from routine to novelty - uniba.sk · plasma to milk. it is mainly in a casein-bound state at...

H2020-MSCA-RISE

WORKSHOP

June 11, 2018

FROM ROUTINE TO NOVELTY ANALYSIS METHODS IN DAIRY INDUSTRY FORMILK 690898 PROJECT Innovative technology for the detection of enzyme activity in milk

RESEARCH CENTRE FOR NATURAL SCIENCES HUNGARIAN ACADEMY OF SCIENCES INSTITUTE OF MATERIALS AND ENVIRONMENTAL CHEMISTRY

HUNGARIAN DAIRY RESEARCH INSTITUTE

PROGRAM

WELCOME 9.00 – 9.15 Zsófia Keresztes Functional Interfaces Research Group, Research Centre for Natural Sciences, Hungary

INSPIRATION AND NOVELTIES IN SENSOR DEVELOPMENTS Chair: Tibor Hianik

9.15 – 9.45 Joseph Wang Center of Wearable Sensors, University of California San Diego, USA Biosensors go wearables: towards lab on the skin or in the mouth Hordozható bioszenzorok: laboratórium bőrön és szájban 9.45 – 10.15 Michael Thompson Department of Chemistry, University of Toronto, Canada Device and measurement advances for high frequency acoustic wave detection of biochemical events Eszköz- és méréstechnikai újdonságok biokémiai folyamatok nagyfrekvenciás akusztikus hullám spektroszkópiai vizsgálatában 10.15 – 10.45 Róbert E. Gyurcsányi MTA-BME Chemical Nanosensors Research Group, Budapest University of Technology and Economics, Hungary Synthetic receptors for selective recognition of proteins and viruses Fehérjék és vírusok érzékelése szintetikus receptorokkal

COFFEE BREAK Chair: Attila Bóta

11.15 – 11.45 Nóra Adányi Department of Food Analysis, National Agricultural Research and Innovation Centre, Food Science Research Institute, Hungary Label-free nanosensors for agro-environmental and food safety Jelölésmentes nanoszenzorok a mezőgazdasági-környezetvédelmi és élelmiszerbiztonsági vizsgálatok szolgálatában 11.45 – 12.15 Tibor Hianik (FORMILK coordinator) Division of Biophysics, Departments of Nuclear Physics and Biophysics, Comenius University in Bratislava, Slovakia Detection of proteases activity at biomimetic surfaces using electrochemical, acoustic and AFM methods Fehérjebontó enzimek érzékelése biomimetikus felületeken elektrokémiai, akusztikus és atomierő mikroszkópiás módszerekkel

LUNCH

STANDARD ANALYSIS METHODS AND TECHNOLOGICAL DEVELOPMENTS IN DAIRY INDUSTRY Chair: Róbert Kocsis 13.00 – 13.15 Carlo Volpe General Director, Óvártej Ltd., Hungary The premium cheese production on the worldmarket A minőségi sajt gyártása a világpiacon 13.15 – 13.30 Norbert Andruška LEVICKÉ MLIEKÁRNE a.s., Slovakia Ensuring quality from dairy farm to finished product A minőség biztosítása a tejtermelő farmoktól a késztermékig 13.30 – 13.45 Gábor Császár Deputy General Director, HDRI Ltd., Hungary Magyar Élelmiszerkönyv 1-3/51-1 számú előírásának változásai, különös tekintettel a tejtermékek egyes vizsgálati módszereire és az európai szabályozásra Changes in the regulations of the Hungarian Food Book 1-3 / 51-1, with special regard to certain test methods for dairy products and the European regulations 13.45 – 14.00 Attila Hucker General Deputy Head of Laboratories, HDRI Ltd., Hungary Laboratóriumi vizsgálatok lehetősége és szerepe a tejiparban Roles and possibilities of laboratory tests in dairy industry 14.00 – 14.15 Réka Sarok Laboratory Engineer, HDRI Ltd., Hungary Fehérje frakciók meghatározása RP-HPLC módszerrel Determination of protein fractions by RP-HPLC 14.15- 14.30 Zoltán Zapletál Business Development Manager, HDRI Ltd., Hungary Fehérjebontás szerepe a sajtgyártás során The role of protein breakdown during cheese production 14.30 – 14.45 Katalin Szabó Head of Physico-Chemistry Laboratory, HDRI Ltd., Hungary Tejcukor mennyiségi meghatározására alkalmas vizsgálati módszerek szakmai áttekintése Overview of test methods suitable for quantitative determination of lactose 14.45 – 15.00 Gábor Szafner Research Associate, HDRI Ltd., Hungary Laktózbontás alkalmazása a tejipari gyakorlatban Application of lactose decomposition in dairy practice

