile substances that are odorous and sensed by

olfactory receptors in the nasal passage. Flavour is the combination of these two classes

(Hendrix and Hendrix. 1996). Sensory evaluation is a way of making a connection

between chemical changes and acceptability of the juice.

Kaanane et al. (1982) used at least 25 untrained panalists from among university

personnel. Samples were compared by a triangle test. Results showed no statistically

significant difference between concentrate stored at -1 8 O C and those stored at 5, 12 and

1 7 O C for 1 7, 10, and 8 months, respectively. After these periods, off-flavours developed

which were associated with a caramel-like taste. Marcy ei al. (1984) asked six trained

panelists to score samples in comparison with a reference juice (store at -1 7 OC). Panelists

were unable to detect a significant difference in orange juice concentrate stored at -12.2

and -6.6 "C during the 12 month of storage. Concerning the juice concentrate stored at - 1.1 and 4.4 O C , the scores of the panel were significantly different after 9 and 5 months

respectively. In 1989, Marcy et ui. determined sensory quality with an experienced 12

member panel by a difference test where ( 0 ) indicates no significant difference and (+)

indicates a significant difference from the control (stored at -1 8 OC). Analysis of the juice

concentrate showed that there was no significant difference from the control when stored

up to 6 months at 4 "C. On the other hand. the samples stored at 15 and 22 OC were

significantly different after 2 months of storage. Bokhari et a/. (1995), used a panel of

eight judges for evaluating the juice for colour and flavour. They found that more loss in

colour and flavour was observed at high temperatures as compared to low temperatures in

all the packaging materials.

CHAPTER 3

MA TERIALS AND METHODS

3.1 Experimental design

The research project started in May 1997 using Valencia orange juice from

Mexico. The samples were divided into three categories: (A) unpasteurized orange juice

stored at -1 8 O C in glass bonks, (£3) pasteurized orange juice stored at -18 O C in glass

bottles and. (C) pasteurized orange juice aseptically packaged in polyethylene bags

containing 1 1 85 Kg of juice stored at +lUC.

Pasteurized and unpasteurized samples were stored frozen (-18 O C ) for eight

months (May 1997 - December 1997). In addition. pasteurized samples were also

aseptically packaged and stored at + I OC in polyethylene bags (see Figure 5). At specific

time intervals, samples were analyzed for quality parameters.

To have a complete study of the effect of the three storage conditions; nine quality

parameten were analyzed as showed in Table 4. Analyses were carried out once a month

for a period of eight months. Certain of the nine parameters changed rapidly. especially

with the thawed unpasteurized juice. An order was established for analyses according to

relative importance.

Figure 5. Experimental design of the three storage conditions.

FRESH ORANGE JUICE

I Unpasteurized I

Frozen Glass bottle

-1 8OC

I Aseptically packagedl

Table 3. Parameters measured.

I PARAMETERS MEASURED I ORDER OF ANALYSIS 1 1 Sedimentation of the pulp

I

I 1 I . . 1 Cloud

I

I - 7 I

I Ascorbic acid I 5 I

b 1

1 Colour I 6 I

Volati les Viscosity and density

1 I

Sugar I 7 Organic acids 8

I

Sensory evaluation* 9 J

*The sensory analysis were perfirmed in parallel at ALessonde Inc. with on expert panel.

3 4

Each month. 4200 rnL (12 bottles of 350 mL) of frozen pasteurized, frozen

unpasteurized and aseptically packaged (sampled from a bin) orange juice were necessary

for the analysis. Sensory evaluation required 2 100 mL (6 bottles of 350 mL), and 2 100

mL (6 other bottles of 350 rnL) were required for the physico-chemical analysis to

perform one series of analysis on one juice. The 2100 mL (6 bottles) of each storage

condition for physico-chemical analyses were mixed by two to produce a total of 3 bonles

of 700 mL. On each bottle, the analysis were performed two times for a total of six

measurements per parameter, per storage condition, per month.

3.2 Sampling of orange juice at Mexico

The fruits were harvested in Nuevo Leon (Mexico), hand picked fruits were

brought by trucks. fiom the groves to the processing plant and extracted by Brown Citrus

Machinery. These extractors use a reaming action to extract juice from citrus h i t . except

for the model 1 100. The Brown model 1100 has a feeder that places the fruit into three

single lanes as it enters the extractor. For the production 8 emacton; model 400 and one

extractor model 1 100 were used. The capacity of the plant was 15 tons of fruits per hour.

After extraction. the juice was finished, to separate cloudy but othenvise clean

juice. fiom pulp. rag, seed and pips. After finishing, the juice was pumped into a holding

tank. The process was well defined by Rebeck (1995). Part of the juice was placed in a

250 liters tank and the sample of unpasteurized juice was placed in bottles. Three

hundred (300) bottles of 350 mL were prepared for the experiment. The balance of juice

was pasteurized (95 OC, 15 seconds) with a TETRA ASEPT system and transferred into

three stainless steel tanks of 5500 liters capacity connected together. The juice was mixed

and the bins were filled. This system was used to fill aseptically (4 O C ) the 1 1 85 Kg bins.

The bins were kept at 1 OC . In total, 12 bins were prepared for the experiment. One of

the bins was opened in a stainless steel tank and then the pasteurized juice was transferred

into glass bottles. The bottles were then frozen at -18 O C . This juice was kept at this

temperature before and during shipping by truck. All the tanks used in this process are

washed, sterilized and aseptized. The juice remained more than one month in Mexico

before its arrival at Rougemont. The juice was processed on the 13" of March and

arrived on the 24" of April at A. Lassonde Inc. (Rougemont. Qc.). The storage

experiment began on the 14" of May 1997 (month 1).

3.3 Sample preparatim for analytical analyses

Frozen orange juice samples were immersed in a warm water bath at 45 OC

(Shaker Bath. Orbit LAB-LINE. model no 3540) with continuous shaking in order to

achieve 25 O C (Merin and Shomer. 1984). About 30-35 minutes was necessary with a 100

RPM shaking to reach the desired temperature. All the nine analyses were carried out at

ambient temperature. The aseptically packaged juice in bins was homogenized for 5-7

minutes and was equilibrated at ambient temperature prior to analysis.

3.4 Artal)ticai methods

Ail the analyses were performed with water obtained from a Milli-Q Water

System (Millipore Corporation. Beddford. MA.).

3.4.1 Sedimentation of the pulp

A well mixed orange juice (100 mL) was placed in a 8" graduated centrifbge tube

(100 rnL, Kimble Glass Inc., US). After 0, 1,2 and 3 hours, a visual measurement of the

height of sedimentation of the pulp in mL was performed (Merin and Shomer, 1984).

3.4.2 Cloud measurement

About 2 mL of the supernatant above the pulp sedimentation was taken with a

pasteur pipette and placed in a colorimeter cell. The % transmittance at 650 nm on a

colorirneter (Spectrophotorneter DU 640, Beckman, US) was measured. Water was w d

for the blank (100 % T). Six readings were collected for each sample. to compensate for

the variation of the response lamp. The measure was taken at 0. 1.2 and 3 hours with the

same sample to follow the evolution of the cloud. After the measurement, the liquid was

carehlly transferred back in the graduated sedimentation tube to avoid changes in the

measurement of sedimentation of the pulp. This method was adapted from Carter. 198 1.

3.4.3 Volatiles

The volatile aroma were measured by GC-MS. and the very volatile components

were quantified by headspace coupled with a GC-FID.

3.4.3.1 Gas chromatography coupled with a mass spectrometer (GC-MS)

A. Jnternal ~~d mixture

Phenol-dS (9.2 mg) was accurately weighed into a 1.5 rnL vial with a screw cap

and 1.0 mL of diethyl ether (Nanograde, Mallinckrodt, Kentucky, US) was added and the

vial was capped immediately. The vial was agitated until all the solids were dissolved.

With a clean 10 pL syringe, 10 pL of cyclohexanone-d4 and 10 pL o f butanoLd9 were

added through the septum. The syringe was washed before and after the addition to avoid

cross contamination. The cap was changed (new septum) and the vial was lightly mixed.

The internal standard was stored at 4 "C.

The three internal standards were purchased from CIL, Cambridge Isotope

Laboratory (Andover, MA, US). This internal standard mixture was used to correct for

the extraction efficiency of the ether and to check the variation of the retention times in

the GC-MS (Finnigan MAT software).

Primary srock solution

Diethyl ether (10 rnL) was added to a 100 mL volumetric flask. According to

Table 5. the appropriate amount of high purity standards were added to the volumetric

flask with a clean 10 pL syringe. Diethyl ether (1 mL) was used to wash the standards

into the flask. The 100 rnL flask was capped after each addition. When all the volatile

standards were added. the volume was diluted with diethyl ether to 100 mL and labeled as

standard #4.

Secondary standard sohions

The primary stock solution (standard #4) was diluted as foliows using a 10 mL

volumetric flask:

standard # 1 1.0 mL of the primary stock solution diluted to 10 mL with diethyl ether:

standard #Z 2.0 mL of the primary stock solution diluted to 10 mL with diethyl ether;

standard #3 5.0 mL of the primary stock solution diluted to 10 mL with diethyl ether.

For the analyses on GC-MS, 1.0 rnL of each standard (standard 1 to 4) was

pipetted into an 1.5 mL auto-sampler vial and 10 pL of the intemal standard were added.

The vials were immediately capped and mixed thoroughly on a Vortex for 10 seconds.

The external standards were used to establish a calibration curve (GC responses vs

concentration) to quantify the unknown concentration in the samples. A Finnigan MAT

software quantified automatically each volatile component with associated calibration

curve. As shown in Table 5, the standards were composed of 36 compounds. An

example of the GC-MS chromatogram showing the 36 volatile standards is illustrated in

Figure 6 .

Table 5. Volatile standards for the primary stock solution (100 mL).

Compounds I Density I pL I Std #J I Std #3 I Std#2

Methyl butyrate 99%*

tdmL) 0.898

( I R)(+)Alpha- pinene 98%* Ethyl butyrate 99%* Hexanal 98 %* (+)Sabinene 99%' 3-Carene 90%* Alpha- phellandrene* Beta-myrcene* Heptanal95 %* d-Limonene (G-R Inc. Om.. CA)

?-methy I-butan01 99+9/0* 3-methyl-butanol 99+%* Trans-2-hexenal 95+%* (S)(7)2-Hexanol 99%' Gamma-terpinene 9 5-e0h Octanal99%* LHexanol98+%* 3-Hexen-l -ol 98%. Nonanal95%* 2-4- dimethylstyrene 97 %* Furfiml* Decanal95 %@ (f )LinalooI 97 Oh* 1 -0ctanoP Terpinen-4-01 96?/0* Ethyl-3-hydroxy- hexanoate 98+%*

0.858

0.878

0.834 0.844 0.860 0.852

0.80 1 0.820 0.844

0.815

0.809

0.846

0.8 14

0.849

0.82 1 0.8 14 0.846

.-

4

17.16 6.864

17.56 7.024

16.68 6.672 16.88 6.752 2 1.50 8.600 I 7 .04 6.8 16

17.96 7.184 16.36 6.544 63 3 253.2

16.30 6.520

16.18 6,472

16.92 6.768

16.28 6.5 12

16.98 6.792

1 6.42 6.568 16.28 6.5 12 16.92 6.768

L

1

0.827 0.910

1.160 0.830 0,870

4

4

4

(IJdmL) 35.92

34.32

35.12

33.36

I

4 4

4 4 4

( W m L) ( ~ g J m L) 1 7.96 7.184

4 I 33.76

1

33.08 3624

46.40 33 2 0 34.80

0.827 0.933

0-974

5 4

4 4

150

4

4

4

4

4

4 4 4

I

16.54 18.12

23 20 16.60 17-40

33.08 37.32

38.96

4 4

4

43 .OO 34.08

35.92 32.72 1266

32.60

32.36

33.84

32.56

3 3.96

32.84 32.56 33 -84

16.54 18.66

19.48

Figure 6. GC-MS chromatogam of the 36 volatile standards used in the analysis of

orange juice samples.

*From Aldrich Chemical Company Inc, Milwaukee, WI, US; unless otherwise specified. **Solid or viscous sample were weighed. The order in the Table 5 is the order of the retention time of the elution of the compoundr fiont the G{ column (DB W.L't;3.

18.66

19.18

7.464

7.672

7.1 12

10.00

10.00

14.72

1.98

2.00

2.00

37.32

38.36

3.732

3.836

3.556

5 .OO

5 .OO

7.360

0.99

1-00

I .OO

Alpha-terpineoi 98%* (RK-)Cawone 98%. Ciaal (cis and trans) 95 %' (S)(-)perilladchyde 92a/08 24decadicnal 85%. Valcnccne ( A ~ S Organics. N-J.US) 4-Viny 1-guaiacol 97'10 ( h c a s t t r Sy nthcsis fnc.N-HUS) HMF 99% (Sigma Chcrnicds Co. MO. US) Vanillin (RP chirnic .France1

0.930

0.959

4

4

3 5.56

50.00

50.00

0-889

1 -00

1 .OO

0.920

liq*

solida*

solid* *

17.78

25.00

25.00

4

5

5

8

-

-

73.60

9.90

10.0

10.0

36.80

4.95

5.00

5.00

C. a m ~ l e preparat~on

NaCl (5.0 g) (U.S.P., BDH Inc., Toronto, Canada) was added to a screw cap tube

(1 5 mL), followed by well mixed orange juice (10.0 mL). A 10 pL of the internal

standard solution was added into the tube before capping. The tube was shaken until the

salt is totally suspended. The juice was extracted by shaking vigorously for 60 seconds in

diethyl ether (1.0 mL) and centrifuged at 3410 x g for 2 minutes (Fisher Centrific

Centrifuge. Model 225, rotor 04-978-54). The tube was shaken another 60 seconds and

centrifuged again for 5 minutes. The supernatant (200 pL) was transferred to a 1.5 rnL

auto-sampler vial with a reduced volume insert. The vial was capped immediately.

Depending on the compound. retention time (GC) and specific ions (MS) were used ro

identify the peaks.

0. * .

c o n d ~ t ~ o n ~

A Varian 3400 GC coupled with a Finnigan Mat Incos 50 MS (scan range: 35-

250 m u . scan rate: 0.43 scans/sec) was used for the GC-MS analysis. The GC column

used was a 0.25 mm X 30 m with 0.25 pm DBWAX film. The flow rate was 1.0 mL/min

for a split ratio of 50: 1. The injection volume was 1 .O pL and the carrier gas was helium.

The temperature of the injector was 250 O C and 225 "C for the transfer line. The column

initial temperature was 40 O C and was increased to 90 O C at a rate of 3 '&in. After 1

rnin the temperature was M e r increased to 120 O C at a rate of 10 "%in and kept at 120

OC for 2 min. Then. the temperature was increased to 200 OC at a rate of 20 O h i n and

kept at 200 OC for 5 min. The total run time was 30 minutes.

3.4.3.2 Gas chromatography with a headspace injector (Headspace-GC-FID)

A* lnternal standard mixture

A 100 pL of 1-propanol(99.5+%, ACS reagent Aldrich Chemical Company Inc,

Milwaukee, WI, US) was added to a 1.5 screw cap vial with 900 pL of water. The final

concentration of 1 -propano1 was 160 ppm when 10 pL were added to 5 mL of juice.

8.

Water (5.0 mL) was added to a volumetric flask (25 mL) and appropriate amounts

of the following compounds were added and diluted to volume with water:

a. 8000 pL of ethanol (anhydrous Accusolv. Anachemia. N-Y. US)

b. 400 pL of methanol (optima, Fisher Scientific. Nepean, Ont.. CA)

c. 80 pL of acetaldehyde ( 99%. Aldrich Chemical Company Inc. Milwaukee. US)

d. 3 pL of ethyl acetate (HPLC. Aldrich Chemical Company Inc. Milwaukee. US)

When the juice samples ( 5 mL) spiked with 25 pL (#I) and 50 pL (#2) of the

primary stock solution, the final concentration obtained (pL/mL) were as follows:

Primary stock solution

Acetaldehyde

Methanol

Ethanol

Ethyl acetate

C. Sample pre~antion

A well mixed juice (5.0 mL) was added to each of three vials (8 mL) followed by

10 pL of the internal standard solution (I-propanol). The first vial was capped. A 25 pL

and 50 FL of the primary stock standard solution were added respectively to the second

and the third vials and then they were capped. A calibration curve was drawn (area-

response factor vs concentration of the standard) with the three values and the unknown

concentrations (no spike) is the point which crosses the x-axis.

D. + *

eads~ace-GC condttms

A Hewlett Packard 5890 GC with a HP 7694 headspace injector (oven at 65 OC,

loop at 200 "C and 15 min for vial equilibration) was used for the Headspace-GC analysis.

The GC column used was a 0.25 mm X 30 m with 0.25 pm DBWAX film. The GC

column flow rate was 1.0 mL/min for a split ratio of 30:l. The injection volume

(headspace gas) was 1.0 rnL and the carrier gas was helium (nitrogen for the make-up

gas). The temperature of the injector was 250 'C and 150 OC for the m s f e r line. The

initial column temperature was 20 O C for 5 min and was increased to 50 O C at a rate of 5

'Ginin. Immediately, the temperature was further increased to 220 "C at a rate of 20

'Omin and kept at 220 'C for 5 rnin. The total run time was 35 minutes.

3.4.4 Viscosity and density measurements

In a 50 mL centrifuge tube, 40 mL of well mixed juice was centrifuged at 1925 x g

for 10 min (GS-6 Centrifuge, Beckman Instruments Inc., Palo Mto, CA). For the

viscosity measurement, 10.0 mL of the supernatant was transferred to a viscosirneter

(100, 484-485-4861, Cannon-Fenske routine viscosimeter, Cannon Instrument Co., PA.

US). The viscosimeter had been conditionned in a thennostated bath at 40 OC. The time

that it took the juice to pass between two ftved points was noted.

Viscosity = constant of the viscosimeter at 40 OC (cSt/s) X Time (s).

For the density, a pycnometer (25.0 mL) was weighed accurately (1) and 25.0 rnL

of the centrifuged juice was placed in it. The flask and the juice were re-weighed

accurately (2). The weight difference divided by the volume gives the density of the

centrifuged juice, according the following equation.

Density = Weight (2)(g) - Weight (l)(g) / Volume of pycnometer (25.0 mL)

3.4.5 Ascorbic acid

A 1% ( W m starch (2.5 mL) (Starch. soluble. BDH. Analar) solution was added

to a well mixed juice (20.0 rnL) in a 100 mL flask. From a burette, Iodine solution

(0.02N) (Iodine. N/50 solution certified 0,0205-0.0195 N. Fisher Scientific) was added in

a slow stream while gently swirling the flask. At the point where the solution showed a

strong biue-purple coloration that fades. the iodine was added one drop at a time. The

endpoint was indicated when one drop produced a blue-purple colour that persisted for at

least 30 seconds. In orange juice, the endpoint produced a light green coloration.

The following equation was used to determine the mg of ascorbic acid per 100 mL of

juice:

ascorbic acid concentration (mgA00rnL) = rnL of 0.02 N iodine X 8.8 1

To confirm the validity of the method, a standard solution of 30 mg of ascorbic

acid/100 mL of water was prepared (L-ascorbic acid, Sigma Chemical Co, MO, US) and

titrated with the solution of iodine.

Note: O.OZN Iodine should nor be storedfor more than 2 months.

3.4.6 Colour measurement

A juice sample (50.0 mL) was transferred to a specimen cell (1.5" Round?

LYSACK SALES, Associated LTD., Etobicoke, Ont., CA). A reading on a Hunter LAB

instrument (Hunter Associated Laboratory, Inc.. Virginia, US) was taken after calibration

of the instrument with black and white plates (HunterLAB color standard). The

HunterLAB was selected for a ON5 mode, 0.5 area view and 1.75 port size. The L. A and

B values were noted. Three readings were carried out per sample.

3.4.7 Sugars and organic acids

3.4.7.1 High performance liquid chromatography (HPLC)

A. tandard suwr solmng

D-Sucrose (500.0 mg), D-glucose (250 mg) and of D-fructose (250 mg) (AnaIar.

BDH Inc.. Toronto) were added to a 100 mL volumetric flask. The sugars were dissolved

and the flask was filled to the mark with water.

B.

Citric acid (150.0 mg) (Analar, BDH Inc., Toronto) and L(-)malic acid (50 mg)

(Sigma Chemical Co. MO. US) were added to 100 mL volumetric flask. The organic

acids were dissolved and the flask were filled to the mark with water.

C.

Standard calibration curves were drawn for each sugar and organic acid. A Waten

s o b a r e calculated calibration c w e s with accuracy limit for each quantified

components.

D.

Well mixed juice (10.0 mL) was centrihged for 5 minutes at 3410 x g (Fisher

Centrific Centrifuge, Model 225. rotor 04-978-54). The supernatant (5.0 mL) was

pipetted into a 100 mL volumetric flask and diluted with water. A SepPak C 18 (Sep-Pak

Plus cartridges. Waters Corporation, Milford. MA.) was conditioned with 5 mL of

methanol followed by 10 mL of water. After the conditioning. 5 mL of the diluted

supernatant juice was passed through the SepPak Cl8. The first 2 mL of the juice was

rejected to avoid dilution of sugars and organic acids by water. The recovered juice was

filtered through a 0.45 pm filter (Millex-HVl3. Millipore. Millipore Corporation.

Bedford. MA ) and placed in a sample vial for HPLC analysis. The standards of sugars

and organic acids were processed in the same manner as the juice samples to measure the

recovery. The percent of recovery for the sucrose, glucose and fructose were >99%.

>99% and 98% respectively. And for the citric and malic acids. the percent of recovery

were 99% and >97% respectively.

E. 1 C cond~t~on~ .

A Waten HPLC (Milford. MA, US) equipped with a Model 510 pump and an

autosampler (717 plus) was used for the KPLC analysis. The mobile phase was a

degassed solution of sulfuric acid 0.01 N (290 pL of H2SOdliter of water) (Analar, BDH

Inc.. Toronto) filtered through a 0.45 pm filter. The HPLC column used was an

Interaction polymetric Ion 300 (Inter Action Chromatography) with a BioRAD cation H+

as a precolumn. The HPLC column flow rate was 0.4 W m i n and the injection volume

was 1 5.0 pL. Solute elution was monitored using a fixed wavelength at 2 14 nm (Waters

484 tunable Absorbance) for the organic acids, and a differential rehctometer (Water

410) for the sugars. The total run time was 20 minutes. This method was adapted from

Doyon er a/.. 199 1.

Well mixed juice (1.0 mL) was placed in the cell of a digital rehctometer

(RFMM 80.0-95 % sugar, Bellingham & Stanley Limited. England). The reading of the

per cent of sugPr and the temperature was noted. The instrument was adjusted to zero with

water before each measurement.

3.4.7.3. Titratable acidity

Well mixed juice (35.0 mL) was added to a 250 mL beaker. The juice was diluted

with about 50 rnL of water. The juice was then titrated with NaOH (1.0 N) (Sodium

hydroxide. 1.000 * 0.002 N. VWR Scientific) using a pH meter (Coming pHmeter 140).

The titration with 1 .O N sodium hydroxide was followed until the equivalence point or pH

of 8.1 was reached. The rnL of NaOH used was noted. Because of the difficulty in

obtaining a precise endpoint (pH 8.2) with the pH meter method. a solution of

phenolphthalein (ACS, Fisher) I % H in ethanol as an indicator was used to verify the

endpoint. With the small variability in the density measurements, a standard value of

1.04 was used in all calculations of the titratable acidity to avoid a source of variation.

This method was adapted from Carter (198 1). Percent of acid was calculated

using the following equation:

% acid = {(N NaOH X volume (mL))/1000) X {100g/(mL juice X density)) X G.E.W.

(g/ 1 OOmL)

N = Normaliiy of NaOHsolution

G. E. W. = 6404for orange juice (molecular weight oj'the citric acid}

Measured density or a standard density of I . 04 was used for the calculation.

3.4.8 Sensory evaluation

The sensory evaluation was performed in parallel with the chemicd analysis by an

expert panel (12 persons) under the supervision of a sensory evaluation expert. A

descriptive quantitative profile with continuous scale limited to 0 to 130 was used. A

sample questionnaire is presented in the Appendix A.

The tests were performed on Tuesdays and Thursdays of the same week that the

samples were analyzed for the other nine parameters. Each panel member received a 50

rnL of juice in a glass (1 bottle for 4 persons). The samples were kept at 1 O C and at -18 0 C depending on the initial condition of processing. Twenty four hours (24 hrs) before

analysis, the samples were stocked in a refrigerator at 8.5 O C to equilibrate the

temperature to 10 O C for the sensory evaluation. The bin samples were frozen, if

necessary, until Monday and Wednesday to be thawed at the same time of the froten

samples. The frozen samples were immersed in a thennostated water bath at 40 'C for 30

minutes (Waterbath 183, Precision Scientitic Inc.) before the test.

3.4.9 Statistical treatment

Statistical treatments were performed on the data obtained under each condition

for months 1 to 8. A One-way analysis of variance (ANOVA) was not possible because

this method assumes populations with equal standard deviations and equal number of

samples. In most cases. in this study, a difference was detected by the Bartlen's test for

homogeneity of variances and some data were rejected according the Chauvenet's

criterion for rejecting a reading (Holman and Gajda. 1984). The Kruskal-Wallis non-

parametric ANOVA test was performed with the Instat software to establish significantly

different measurements. Statistical treatment (ANOVA) were performed on all data.

However. only relevant data are presented. Because of the variations in data among the

months of analysis, a t-test was performed on each parameter to compare the means of the

first and the last month of analysis. The critical value of the 1 t I for P value of 0.0 1 (99%

confidence interval) for a 5 degrees of freedom was 4.03 (Miller and Miller. 1988).

Appendix B shows different examples of the statistical calculations.

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Effect of storage condition on individual parameters

Parameters monitored during storage including density, cloud, sedimentation of

the pulp, sugars, organic acids. ascorbic acid, viscosity, colour. volatiles and sensory

analysis. are presented and discussed separately. Parameters which did not show

significant changes during the storage period are discussed first.

4.1.1 Density

Measurements of the den sity of the orange juice were performed monthly as

described in the Materials and Methods section. The results of the density measurements

are presented in Table 6.

A t-test analysis of the density data showed significant differences for juices

stored under conditions A and C ( I t I equaled 5.1 9 (A), 2.63 (B) and 6.75 (C)). However.

when the data were analyzed for a linear trend. no significant constant increase or

decrease were found. The data points were varied within one standard deviation of the

mean. Close to zero slopes (Y z b) were calculated and poor regression coeffcients

indicated no trend in the data among the eight months of analysis (see Table 7).

Table 6. Density value' of orange juice over an eight month storage period.

Condition A Condition B Condition C

Mean (g/mL) Standard Mean (g/mL) Standard Mean (g/mL) Standard Month deviation deviation deviation

I 1 .090* 0.002 1.105 0.005 1 SO4 0.002 2 1.1 14 0.0 14 1.120 0.0 13 1.107** 0.002 3 1.1 12 0.016 1.129** 0.0 19 1.121 0.02 1 4 I. 105 0.003 1.107 0.002 1.104** 0.00 1 5 1.105 0.005 1.107'" 0.003 1.104 0.005 6 1 SO0 0.007 1.097 0.006 1.103** 0.002 7 1.1 14 0.022 1.1 13 0.034 1.122 0.024 8 1.106 0.005 1.1 lo** 0.002 1.1 10 0.00 1

*Calculated with only three values. **Calculated with only five values. Chauvenet's criterion. I Average of six measurements. unless otherwise specified. Condition A: Not pasteurized orange juice stored at -1 8°C in glass bottles. Condition B: Pasteurized orange juice stored at 48*C in glass bottles. Condition C: Pasteurized orange juice stored at +l°C in polyethylene bag.

Table 7. Equation of the regression curves and coefficients of regression (R') of the

density of orange juice over an eight month storage period.

Condition Equation R' A Y = 0.0009 X + 1.1013 0.08320 B Y =-0.001 X + 1.1161 0.07663 C Y = 0.0008 X + 1,1058 0,06 195

The data tabulated in Table 6 did not present changes over time and showed no

difference at the beginning of the study between the juice stored under the three

conditions of storage (A, B and C). No significant increase or decrease in juice density

was found among the dam Changes of density due to different processes or storage times

have not been reported also in the literature.

4.1.2 Cloud and sedimentation of the pulp

Sedimentation of the pulp and cloud were measured by visual observation and

spectrophotometric readings. These two parameters are important for the appearance and

mouth feel of the orange juice. The results are presented in Tables 8 and 9. Orange juice

stored under conditions B and C did not show any sedimentation or loss of cloud.

Table 8. Sedimentation of the (measured at 3 time intervals) of orange juice

over an right month storage period.

Condition A I hour 2 hours 3 hours

Mean (mL) Standard Mean (mL) Standard Mean (mL) Standard Month deviation deviation deviation

1 8 3 9 0.7 62.01 0.0 55' I .4 2 66.7 9 -3 58.3 6.1 55.2 3 -8 3 58.6** 0.5 55.8 I .3 54.0 1.1 4 62.0 4.7 56.2 2.8 54.2 1.6 5 69.0 4.7 60.8 2.8 57.2 1.9 6 58.8 2.5 54.8 1.7 54.0 1.4 7 56.3 1.6 55.2 1 .Z 54.4* * 1.1 8 S 8 . P 0.5 55 .O 1.4 54.7 1 .4

*Calculated with only three values. **Calculated with only five values. Chauvenet's criterion. L Average of six measurements, unless otherwise specified.

Table 9. Cloud stability1 (measured at 3 time intervals) of orange juice over an

eight month storage period.

Condition A 1 hour 2 hours 3 hours

Mean (%T) Standard Mean (%T) Standard Mean (%T) Standard Month deviation deviation deviation

1 44.32* 1.84 55.76' 3.63 6 1.67* 2 -94 2 54.89 3 .OO 54.93 1.08 59.39 2.6 1 3 44.53 4.32 50.19 4. 14 52.84 4.25 4 48 2 2 0.70 55.12 2.70 58.29 2.93 5 56.78 7.95 61.01 3.85 63.24 1.54 6 56.2 1 5.57 55.39 3 .03 58.86 3.8 1 7 53 -63 1.51 58.78 1.22 60.49* * 0.75 8 57.07 - 3 r 61 57-89" 0.3 1 60-64 1 .58

*Calculated with only three values. **Calculated with only five values. Chauvenet's criterion. ' Average of six measurements. unless otherwise specified.

The data shown in Tables 8 and 9 are for orange juice samples stored under

condition A. Despite the variations in the data, a decreasing trend during the three hours

of measurements was observed. Moreover. after 3 hours. the monthly measurements o f

the unpasteurized orange juice showed a sedimentation of almost 50 %. A similar

phenomenon was observed for the measurement of the cloud. After three h o r n of

observation. the percent of transmittance increased by about 10 %. These observations

may be important to predict the stability of orange juice after thawing and processing.

Sedimentation of the pulp and cloudiness are important factors for mouth feel and

appearance. The presence of pulp modifies not only the intensity but also the balance of

the overall aroma.

4.1.3 Sugars

Brix measurement

Sugars comprise 70-85 % of the total soluble solid material in orange juice. Total

sugar was measured as 'Brix. The OBrix measurement was performed with a digital

refiactometer. The results of the O~rix measurements are presented in the Table 10.

Table 10. 0 Brix value' of orange juice over an eight month storage period.

Condition A Condition B Condition C

Mean (%) Standard Mean ( O h ) Standard Mean (%) Standard Month deviation deviation deviation

1 11.5 0.3 12.0 0.1 11.4 0.1 2 11.1 0.3 11.5 0.2 11.8 0.3 3 11.1 0.4 1 1.2** 0.3 10.6 0.5 4 1 1.5** 0.2 11.7 0.2 1 1.9** 0.1 5 11.1 0.5 11.6** 0.1 10.8 0.5 6 10.5 0.5 1 f .O** 0.4 11.4 0.3 7 11.2 0.3 1 I.8** 0.3 11.8 0.2 8 1 1.9** 0.1 12.2 0.1 12.1S* 0.0

**Calculated with only five values. Chauvenet's criterion. I Average of six measurements. unless otherwise specified.

Table 10 shows the results obtained over an eight month period. For the OBrix

value, no change in concentration with time of storage or method of processing was

observed. Linear equations of the regression curve and coefficients of regression were

calculated from the data (see Table 1 1).

Table 1 1. Equation of the regression curves and coefficients of regression (R') of the

'Brix of orange juice over an eight month storage period.

Condition Equation RL A Y = 0.0131 X + 11.2 0.006 1 1 B Y = 0.2620 X + 11.5 0.0263 1 C Y = 0.0738 X + 11.1 0.1 1355

A t-test analysis of the "Brix data showed significant difference only for the juice

stored under condition C ( 1 t 1 equaled 2.1 1 (A). 2.08 (B) and 13.58 (C)). A standard

deviation of zero for the last month can explain this result. However. when the data were

analyzed for a linear trend. no significant constant increase or decrease was found for any

of the stonge conditions. There was no linear correlation between the time of stonge and

the " ~ r i x value. The variability in the data may be due to random errors associated with

the refkctometer used to measure the 'Brix.

The literature also indicates no change in "Brix values of orange juice during

storage.

HPLC measurements

The main sugars found in orange juice (D-sucrose. D - ~ ~ U C O S ~ and D-fructose) were

measured by HPLC according to procedure described in the Materials and Methods

section. The results of the HPLC measurements for each sugar (D-sucrose, D - ~ ~ U C O S ~ and

D-fructose) are presented in Tables 12, 13 and 14. An example of an HPLC

chromatogram showing the three sugars is illustrated in Figure 7.

Table 12. Sucrose concentration' of orange juice over an eight month storage period.

Condition A Condition B Condition C

Mean Standard Mean Standard Mean Standard Month (g/lOOrnL) deviation (g/100mL) deviation (g/100mL) deviation

1 4.89 0.09 5.15 0.07 4.90 0.07 7 4.92 0.04 5.17 0.07 5.15 0.08 3 5.17 0.14 5.37 0.12 5.09 0.14 4 5.2 1 0.08 5.32 0.08 5.36 0.06 5 4.71 ** 0.07 4.85 0.12 4-50 0.12 6 4.96*+ 0.02 5.06 0.14 5.08 0.1 1 7 4.87 0.08 5.02 0.06 4.9 1 ** 0.03 8 4.90 0.02 5.10 0 .04 4.9 1 0.05

**Calculated with only five values, Chauvenet's criterion. I Average of six measurements. unless otherwise specified.

Table 13. Glucose concentration' of orange juice over an eight month storage period.

Condition A Condition B Condition C

Mean Standard Mean Standard Mean Standard Mon tb (g/100mL) deviation (gf 100mL) deviation (g/100rnL) deviation

1 1.97 0.0 1 1.99 0.05 1.90 0.02 3 - 1.85 0.04 1.89 . 0.02 1 -90 0.04 3 1.98 0.04 2.00 0.05 1.91 0.06 4 1.97 0.03 2.00 0.03 2.0SS* 0.0 1 5 1.82 0.06 1.84 0.03 1.77 0.04 6 1.91 0.02 1.93 0.07 1.99 0.07 7 1.95 0.04 1 -97 0.02 2.0 1 ** 0.00 8 1.88** 0.0 1 1.93 0.02 1.97 0.03

**Calculated with only five values, Chauvenet's criterion. I Average of six measurements, unless otherwise specified.

Table 14. Fructose concentration1 of orange juice over an eight month storage

period.

Condition A Condition B Condition C

Mean Standard Mean Standard Mean Standard Month (g/lOOmL) deviation (g1100mL) deviation (gI100mL) deviation

1 2.34 0.04 2.40 0.33 2.3 1 0.02 2 2.25 0.04 2.29** 0.0 1 2.34 0.07 3 2.36 0.03 2.40 0.04 2.30 0.04 4 2.33** 0.03 2.3 4 0.03 -. 7 40 0.05 5 2.27 0.02 2.29 0.04 - 7 .-- 77** 0.04 6 2.3 8 0.03 2.40 0.09 2.52** 0.04 7 2.42 0.03 2.47* * 0.03 2.50 0.04 8 2.3 1 0.02 2.34** 0.0 1 2.3 8 0.02

"Calculated with only five values, Chauvenet's criterion. I Average of six measurements, unless othenvise specified.

Figure 7. HPLC chromatogram of the three sugars in orange juice processed by condition

A at the last month (8) of storage (D-sucrose tR = 1 2.8 76 min. o-glucose rR = 1 5.550 min

and D-fructose tR = 17.1 1 7 min).

Data fiom the HPLC analysis of sugars (Tables 12- 14) showed some variability

over the eight months of analysis. The data appeared to increase or decrease as a block

regardless of the storage condition. A t-test was performed (see Table 15) with the data

tabulated in Tables 19 and 20.

Table IS. Results obtained from the t-test for each sugar contained in orange juice.

Condition I t 1 value

SUCROSE A 1.68 B 4.65 C 6.02

GLUCOSE A 14.4 1 B 2.8 1 C 4.93

FRUCTOSE

Significant differences between the means of D-sucrose concentration was found

for the juices stored under conditions B and C and similarly for D-glucose concentration

in juices stored under conditions A and C. However. when the data were analyzed for a

linear trend. no significant constant increase or decrease was found in all cases. Close to

zero slopes (Y z b) and poor regression coefficients were calculated for the data obtained

for the three storage conditions (see Table 16).

Table 16. Equation of the regression curves and coefficients

three sugars of orange juice over an eight month storage period.

of regression (R') of the

Condition Equation R SUCROSE

A Y = -0.0156 X + 5.02 0.05463 B Y = -0.0298 X + 5.26 0.19376 C Y = -0.0240 X + 5.10 0.0550 1

GLUCOSE A Y = -0.006 X + 1.94 0.05499 B Y = -0.005 X + 1.96 0.03903 C Y = 0.012 X + 1.88 0.1 1157

FRUCTOSE A Y = 0.008 X + 2.30 0.1 1107 B Y = 0.005 X + 2.34 0.04082 C Y = 0.021 X + 2.28 0.25797

The conclusion was the same for the three sugars. No linear trend could be

observed (almost zero slope). and the variation in the data could not be correlated linearly

with the time (R' very low). Therefore. there was no linear association between time of

storage and sugar concentrations.

4.1.4 Organic acids

Titratable acidity measurements

The measurement of the acidity of the orange juice is ofien expressed as titratable

acidity. This method is rapid and gives a general percentage of organic acids in juice.

Titration with NaOH was used to perform this measurement. The results of titratable

acidity measurements are presented the Table 17.

A t-test analysis of the titratable acidity data showed significant difference only

for juice stored under condition A ( I t 1 equaled 7.35 (A), 3.34 (B) and 2.75 (C)).

However, when the data were analyzed for a linear trend, no significant constant increase

or decrease was found in each condition.

Table 17. Titratable acidity measurement1 of orange juice over an eight month

storage period.

Condition A Condition B Condition C

Mean (%) Standard Mean (%) Standard Mean (%) Standard Month deviation deviation deviation

1 0.78 0.0 1 0.78 0.0 1 0.74 0.0 1 2 0.76 0.02 0.78 0.02 0.77** 0.0 1 3 0.77** 0.00 0.77" * 0.00 0.74" * 0.00 4 0.75 0.0 1 0.76 0.0 1 0.77 0.0 1 5 0.76 0.0 1 0.77 0.0 1 0.73 0.0 1 6 0.74** 0.0 1 0.76 0.0 1 0.77" * 0.00 7 0.75 0.0 t 0.75** 0.00 0.77 0.0 1 8 0.75 0.0 1 0.76 0.0 1 0.76 0.0 t

**Calculated with only five values. Chauvenet's criterion. 1 Average of six measurements. unless othenvise specified.

Table 18. Equation of the regression curves and coefficients of regression (R') of the

titratable acidity measurement of orange juice over an eight month storage period.

Condition Equation R A A Y = -0.004 X + 0.78 0,59834 B Y = -0.004 X + 0.78 0.72637 C Y = 0.002 X + 0.75 0.10812

Linear equations of the regression curve and coefficients of regression were

calculated from the data (see Table I I). The slopes of the regression curves were close to

zero and no real trend was observed. Some variability appeared in the three conditions

over the eight months. Regression coefficients were low and indicated that no linear

trend can be observed.

Table 17 is the data obtained fiom the colorimetric titration. The end point was

more stable with the method using phenolphthalein than the method with a fixed pH end

point (pH = 8.2).

The literature has also indicated that there is no change in titratable acidity of juice

during storage period.

4.1.4.2 HPLC measurements

Organic acids in orange juice consist primarily of citric and malic acids. These

two acids were quantified by HPLC measurement. The results of the HPLC analysis are

presented in Tables 19 and 20. An example of a HPLC chromatogram including the two

organic acids is illustrated in Figure 8.

Table 19. Citric acid concentration' of orange juice over an eight month storage

period.

Condition A Condition B Condition C

Mean Standard Mean Standard Mean Standard Month (mg/lOOrnL) deviation (mg/lOOmL) deviation (mg/lOOrnL) deviation

1 958.70 27.83 958.85 97.17 965.20 1 3 -69 3 - 894.48 19.76 922.63" 5 .07 932.16 25.06 3 989.25 * * 3.88 I 005.52** 6.12 957.36 10.85 4 1055.33 12-86 1074.60 12-79 1089.7 1 1 1.29 5 947.78 33.05 971.57 20.54 9 18.63 18.95 6 998.33 9.3 3 1012.80 33.69 1033.74** 15.62 7 962.42 18.13 980.89 1 3.23 980.77 7.66 8 979.57 8.70 1006.95 7.6 1 99 1.58 4.93

"Calculated with only five values, Chauvenet's criterion. Average of six measurements. unless otherwise specified.

Table 20. Malic acid concentration' of orange juice over an eight month storage

period.

Condition A Condition B Condition C

Mean Standard Mean Standard Mean Standard Month (mg/lOOmL) deviation (mg/lOOmL) deviation (mgI100mL) deviation

1 77.96* 2.27 162.23 4.85 158.55 2.95 2 157.40 4.4 1 160.80** 1.39 163.88 6.36 3 160.85 3 -03 164.3 7 2.09 159.42 2.07 4 165.67 1.89 170.16 5.37 1 72.72* * 0.95 5 1 65.69 6.99 170.55 3.32 158.18 7.05 6 167.78 3.66 171 -66 7.40 177.35** 3.30 7 140.10 4.55 164.6 1 2.83 164:94 2.65 8 161.78 1.85 165.84 1.33 156.02 11.14

*Calculated with only two values. **Calculated with only five values. Chauvenet's criterion. I Average of six measurements. unless otherwise specified.

Figure 8. HPLC chromatogram of the two organic acids in orange juice processed by

condition A at the last month (8) of the norage period (citric acid tR = 14.167 min and

malic acid tR = 17.267 min).

Data from the HPLC analysis of organic acids (Tables 19 and 20) showed some

variability over the eight months of analysis. The data appeared to increase or decrease as

a block regardless of the storage condition. A t-test was performed (see Table 21) with

the data tabulated in Tables 19 and 20. Significant differences between the means of

citric acid concentration was only found for the juice stored under condition C. However,

when the data were analyzed for a linear trend, no significant constant increase or

decrease was found (see Table 22).

Table 2 1.

juice.

Results obtained from the t-test for each organic acid contained in orange

Condition I t 1 value

CITRIC ACID

MALIC ACID

Table 22. Equation of the regression c w e s and coefficients of regression (R') of the

two organic acids of orange juice over an eight month storage period.

Condition Equation R A CITRIC ACID

A Y = 4.8271 X + 951.5 1 0.06593 B Y = 6.5096 X + 962.43 0.12610 C Y = 5.7830 X + 957.62 0.0646 I

MALIC ACID

The conclusion was the same for the two organic acids. No linear trend could be

observed (almost zero slope), and the variability in the data could not be correlated

linearly with the storage time (R' very low). Therefore, there was no linear association

between time and organic acids concentrations.

4.1.5 Ascorbic acid

The ascorbic acid was measured by titration of juice samples with a solution of

iodine (0.02 N) according to the experimental section. The results of the measurements of

the concentration of ascorbic acid are presented in Table 23 and illustrated in Figure 9.

Table 23. Ascorbic acid concentration of orange juice over an eight month storage

period.

Condition A Condition B Condition C

Mean Standard Mean Standard Mean Standard Month (mg/lOOmL) deviation (mgA00mL) deviation (mg/lOOmL) deviation

1 54.77 0.23 54.9 1 0.23 50.95 0 -23 7 - 52.94* * 0.20 52.05 1.35 50.44 0.72 3 52.42 0.56 51.91 0.43 49.04 0.45 4 53.08 0.46 52.42 0.28 48.82 0.94 5 52.79 0.30 52.41 ** 0.00 49.0 1 0 .27 6 53.37 0.5 1 5 1.83 0.87 48.71 ** 0.50 7 52.49 0.33 52.20 0.82 50.00 1.03 8 53.59 0.53 52.49 0.33 48.16 0.66

**Calculated with only five values. Chauvenet's criterion. I Average of six measurements, unless otherwise specified.

Table 24. Equation of the regression curves and coefficients of regression (R') of the

ascorbic acid concentration of orange juice over an eight month storage period.

Condition Equation R; A Y = -0,095X + 53-6 1 0.0939 1 B Y =-0.19SX + 53.41 023295 C* Y = -0,373X + 50-85 0.80 167

excluding data for the seventh month

Figure 9. Ascorbic acid concentration of orange juice over an eight month storage period

56

0 1 2 3 4 5 6 7 8 9

Month

Linear equations for the regression curves and coefficients of regression were

calculated from the data (see Table 24). A plot of the concentration of ascorbic acid as a

hction of time (months) is shown in Figure 9. Inspection of Table 24 and Figure 9

indicates that if the data for the first month is ignored, no trend can be observed in

ascorbic acid concentration for juices stored under conditions A and B. Although the

efficiency of the measurement of ascorbic acid concentration by titration is about 99 %

(measured with a standard of ascorbic acid). However, the titration method is associated

with difficulty of observing the end point in the orange juice. In fact, the juice becomes

lightly greenish. The higher values of the first month were probably due to experimental

enon of determining correctly the end point.

A t-test was performed with the data tabulated in Table 23. For juices stored

under conditions A and B, the first month was neglected. The comparison of the two

means showed a significant difference only for juice stored under condition C ( / t 1 equaled 2.78 (A), 0.78 (B) and 9.76 (C)). The data From juice stored under the third

condition (C) was also analyzed by a non-parametric ANOVA. Significant changes

(P<0.0001) were also found between different months of storage under condition C. The

Durn's test indicated that the first month data was significantly different from the fourth.

sixth and eighth months. Similarly, the second and seventh months data were also

significantly different. Regression equations were calculated using the data from all the

months (Y = -0.1796X + 50.07 and R squared equaled 0.24431) and also when the

seventh month was excluded (Y = -0.373 1X + 50.8499 and the coefficient of regression

was 0.801 67: Table 24). The Figure 9 shows the data without the seventh month for juice

stored under condition C. The slope presented a decreasing trend and the coefficient of

regression showed a negative relation between the time and the concentration of ascorbic

acid. indicating that the level of ascorbic acid present in the juice changes (decreases)

during the storage period. These changes may be due to the degradation of ascorbic acid.

primarily by the oxygen contained in the juice and by anaerobic non-enymatic reactions.

in the polyethylene bag stored at +1 OC. The ascorbic acid concentration dropped by 5 %

during the eight months of storage. The low temperature of storage (+l°C) minimized the

loss of ascorbic acid. A retention of 95 % of the original ascorbic acid concentration

during the storage period is sufficient to keep the nutritive quality of the juice.

The second month of each storage condition was examined to determine the

presence of any initial differences between the three conditions before the study period.

The D m ' s test indicated significant differences only between conditions A and C (PC

0.0 1). The initial significant difference between juice stored under condition A versus the

condition C is probably due to the degradation of ascorbic acid by oxygen contained in

the juice in the polyethylene bag at the beginning of the experiment. The decrease of

ascorbic acid probably occurred in the first week after the packaging. In the frozen juices

(conditions A and B), the reaction with the oxygen and the ascorbic acid molecule was

very slow. Pasteurization can also have a negative initial impact on the concentration of

ascorbic acid.

4.1.6 Viscosity

The viscosity measurements were performed on the orange juice on a monthly

basis during the eight months of storage. A 10.0 mL of centrifuged juice was placed in a

viscosimeter according to Chapter 4. The results were expressed in centi-Stoke (cSt =

mrn2/s). The results of the viscosity measurements are presented in Table 25. and

illustrated in Figure 10.

Unfortunately, the juice was not handled properly during the analysis of the first.

sixth and the seventh month samples. As a result the samples were refrozen and analyzed

a few months later. Consequently, the data obtained for these months were affected by re-

freezing and the results were not reliable (see Table 25). Data from these three

measurements (shown between brackets) were removed From the analysis and not

included on Figure 10 in the subsequent equations or calculations.

Table 25. Viscosity measurement' of orange juice over an eight month storage

period.

Condition A Condition B Condition C

Mean (eSt) Standard Mean (cSt) Standard Mean (cSt) Standard Month deviation deviation deviation

1 (1.1777*)~ 0.0047 (1.6407)' 0.1 101 (1.5 1 57)L 0.0575 2 0.93 19 0.0077 1.3 138 0.0 106 1 -234 1 0.0053 3 0.950 1 0.0 1 52 1.3336 0.0095 1.1876 0.0 157 4 0.9449 0.01 10 1.3 180 0.0082 1.2 157** 0.009 1 5 0.9367 0.0050 1.3141 0.0 100 1.1700 0.0048 6 (0.9094**)' 0.0004 (1.1763)' 0.0 158 (1.0882)' 0.0 192 7 (0.8969**)' 0.0068 (1.2096**)' 0.01 33 (1.1 9 8 ) 0.0193 8 0.9458 0.0092 1.3 109 0.0060 1 .I09 1 * * 0.0200

*Calculated with only two values. **Calculated with only five values, Chauvenet's criterion. I Average of six measurements, unless othenvise specified. ' Data unreliable due to improper manipulation.

A t-test analysis of the viscosity measurement data showed no significant

differences for all three conditions of storage ( 1 t I equaled 2.85 (A). 0.58 (B) and 2.97

(C)).

Table 26. Equation of the regression curves and coefficients of regression (R~) of the

viscosity measurement of orange juice over an eight month norage period.

Condition Equation R' A Y = 0.00 1 X -t 0.9373 0.10678 B Y = -0.002 X + 1.3263 0.227 18 C Y = -0.003X + 1.2150 0.05937

Figure 10. Viscosity of orange juice over an eight month storage period

1 2 3 4 5 6 7 8 9

Months

Linear equations of the regression curve and coefficients of regression were

calculated from the data (see Table 25). The graph of the viscosity (cSt) as a function of

time (months) shows a close to zero slope for the regression curve for dl the three

conditions. With the low significant differences in time and the slopes of the regression

curve of the three conditions of storage (A, B and C), no linear trend could be detected

over time. The t-test has confirmed this with t values less than 4.03 for all three

conditions. No increase or decrease in juice viscosity was found in this study. This

results are consistent with the literature data.

The Kruskal-Wallis ANOVA indicated a significant difference (P < 0.0001) in the

initial viscosities of the three conditions of storage (see Figure 10). The D u d s test

indicated that the difference was between the juices stored under conditions A and B

(considering the second months). The viscosity of juice stored under condition A was 30

% lower than the condition B. Lower viscosity for the unpasteurized orange juice is

expected because of the enzyme pectin methyl esterase was not deactivated and continued

to be effective before the complete freezing of the juice. A faster freezing could minimize

this effect. The two pasteurized orange juices differed only by 10 % in viscosity.

Probably. freezing increased the viscosity of orange juice however, no reference to this

phenomenon was found in the literature. The freezing storage could cause. on a long time

basis. problems in texture and mouth feel.

4.1.7 Colour

A phenomenon named browning can arise when orange juice is stored under

inappropriate conditions. The colour of the orange juice was measured with a Hunter

LAB instrument. The L value measures the lightness of the juice. This value is the most

important reading to measure a possible darkening of the juice. The results of the colour

measurements are presented in Table 27 and illustrated by Figure 1 1.

Table 27. Colour measurement1 (L value) of orange juice over an eight month

storage period.

Condition A Condition B Condition C

Mean Standard Mean Standard Mean Standard Month deviation deviation deviation

1 63-16 0.28 67.22 0.07 66.25 0.1 1 2 62.74 0.39 67.44 0.1 1 67.3 1 0.26 3 61.94 1.19 66.98 0.53 65.27 0.66 4 62.56 0.33 67.28 0 2 0 66.60 0.14 5 62.62 0.4 1 67.23 0.10 65.54** 0.19 6 61.70 0.87 65.96 0.6 1 64.4 1 0.57 7 61.98 0.59 67.19 0.3 1 63.74 0.30 8 6 1.98* 0.43 66.08 0.76 64.10** 0.36

*Calculated with only two values. **Calculated with only five values, Chauvenet's criterion. 1 Average of' six measurements. unless otherwise specified.

a A 1-test analysis of the L value data showed significant differences for juices

stored under conditions A and C ( ( t 1 equaled 5.33 (A). 3.65 (B) and 14.06 (C)).

However, when the data was analyzed for a linear trend. only juice stored under condition

C showed a decreasing trend.

Table 27 and Figure 1 I showed little variation in the L value for juices stored

under conditions A and B, but no major trends could be observed. On the other hand.

juice stored under condition C showed a decrease in the L value with time. The plot of L

value as a function of time showed a slope close to zero for the regression c w e for

condition A and B (Table 28).

Table 28. Equation of the regression curves and coefficients of regression (R') of the

colour measurement (L value) of orange juice over an eight month storage period.

Condition Equation RL A Y =-0.151X + 63.02 0.53870 B Y = -0.147 X + 67.58 0.39585 C Y = -0.435 X + 67.36 0.7050 1

Linear equation of the regression curves and coefficients of regression were

calculated from the data in Table 28. The juices stored under conditions A and B had

lower slopes and lower values of coefficients of regression than the juice stored under

condition C. The storage conditions A and B did not show any significant changes during

the storage. However, the juice stored under condition C revealed a decreasing trend over

time in the value of L. The coeficient of regression indicated a variability in the

measurement of this parameter.

The value of L of the juice stored under condition C showed a slight trend to

decrease as seen with the t-test. An analysis by Kruskall- Wallis non-parametric ANOVA

showed a significant difference in the data (P < 0.000 1). The D m ' s test indicated larger

differences between months. Comparison of the first month's value with the last month's

indicates that the juice had retained its colour almost at 97 % value. This change was not

considered important for the overall colour of orange juice.

Figure 11. Colour measurement (L value) of orange juice over an eight month storage period

0 1 2 3 4 5 6 7 8 9

Months

The first month's data was analyzed to find initial differences between the three

conditions of storage. The Kruskd-Wallis ANOVA indicated significant differences (P <

0.0001) between the three conditions of storage at the first month of analysis. The

Dunn's test indicated a significant difference between juices stored under conditions A

and B. This difference is obvious in Figure 1 I. The unpasteurized juice (condition A)

was darker than juices stored under conditions B and C. This fact was contrary to the

expected results because the heat treatment is more favorable to darkening of the orange

juice. However, this may be due to the interference of the pulp during the measurement.

4.1.8 Vola tiles

Volatiles were the most important parameters measured in this study. Some of the

volatile compounds were degraded during heat treatment and produced undesirable

components during the storage period. Changes in flavour compounds were monitored by

gas chromatography (GC) analysis. One GC system was coupled with a mass

spectrometer (MS) and the other with headspace injector. These two systems combined

permitted the quantification of 40 volatile compounds. The GC-MS analysis of volatiles

were performed during the first six months (problems with power failures during analyses

in the last two months caused loss in sensitivity in the MS multiplier and data were

therefore discarded). However. data from the six months were sufficient to observe trends

in the volatiles during storage. Only the volatiles that are most relevant are presented in

this chapter. Some volatiles did not change such as Iinalool, some others were on the

limit of detection such as vinyl guaiacol, and others were not detected like furfural. HMF

and h e o l . An example of the data obtained for the first month is presented in Table 29

(see Appendix C for huther examples).

Table 29. Volatile concentration of orange juice for the first month of storage.

CONDITION A B C

methyl-butymte a- pinene ethy 1-butyrate hexanal sabinene 3 -carene a-phellandrene P-myrcene heptad limonene 2&3 - methy 1-butano 1 2-hexenal 2-hexanol y-terpinene octanal 1 -hexand 3-hexen-1 -01 nonanal dimethyl-styrene fUrfirra1 decanal linalool octanol terpinene-4-0 1 hydroxy -ethy 1-hexanoate a-terpineo 1 valencene geranial & carvone perillaldehyde 2-2-decadienal vinyl guiaicol hydroxymethyl furfUral vanillin

CONDITION A B C

Headspace-GC @%mL) (ug/mL) (ug/mL) acetaldehyde 9.67 8.45 8.56 ethyl acetate 0.33 0.23 0.20 methanol 50.55 35.37 29.00 ethano 1 607.35 636.69 592.52

ND, not detected. *limit of detection.

The following section discusses the five volatiles which showed the most

interesting differences. Volatile that showed significant differences under different

storage conditions were methanol, 1-hexanol. P-myrcene. limonene and a-terpineol.

Methanol was the second most abundant alcohol in orange juice after ethanol. Its

contribution to the overall taste of juice has not been determined according to the

literature. The results of the methanol analyses are presented in Table 30 and illustrated

in Figure 12. Data obtained for the methanol concentration of the juice samples showed

some variability, especially for juice stored under condition A. the standard deviation of

the concentrations were large. This fact may be explained by the interference of the pulp

present in some samples. The pulp appears to have complicated the measurement of

volatile compounds. The dispersion of the volatile components between the pulp and the

serum can be affected by the aggregation of the pulp found particularly in samples stored

under condition A.

Table 26. Methanol concentration' of orange juice over an eight month storage

period.

Condition A Condition B Condition C

Mean Standard Mean Standard Mean Standard Month (ug/mL) deviation (ug/mL) deviation (uglmL) deviation

1 50.55** 7.29 35.37** 0.98 29.00* 7.19 2 49.86** 3.64 36-21 1.80 35.62** 5. I8 3 53.01S* 2.30 33.91 2.08 32.19** 0.29 4 52.54** 2.42 3 5 -54 3.93 36.65 1.49 5 50.99 12.83 37.09* * 0.44 3 3.40 1 .58 6 55.12 6.82 37.05 0-94 38.03"" 0.52 7 59.46 12.29 35.88 1. I5 3 5.3 3 2.04 8 47.05** 3.79 35.80** 1.99 3 5 .27+ * 1.14

"Calculated with only five values. Chauvenet's criterion. I Average of six measurements. unless otherwise specified.

The t-test analysis of the methanol data showed no significant differences for all

the three conditions of storage ( I t I equaled 0.95 (A), 0.43 (B) and 1.93 (C)).

Table 3 1. Equation of the regression curves and coefficients of regression (R') of the

methanol concentration of orange juice over an eight month storage period.

Condition Equation R; A Y = -0.004 X + 0.78 0.59834

The graph of the concentration of methanol as function of time showed close to

zero slopes and poor coefficients of regression for all the three conditions of storage (see

Figure 12) indicating no linear trends. The t-ten has confirmed this with values less than

4.03 for all the three conditions of storage. No increase or decrease in methanol

concentration in orange juice was found in this study. Despite the addition of an internal

standard. the headspace data were found to be very variable.

A difference was observed between the unpasteurized (A) and the pasteurized (B

and C) orange juices. A non-parametric ANOVA (Kruskal-Wdlis) was performed on the

initial data. Significant changes were found at P < 0.0001. The Dunn's test showed a

significant difference between juices stored under conditions A and C. Methanol

concentrations in the samples processed by condition C were approximately 45 % that of

those processed by the condition A. Microbial activity before the complete freezing of

the orange juice may be responsible for these differences.

Figure 12. Methanol concentration of orange juice over an eight month storage period

I 1 I 1 1 I I I

0 1 2 3 4 5 6 7 8 9

Months

Although the role of methanol in the overall flavour of orange juice is not

described in the literature, it could be speculated that it may enhance some of the

desirable flavour notes of the orange juice.

1-Hexanol is another volatile whose contribution to the orange flavour has not

been clearly determined. Diin et al. (198 1) have classified it as a detrimental alcohol. No

other references were available in the literature. The results of the I-hexanol analyses are

presented in Table 32 and illustrated at Figure 13.

Table 32. I-Hexanol concentration' of orange juice over a six month storage period.

Condition A Condition B Condition C

Mean Standard Mean Standard Mean Standard Month (ugImL) deviation (ug/mL) deviation (ug/mL) deviation

1 0.88 0.06 0.34 0.0 1 0.08 0.06 2 0.94 0.04 0.28** 0.03 0.12 0.04 3 0.85 0.03 0.41** 0.0 1 0. 19** 0.0 1 4 0.88 0.10 0.35** 0.0 1 0.28 0.02 5 1 .OO 0.05 0.42 0.0 I 0.19 0.0 Z 6 0.72 0.07 0.35 0.03 0.35 0.02

**Calculated with only five values. Chauvenet's criterion. I Average of six measurements, unless otherwise specified.

A t-test analysis of the I-hexanol data showed significant differences for juices

stored under conditions A and C ( 1 t 1 equaled 4.18 (A), 1.29 (B) and 9.98 (C)). No linear

trend was observed in juices stored under conditions A and B even though the t-test

showed a significant difference in the two means for juice stored under condition A.

Table 33. Equation of the regression curves and coefficients of regressior. (R') of the

1-hexanol concentration of orange juice over an eight month storage period.

Condition Equation R; A Y = -0.0 17X + 0.93 0.1 1179 B Y =O.OllX + 0.31 0.18355 C Y = 0.047X + 0.04 0.77968

The graph of the concentration of 1-hexanol as hc t ion of time (months) showed

close to zero slopes and poor coefficients of regression for the three conditions. No linear

correlation or trend between the concentration of 1-hexanol and time was found (Figure

15). The juice stored under condition C seemed to increase slightly with time, but the

concentrations were at the detection limit, and the little increase was not significant.

Differences were observed between the initial concentrations of 1 -hexan01 in

juices processed under the three conditions of storage. A non-parametric ANOVA

(Kruskai-Wallis) performed on the first month data indicated the presence of significant

changes (P < 0.0001). The D m ' s test also showed significant differences between

samples of juice stored under conditions A and C. Pasteurization may be responsible for

the initial difference of 90 % between the conditions C and A (see Figure 13). Samples

processed by condition B had approximately 60 % less I -hexan01 than samples processed

by condition A. The pasteurization and/or microbial activity may interfere with the levels

of I-hexanol. Although the literature indicates that 1-hexanol is undesirable in orange

juice. However. sensory analysis (see section 4.1.9) favored storage condition A.

Figure 13. 1-Hexanol concentration of orange juice over a six month storage period

0 1 2 3 4 5 6 7

Months

The P-myrceae was the second most abundant terpene found in the orange juice

samples. It is recognized to be important in orange juice flavour with its fruity aroma and

sweet balsamic-herbaceous taste at below 10 ppm levels (Arctander, 1969). The results of

the P-myrcene analyses are presented in Table 34 and illustrated in Figure 14.

Table 34. P-Myrcene concentration of orange juice over a six month storage period.

Condition A Condition B Condition C

Mean Standard Mean Standard Mean Standard Month (ug/mL) deviation (ug/mL) deviation (ug/mL) deviation

I 2,17** 0.10 2.02 0.06 1.89 0.02 2 2.02 0.22 1 -79'' 0.05 1.64* * 0 .03 3 2.37* * 0.25 2.04* * 0.02 1.65 0.09 4 2.58 0.50 1.94 0.08 I .58* * 0.0 1 3 2.3 5 0.26 1.97** 0.05 1.39 0.26 6 1.90 0.10 1.60** 0.02 1.22 0.03

*Calculated with only five values. Chauvenet's criterion. 1 Average of six measurements, unless otherwise specified.

The P-myrcene is a non-polar component associated with the pulp rather than with

the serum of juice. As mentioned above, the aggregation of the pulp seems to interfere

with the quantification of pulp-associated components such as P-myrcene. As a result the

data obtained from the analysis were variable. especially for juices stored under condition

A. A t-test analysis showed significant differences for all the three conditions of storage

(A. B and C) ( 1 t 1 equaled 4.39 (A), 15.40 (B) and 45.06 (C)). However. these

differences between the two means were affected by the variability in the data. No linear

trend was observed in juices stored under conditions A and B even if the data for the t-test

presented a significant difference in the two means.

Table 35. Equation of the regression curves and coefficients of regression (R') of the

P-myrcene concentration of orange juice over an eight month storage period.

Condition Equation R' A Y = 0.004 X + 2.25 0.00 103 B Y = 0.047X + 2.06 0.27657 C Y =-0.1 19X + 1.98 0.92593

The graph of the concentration of P-myrcene as function of time (months) showed

close to zero slopes and poor coefficients of regression indicating the absence of a linear

correlation between the concentration of P-myrcene and the storage time for conditions A

and B. The concentration of P-myrcene in samples processed by the condition C

decreased slightly with time. A non-parametric ANOVA (Kurskal-Wallis) was performed

on the data processed by juice stored under condition C. Significant changes were found

between the different months (P < 0.0001). The Dunn's Multiple Comparison test

indicated differences between the data of the first month and the fifth and similarly

between the first and the sixth.

Figure 14. Beta-myrcene concentration of orange juice over a six month storage period

0 1 2 3 4 5 6 7

Months

A non-parametric ANOVA (Kruskal-Wallis) was performed on the initial data

from the first month of analysis to see if any initial difference can be observed due to the

method of processing. Significant differences were found (P < 0.0001). The D m ' s test

showed significant differences between juices stored under conditions A and C. Sorption

of the myrcene by the LDPE (low density polyethylene) was probably responsible for this

difference. The sorption of the P-rnyrcene continued with time of storage until the sixth

month. No degradation mechanism of P-myrcene has been reported in the literature, and

for this reason ail the losses are attributed to the sorption by the LDPE. A decrease of

about 35 % was noted between concentrations of P-myrcene at the beginning and at the

end of the analysis for condition C. The loss of 35 % by sorption due to LDPE was in

agreement with the literature (up to 40 % sorption by the LDPE). The temperature of

storage may have influenced the time taken for this reaction.

The limonene was the most abundant volatile component in orange juice. This

terpene accounted for almost 95 % of all the volatiles measured. The limonene has a low

aroma of citrus. Its contribution to the total flavour of orange juice is low and is

recognized to be an important precursor of ot'flavours through degradation to a-

terpineol. The results of the limonene analyses are presented in Table 36 and illustrated

in Figure 15.

The analysis of limonene was complicated due to relatively high levels found in

the orange juice compared to the rest of volatiles. The simultaneous extraction of all the

volatiles presented problems during quantification. In order to have sufficient

concentrations of the less abundant volatiles, the extracts were concentrated ten times.

The analysis by GC-MS of the extract showed saturation of the signal for the limonene.

As a result a new calibration curve was developed for limonene with lower

concentrations. A quadratic equation was fitted to calculate the concentration of limonene

in the juice samples.

Table 36. Limonene concentration1 of orange juice over a six month storage period.

Condition A Condition B Condition C

Mean Standard Mean Standard Mean Standard Month (ug/mL) deviation (ughL) deviation (ug/mL) deviation

1 193.73** 11.41 1 76.03 9.92 159.12** 4.88 2 181.1 1 17.2 1 173.29 5.82 158.1 1 4.22 3 1 92.00* * 27.22 169.34** 4.32 1 3 7.22 13.65 4 1 83 .OO 25.23 153 -67 8.69 128.69 4.78 5 (234.20)? 26.62 (202.34)' 1 1.26 (146.16)' 5.3 1 6 189.62 9-77 172.30 6.62 129.98 5.10

**Calculated with only five values, Chauvenet's criterion. I Average of six measurements, unless otherwise specified.

Data unreliable due to instrumental malfunction.

A t-test analysis of the limonene data showed significant differences for juice

stored under condition C ( I t I equaled 0.93 (A). 1.25 (B) and 11.00 (C)).

Table 37. Equation of the regression curves and coefficients of regression (R') of the

lirnonene concentration of orange juice over an eight month storage period.

Condition Equation R A Y = -0.3 11X + 188.89 0.0 1 155 B Y = -1.60X + 174.05 0.12010 C Y = -6.78X + 164.32 0.7600 1

The graph (Figure 15) of the concentration of limonene as bct ion of time

(months) shows no linear wnd of limonene concentration with time for juices stored

under conditions A and B. Regression analysis also indicated close to zero slopes and

poor coefficients of regression for juices stored under conditions A and B (Table 37).

(The limonene

calculations - spectrometer).

concentration obtained for the fifth

Figure 15 and Table 37 - due to

month was excluded in the above

technical problems with the mass

Figure 15. Limonene concentration of orange juice over a six month storage period

0 I 2 3 4 5 6 7

Months

Like P-rnyrcene, the Iimonene is a non-polar component associated with the pulp

rather than with the serum of the juice. Differences found in the pulp consistency may

explain the difficulty to obtain reproducible measurements. sIts appears to be easier to

extract the volatile component associated with the serum than with the pulp. The

concentration of limonene in juice stored under condition C decreased with time

according the Figure 15, and confirmed by the t-test. A non-parametric ANOVA

(Kurskal-Wallis) was also performed on this data. Significant differences were found

between the different months (P < 0.0001). The Dunn's Multiple Comparison test

indicated differences between the first and the fourth and also between the first and the

sixth months among others.

The first month data were analyzed to detect initial differences among the three

sample types due to sorption or pasteurization. A non-parametric ANOVA (Kruskal-

Wallis) was performed on the fiat month data From each group. Significant differences

were found (P c 0.0001). The D m ' s test showed significant differences between juices

stored under conditions A and C. Sorption of the limonene by the LDPE and

pasteurization were probably responsible for these differences. The differences in

limonene concentration between juices stored under conditions A and B can be explained

by the pasteurization. A difference of about 8 % was found between the two means for

the two processes (A and B). The initial sorption of the LDPE can be estimated by

comparing data from juices stored under conditions B and C. A difference of about 8 %

was found between the means of the first months of juices stored under conditions B and

C. The sorption of the limonene probably continued with time until the sixth month.

Another phenomenon was probably occurring during the six months of storage: the

degradation of the limonene. especially to a-terpineol. Adsorption of limonene may lead

to a loss of about 25 % according to the literature but the temperature of storage may

affect the rate of this reaction. The difference in concentration of limonene between the

first month and the sixth month was about 18 Oio. About a total of 30 ppm of limonene

was adsorbed or degraded at a rate of about 5 ppm per month..

The a-terpiaeol is one of the most detrimental degradation products in orange

juice. Its gives a stale, musty or piney aroma at concentrations around 2-3 ppm. In

general, the a-terpineol concentration increases with heat treatment and storage. The

results of the a-terpineol analyses are presented in Table 38 and illustrated in Figure 16.

Table 38. a-Terpineol concentration' of orange juice over a six month storage

period.

Condition A Condition B Condition C

Mean Standard Mean Standard Mean Standard Month (ug/mL) deviation (ug/mL) deviation (ughnL) deviation

1 4.09 0.33 1.43 0.09 1.67** 0.05 2 3.56* * 0.05 1.80 0.06 2.50 0.08 3 4.34* * 0.13 t -55 0.05 2.33** 0.08 4 3.95 0.26 1.40 0.04 3. I5 0.14 5 4.10** 0.08 1.37 0.06 3.13 0.10 6 4.49 0.13 1.15** 0.03 3.46 0.08

*+Calculated with only five values. Chauvenet's criterion. I Average of six measurements, unless otherwise specified.

A t-test analysis showed significant differences in the a-terpineol data for juices

stored under conditions B and C ( I t 1 equaled 2.79 (A), 6.40 (B) and 43.50 (C)).

However, no linear trends were observed in juices stored under conditions A and B

although the t-test showed a significant difference in the two means for condition B.

Table 39. Equation of the regression curves and coefficients of regression (R') of the

a-terpineol concentration of orange juice over an eight month storage period.

Condition Equation R' A Y = 0.093 X + 3.77 0.28493 B Y =-O=OSI X + 1.73 0.49708 C Y = O X 3 X + 1.54 0.88 18 1

The graph (see Figure 16) of the concentration of a-terpineol as h c t i o n of time (months)

showed close to zero slopes and poor coefficients of regression for juices stored under

conditions A and B (Table 39). No decreasing or increasing trends were observed for the

concentration of a-terpineol with time for juices stored under conditions A and B.

However, the concentration of a-terpineol in juice samples stored under condition C

increased with time according the Figure 16 and confirmed by the t-test.

Figure 16. Alpha-terpineol concentration of orange juice over a six month storage period

Months

An non-parametric ANOVA (Kurskal-Wallis) was performed on the data of juice

stored under condition C. Significant differences were found between the different

months (P < 0.000 1). The Dunn's Multiple Comparison test indicated differences

between most of the months. An increasing mnd could be observed for the concentration

of a-terpineol in orange juice after six months of storage. An increase of about 50 % in

a-terpineol concentration was observed (about 1.80 ppm) at the end of the sixth month.

This is equal to the formation of about 0.30 ppm per month and this value represents only

the 6 % of the drop in limonene concentration.

A non-parametric ANOVA (Kruskal-Wallis) was performed on the first month

data to detect any initial differences. Significant differences were found (P c 0.0001).

The D u d s test showed significant differences between juices stored under conditions A

and B. The percent difference between the concentrations of a-terpineol in juices stored

under conditions A and B at the first month was 65 % higher in A. This difference in a-

terpineol concentration between unpasteurized orange juice (A) and pasteurized orange

juice (B) was unexpected because a-terpineol concentration is supposed to increase with

increasing heat treatment, such as during pasteurization. However, the sensory evaluation

data indicated (see the following section) that the juice stored under condition A had the

highest scores for the "fresh pressed" note. This might indicate that the off-flavour of a-

terpineol was masked due to the presence of other unknown components.

4.1.9 Sensory evaluation

Sensory evaluation is essential to correlate chemical and physical changes to the

overall organo leptic properties of the juice. Without sensory evaluation. it is difficult to

assen that one condition was better than the others. The sensory evaluation was

performed by a panel of twelve orange juice experts. The panel judged twelve orange

juice attributes (acidity, sweetness, orange perfume. orange taste. homogeneity, presence

of pulp. fresh pressed, oily. maturity, plastic, oxidation and peely) over the eight month

period. The scores for the first month were not included in the analysis due to

adjustments introduced after the first session such as some attributes were added and the

scale was changed from 100 to 130. The mean scores of each attribute of the sensory

evaluation are presented in Table 40.

Table 40 shows the monthly means (20 to 24 evaluations) of the scores by the

expert judges. The standard deviations are not included in Table 40. Deviations between

the scores of the expert panel were sometimes very large but the data seemed to be

consistent during the period of analysis.

Analysis of the sensory scores indicated that bbmaturity" and "peely" attributes did

not show any significant changes during the storage period for all the conditions. The

results were initially the same and no trend in time was observed. Concerning the

attributes that showed variations. the changes can be divided into two classes: initial

changes and changes due to storage (8 months). The "acidity " attribute was constant in

time but juice stored under condition A seems to have slightly less acidic taste thm the

pasteurized juices (conditions B and C). Similar pattern was observed for the "oily"

attribute. The opposite phenomenon was noted for the "sweetness" attribute. The

unpasteurized juice (condition A) had slightly higher scores for the sweet taste. The

condition A was also noted to have a better "perfine of orange" compared to the

conditions B and C. Figure 17 showed the results obtained for the anribute "orange

taste".

Table 40. Mean scores of sensory evalunliun' of orange j juice over eight months o f storage.

Month Acidity S d n e s s Onnw Orange Homogeneity Presence of Fresh oily Maturity Plastlc Oxidation Pwly pedume taste PU~P P - U ~

Condition A: Not pasteurized orange juice stored at - 18°C' in glass bottles. a

Condition 13: Pasteurized orange juice stored ill - 1 8 " ~ in ~ I ~ I S S bottles. Condition C: Pasteurized orange juice stored at + I"C in polyethylene bag. 'scores are on a scale of*O- 130.

Figure 17. Evolution of the attribute "orange taste" in orange juice over an eight month storage period

1 2 3 4 5 6 7 8 9

Months

The "orange taste" was more pronounced in orange juice stored under condition A

which showed about 30 % higher score compared to condition C. A more appreciable

difference was found for the "fresh pressed" attribute (Figure 18). Orange juice stored

under condition A showed 40 % higher score than condition B and a 50 % higher score

than condition C. Therefore, the juice stored under condition A had significantly better

fresh pressed orange taste. On the other hand, a decreasing freshness was observed in

juice stored under condition C to the extent of 25 %. Consistent with this observation. the

juice stored under condition C also appeared to have higher scores in two attributes:

"oxidation" and "plastic". The former showed a tendency to increase during the storage

period ( 6 5 %). On the other hand, the "plastic" attribute was five times higher in juice

stored under condition C compared to A.

Figure 18. Evolution of the attribute "fresh pressed" in orange juice over an eight month storage period

0 , I i 1 I I 1 I I

1 2 3 4 5 6 7 8 9

Months

The last two attributes, "homogeneity" and "presence of pulp" were significantly

different in the pasteurized and unpasteurized juices. Figures 19 and 20 show

respectively the variation in the "homogeneity" and the "presence of pulp" over a period

of eight months.

Figure 19. Evolution of the attribute "homogeneity" in orange juice over an eight month storage period

1 2 3 4 5 6 7 8 9

Months

The juice stored under condition A was about 50 % less homogenous and had 50

% more pulp than the two other pasteurized orange juices (B and C). The pasteurization

seems to have a stabilizing effect on the pulp matrix. According to Figure 20, a

decreasing trend for the presence of pulp in juice stored under condition C was observed

(decrease of 25 %). This fact could be caused by a variation in texture due to pulp

aggregation.

Figure 20. Evolution of the attribute "presence of pulp" in orange juice over an eight month storage period

1 2 3 4 5 6 7 8 9

Months

4.2 Summary of all the parameters

All changes found in the ten parameters are summarized in Table 4 1.

Table 41. Summary of the changes during the period of storage.

PARAMETER COMMENTS

Density Cloud a

Sedimentation a

Sugars Organic acids Ascorbic acid

Viscosity a

8 Colour

Volatile Methanol a

P-myrcene

Limonene

No change An increase of 10 % in the transmittance of the juice stored under condition A, after 3 hours of monitoring. Sedimentation of 50 % of the pulp particles after 3 hours for juice stored under condition A. No change No change A decrease of 5 % in concentration of ascorbic acid for juice stored under condition C over the storage period. Initial differences: (1) viscosity of juice stored under condition A was 30 % lower than in juice stored under condition B. (2) A difference of 10 % between the two pasteurized orange juices (conditions B and C). 97 % of the colour was retained under condition C (loss of 3 %). Juice stored under condition A was the darkest (screening effect of the pulp particles).

Initial difference in concentration of methanol: orange juice stored under condition A showed 45 % higher concentration than juices stored under conditions B and C. Initial difference in concentration of I -hexanol: orange juice stored under condition A showed 60 and 90 % higher concentrations than juices stored under conditions B and C respectively. Decrease of about 35 % in concentration of p-myrcene for juice stored under condition C. Decrease of 18 % in concentration of limonene for juice stored under condition C (5 ~ ~ d r n o n t h ) .

a-terpineol

10 Sensory analysis Maturity Peely Acidity

Oily

Sweetness

Perfume of orange

Orange taste

Fresh pressed

a

a

0

0

Oxidation

Plastic

Homogeneity

Presence of pulp

a

Increase of about 50 % in concentration of a-terpineoi for juice stored under condition C (0.30 ppdmonth). Initial difference in concentration of a-terpineol: orange juice stored under condition A showed 65 % (4 ppm) higher concentrations than juices stored under conditions B and C*

No change No change Initial difference: slightly less acid taste in juice stored under condition A than in juice stored under conditions B and C. Initial difference: slightly less oily taste in juice stored under condition A than in juice stored under conditions B and C. Initial difference: slightly higher sweet taste in juice stored under condition A than in juice stored under conditions B and C. Juice stored under condition A had a better p e h e of orange than juices stored under conditions B and C. Juice stored under condition A showed a higher initial difference of about 30 % compared to condition C. A 50 % initial difference was observed between the conditions A and C. Loss of freshness of 25 % in juice stored under condition C during storage. Tendency to increase (35 %) in juice stored under condition C over the storage period. Five times higher in juice stored under condition C than in juice stored under condition A. Juice stored under condition A was about 50 % less homogenous than juices stored under both conditions B and C. Unpasteurized orange juice (condition A) contained 50 % more pulp. A wnd to decrease (25 %) in the presence of the pulp in iuice stored under condition A over the storage wriod.

4.3 Correlation between chemical and physical changes and sensory

evduation

Correlation between chemical, physical and sensory analysis was complicated due

to the number of variables that could affect the taste of the orange juice. However some

suggestions can be put forward.

First. lower viscosity and higher rate of sedimentation of orange juice stored under

condition A (unpasteurized orange juice) can be associated with the attributes

"homogeneity" and "presence of pulp". The activity of the PME enzyme could affect the

cloud which suppons the pulp particles. Most of the particles contributing to the

viscosity of the orange juice are pectin molecules. The lack of homogeneity could be due

to a low freezing rate which is associated with large ice crystal formation and

agglomeration of the pulp into larger particles. This phenomena was evident for the

expert panel. The presence of pulp could be advantageous or disadvantageous depending

on the consumer's taste. To minimize the global effect of the enzymes (PME and others).

a faster freezing rate of orange juice could be suggested to overcome this problem in

unpasteurized orange juice.

The attributes "acidity" and "sweetness" were perceived slightly differently in the

unpasteurized orange juice (condition A). It could not be correlated to the chemical

analysis since no significant changes were observed in the sugar and organic acid

contents. The attributes "plastic" and "oxidation" also could not be correlated to chemical

analysis. These off flavours probably leaked, in large part, fiom the packaging material

(LDPE), which could impart the undesirable attributes noted..

The most interesting data were fiom the "fiesh pressed" and "orange taste"

attributes. The high scores of the expert panel given for juice stored under condition A

versus the pasteurized juices (conditions B and C) were important for the evaluation of

this study and for fisher research. These attributes were probably related, in large part,

to the volatile components identified in the orange juice. According to section 4.1.8

(volatiles), some important differences were found among the juices stored under the

three conditions. For orange juice stored under condition A, the concentrations of

methanol and I-hexanol were 45 and 90 % higher respectively than in C. In addition

condition A showed no sorption of volatiles by the packaging material. On the other

hand, the juice stored under condition C which scored low in these attributes, had 18 %

sorption or degradation of limonene and an increase of 0.30 ppm per month in alpha-

terpineol concentration. This obsentation could explain the low scores obatined in the

"fresh pressed" attribute. However. the finding of a high content of an off-flavour

compound alpha-terpineol in orange juice (condition A) which as also rated high in

sensory evaluation was unexpected.

CHAPTER 5

CONCLUSION

Orange juice is prone to rapid chemical deterioration which is highly dependent on

storage temperature. The condition A (unpasteurized frozen orange juice) seems to be the

optimum storage condition with the retention of some important volatile components and

the highest sensory evaluation scores. The condition B (pasteurized frozen orange juice)

is completely discarded as a method of storage because of its high cost and the low

sensory evaluation scores associated with the process. Condition C (the aseptically

packaged orange juice stored at +1 "c) is economically favorable but losses of important

volatile components by sorption on LDPE and degradation during long term storage led to

its lower rating in sensory evaluation. It could be proposed, as a practical alternative that

mixing aseptically packaged pasteurized orange juice with unpasteurized frozen juice. can

generate an orange juice with good sensory qualities and at a lower cost to the consumers.

However, a more rapid freezing can avoid the effect of enzymes and agglomeration of the

pulp particles. Moreover. a method to stabilize the pulp in the thawed unpasteurized

orange juice should also be developed before commercial application of the freezing

method of storage.

Although, in this study, some differences were found in the concentrations of the

volatile components in the three conditions of storage (A, B and C). However, undetected

and very low concentrations of low threshold compounds could influence the aroma

profile. In addition, synergetic effect of different volatiles are still unknown. The effect,

on the flavour profile of orange juice, of the individual volatile components such as

methanol, 1-hexanol, a-terpineol, limonene and myrcene, should be known in order to

have a better understanding of the complex aroma of the orange juice.

Future studies on the possible effects of the microorganisms and enzymes on the

quality of fresh unpasteurized orange juice. could lead to a better understanding of the

reactions that could occur before the complete Freezing of the orange juice. Higher

concentrations of certain volatiles, such as methanol and 1-hexanol. found in the

unpasteurized orange juice could be due to the activity of enzymes and microorganisms.

Finally, research could also be undertaken, to verify the possibility of masking of off-

flavours such as a-terpineoi.

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APPENDIX A

PROFIL DESCRlPTIF Q W T I T A TIF*

NOM:

Prenom: Date:

Nous dons evaluer un ichantillon de jus d'orange. en fonction de l'intensite de ses

caracteristiques do srveurs ET d'arhes. Pour chaque descripteur. on notera l'intensite

perGue sur l'echelle bomee correspondante. Une echelle d'ajout de termes se trouve en

fin de questionnaire. en cas de besoin.

MDIQUEZ LE CODE DE L'ECHANTILLON D E G U S ~ DANS LA CASE

SUIVANTE:

Descri pteur: AC IDE

MTENSI'T$ nulle tres forte

Descripteur: SUC&

MTENSIT~. nulle tres forte

Descripteur: PARFUM D'ORANGE

INTENS 1s nulle tres forte

* The test was conducted in French.

Descripteur: G O ~ T (AROME) D'ORANGE

M T E N S I ~ nulle tres forte

Descripteur: HOMOGENEITE DU N S (TEXTURE)

M T E N S I ~ nulle tres forte

Descripteur: P R ~ E N C E DE PULPE (TEXTURE)

INTENS ITE faible forte

Descripteur: FRAIS PRESSE

MTENS ITE pas du tout tres

Descripteur: HUILEUX

[NTENSITE pas ciu tout tres

Descripteur: MATUN* DE L'ORANGE

MTMSITE pasdu tout

Descripteur: LAMINEPLASTIQUE

INTENSITE pas du tout tres

Descripteur: OXYDATION

IN TENS IT^. pas du tout tres

Descripteur: ZESTE

IN TENS IT^. pas du tout tres

E W P L E S OF STATISTICAL CALCULATIONS

on (Holman and Gajda, 1984)

-

In applying Chauvenet's criterion to eliminate dubious data points. one first calculates the

mean value and standard deviation using all data points. The deviations of the individual

points are then compared with the standard deviation in accordance with the following

information, and the dubious points are eliminated.

Number of readings

n

6

Ratio of maximum acceptable deviation

to standard deviation. dm,&

1.73

The mean (x,,,) and the o value is given by:

Example: Methanol for the processing condition A

Data 40.67.45.35,54.13,59.01,46.35,58.73

Mean 50.7 1

a 7.66

di = 1 40.67 - 50.7 1 1 = 10.04

dmJm = 10.04/7.66 = 1.3 1

The data was not rejected.

Kruskal- Wallis non-~ametric ANOVA (Ins tat software)

Example: Methanol for the processing condition A

Kruskal-Wallis Nonparamedc .4,VOV-4 Ten

Number Sum Mean of of of

Group Points Ranks Ranks

Kmkal-Wallis Statistic KW = 8.279 The exact P value calculation would have taken too Long. - so the ch-square approximate P value is s h o w innesd. The P value is 0.3086. considered not sigzificulr. Variation among column medians is nor sigiircmtiy grever rhan cspcc: by chance.

D m ' s Multiple Comparisons Test

Mean Comparison DiEerence P value

-III-LI---- - ----.-.. 1 vs. 2 3.100 ns P X M i L VS. j -5.000 P>O.OI L vs* 4 -2.000 ns P>0.05 I vs. 5 2.100 ns P>O.Oj L vs. 6 -6.900 ns P>O.O5 I vs. 7 -9.133 n~ P>0.03

I vs. 8 8.600 ns PO.05 2 vs. 3 -8200 n~ PW.05 2 VS. 4 -5.200 ns PO.05 2 vs. 5 -1.100 ns PW.05 2 vs. 6 -10.100 n~ P>O,OS 2 VS. 7 -12.433 ns P>O.OS 2 VS. 8 5.400 ns EW.05 3 vs. 4 3.000 ns bO.05 3 vs. 5 7.100 ns PW.05 3 VS. 6 -1.900 ns PW.05 3 vs- 7 4233 us P~0.05 3 vs. 8 13.600 ns p~I.05 4 1-s. 5 4.100 ns D0.05 4 vs. 6 4900 ns P>O.OS 4 vs. 7 -7.233 ns P>O.OZ 4 vs. 8 10.600 ns P>O.OI' 5 VS. 6 -9.000 n~ P>O,OZ 5 vs. 7 -I 1.323 n~ P>O.O5 5 vs. Q 6.500 ns P>O.OT 6 vs. ' -1.333 ns P>O.OT 6 vs. 8 15.500 ns P>O.OZ 7 vs. 8 17.8; S ns P>O.OZ

These trsis are based on a Gaussian approximation. n e y are only accur for large sarnpie sizes.

Sunmap- of Dan Surnber of

Group Poinrs Median Minimum 4Ia~rnum

t-test (Miller and Miller, 1988)

Taking the null hypothesis that the two methods give the same results. The difference

was measured from the two individual standard deviation sl and s2 by using the equation:

It can be show that "t" is given by:

t = (u,, - I,) / s(l/n, - 1/nz)'"- where 'T' has n, + n2 - 2 degrees of freedom.

Value of "t" for a confidence interval of 99 %, for 5 degree of freedom = 4.03.

Example: Methanol for the processing condition A. B and C between months 1 and 8.

METHANOL First month Last month n Average std n Average std

s Value

t value Value

All the results were below 4.03, therefore no difference were found between the fist and

the last month.

APPENDIX C

CONCENTRATlON OF VOLATILES IN ORANGE JUICE FOR THE

MONTHS 2-6

Month 2 CONDITION A B C

GC-MS (uWmL) ( u g h L) (ughL) methyl-butyrate 0.01 0.0 I 0.0 1 a-pinene ethy Lbutyrate hexanal sabinene 3-carene a-phellandrene Q-myrcene heptanal limonene 2&3-methy I-butanol 2-hexenal 2- hexano 1 y-terpinene octanaI I -hexan01 3-hexen- 1-01 nonanal dimeth y I-styrene firfkral decanai Iinatoo t octanol terpinene-4-ol hydroxy-ethyl-hexanoate a-terpineol valencene geranial& carvone perillaldehyde 2-2-decad ienal viny t guiaicol hydroxymethyi hfiral

acetaldehyde ethyl acetate methanol ethanol 543 -02 580.07 599.23 ND, not detected, *limit of detection.

Month 3

CONDITION A B C

a-pinenc ethy 1-buryrate hexanal sabinene 3-carene a-phellandrene P-rn yrcene heptanal limonene 2&3-methyl-butanol 2-hexenal 2-hexanol y-terpinene octanal 1 -hexan01 3 -hexen- 1-01 nonanal dimethyl-styrene brfural decanal linalool octanol terpinene-4-ol hydroxy-ethyl-hexanoate a-terpineol valencene geranial & carvone perillaldehyde 2-2-decadienal vinyl guiaicol hydroxyrnethy I f irfiral vanillin

acetaldehyde ethyl acetate methanol ethanol ND, not detected.

Iimit of detection.

Month 4

CONDITION A B C

GC-MS (u%m L) ( W m L) ( W m L ) methy 1-butyrare 0.09 0.07 0.07 a-pinene ethy I-butyrate hexanal sa b inene 3-carene a-phellandrene P-rn yrcene heptanaI limonene 2&3-methy I-butanol 2-hexenal 2-hexanol y-terpinene octanal I -hexan0 1 3-hexen- 1-01

nonanal dimethy [-styrene furfural decanal linaloo t octanol terpinene-4-01 hydroxy-eth y I-hexanoate a-terpineol valencene geranial & carvone perillaldehyde 2-2-decadienal vinyl guiaicol h ydroxyrnethy l hrfura1

0.67 0.34 0.34 0.04* N D ND 1 -94 ND

153.37 ND ND ND 0. IO* 0.08* 0.35 0.28 ND ND ND 0.30 1.09 0.23 0.37 2.44 1 .JO 7.1 1 ND

0. Id* 0.02* 0.18 0.02.

acetaldehyde ethyl acetate methanol ethanol 624. 16 627.13 632.23 ND, not detected. +Iimit of detection.

Month 5

CONDlTION A B C

a-pinene ethyl-butyrate hexanal sabinene 3-carene a-phellandrene ernyrcene heptanal limonene 2&3-rnethy i-butanol 2-hexend ?-hexan01 y-terpinene 0. IS* 0. IS* 0.12 octanal I - hexanol 3-hexen- 1-01 nonanal dimethy f-styrene hrfiJmI decanal linalool octanol terpinene-l-01 hydroxy-ethy I-hexanoate a-terpineol valencene geranial dk carvone

vinyl guiaicol hydroxyrnethyl furfirral vanillin Headspace-GC W m L ) (uwm L) (ug/mL) acetaldehyde 11.10 9.62 9.66 ethyl acetate methanol ethanol 648.96 63025 6 19.02 ND, not detected. *limit of detection.

Month 6

GC-MS (uglm L) (u%mL) ( W m L ) methy I-butyrate ND ND ND a-pinene ethy I-butyrate hexanal sabinene 3-carene a-phellandrene P-myrcene heptanal t imonene 2&3-methy l-butanol 2-hexenal 2-hexano l y-terpinene octanal I-hexanol 3-hexen-l-01 nonanal dimethy I-styrene furfirral decanal IinaIool octanol terpinene-4-o l hydroxy-ethyl-hexanoate a-terpineol valencene geranial& carvone perillaldehyde 2-2-decadienal vinyl guiaicol hydroxyrnethyl firfirat vanillin Heads paceGC (~g/mL) ( ~ % m L) (WvmL) acetaldehyde 10.77 10.28 10.33 ethyl acetate methanol ethanol 656.60 628.47 639.06 ND, not detected. *limit of detection.