COFFEE BREAK

FORMILK PROJECT NEW WAYS OF SENSOR APPLICATIONS IN DAIRY INDUSTRY Chair: Zsófia Keresztes 15.30 - 15.50 Marek Tatarko Division of Biophysics, Departments of Nuclear Physics and Biophysics, Comenius University in Bratislava, Slovakia Monitoring of plasmin regulation on immobilised milk protein using acoustic methods Plazmin enzim aktivitásának követése immobilizált tejfehérjén akusztikus módszerekkel 15.50 – 16.10 Loránd Románszki Functional Interfaces Research Group, Research Centre for Natural Sciences, Hungary Comparing plasmin activity measurements at the air/liquid and liquid/solid interfaces Plazmin enzim aktivitás mérések összehasonlítása levegő/folyadék és folyadék/szilárd határfelületeken 16.10 – 16.30 Rian Lynch Laboratory of Physical Chemistry of Biocolloids, University College Dublin, Ireland Application of HR-US spectroscopy for monitoring of hydrolysis of lactose in milks and developments of enzyme formulations for lactose intolerant infants A HR-US spektroszkópia alkalmazása a tejcukor hidrolízisének követésére laktóz intoleráns csecsemők részére előállított enzimkészítményeinek fejlesztése során 16.30 - 16.50 László Trif Functional Nanoparticles Research Group, Research Centre for Natural Sciences, Hungary Protein stabilised nanoparticle assisted detection of proteolytic activity studied with photometric and HR-US spectroscopic methods Proteolitikus aktivitás detektálása fehérje stabilizált nanorészecskék fotometrikus és HR-US spektroszkópiai módszerekkel történő vizsgálatával

Poster presentations related to the FORMILK project will be exposed during the workshop at the lobby next to the lecture hall.

FORMILK projekthez kapcsolódó poszter prezentációk megtekinthetők a workshop ideje alatt az előadóterem melletti aulában.

Research Centre for Natural Sciences, Hungarian Academy of Sciences Anyag- és Környezetkémiai Intézet 1117 Budapest, Magyar tudósok krt. 2. Hungary Magyar Tudományos Akadémia Természettudományi Kutatóközpont Institute for Materials and Environmental Sciences 1117 Budapest, Magyar tudósok krt. 2.

ORAL PRESENTATIONS

ELŐADÁSOK

International workshop "From routine to novelty – analysis methods in dairy industry"

Budapest, June 11, 2018.

Biosensors Go Wearables: Toward Labs on the Skin or in the Mouth

Joseph Wang

Department of Nanoengineering, University California San Diego,

CA 92093, USA,

e-mail: [email protected]

Wearable sensors have received a major recent attention owing to their

considerable promise for monitoring the wearer’s health and wellness. The medical

interest for wearable systems arises from the need for monitoring patients over long

periods of time. These devices have the potential to continuously collect vital health

information from a person’s body and provide this information to them or their

healthcare provider in a timely fashion. Such wearable biosensing platforms provide new

avenues to continuously and non-invasively monitor individuals and can thus tender

crucial real-time information regarding a wearer’s health and wellness. This presentation

will discuss recent advances made by our IEM Center for Mobile Health Systems in the

field of wearable biosensors integrated directly on the epidermis or within the mouth for

various non-invasive biomedical monitoring applications. Particular attention will be given

to non-invasive monitoring of metabolites and electrolytes in sweat, ISF, saliva and tears,

using flexible biosensors, respectively. The preparation and characterization of such

wearable electrochemical sensors will be described, along with their current status and

future prospects and challenges.



Device and measurement advances for high frequency acoustic wave detection of biochemical events

Paul Gavin1 and Michael Thompson2

1Life Sciences Interface, Tyndall National Institute, Cork City, Ireland 2Department of Chemistry, University of Toronto, Canada

Conventional acoustic wave detection based on the so-called quartz crystal microbalance

operated at 10-20 MHz has been employed for the assay of biochemical species in fluid

for many years. In this presentation, we discuss the design and applications of

significantly higher frequency devices. One example is the previously-introduced

electromagnetic piezoelectric acoustic wave sensor (EMPAS) which operates upon the

principle of excitation of close to 1 GHz waves in quartz via an electromagnetic field [1, 2].

Generally the sensor is operated around the 50th harmonic of a formal 20 MHz quartz

crystal. The sensor has been applied to the detection of endotoxin in serum [3],

biomarkers for cancer [4] and, in connection with the FORMILK project, casein interaction

with plasmin [5].

Recently, we have instigated research into high frequency acoustic biosensor detection

based on lithium niobate devices. This piezoelectric material provides a number of

advantages over quartz - it possesses enhanced piezoelectric properties, is easily

fabricated by microelectronic protocols, and can be employed in conjunction with

wireless interrogation. We will describe the design and operation of Love-wave shear

horizontal (SH) devices in terms of their potential for chemical and biosensing. This

technology is based on the imposition of surface acoustic waves via interdigital fingers

placed on the Li niobate substrate. One aspect of this work under development is the

necessary requirement for attachment of biochemical probes to the device surface [6].

Our early efforts in this virgin research field will be evaluated. The overall goal is to use

the technology for the rapid screening of patients for ovarian cancer. References [1] M. Thompson, S.M. Ballantyne, A.C. Stevenson and C.R. Lowe, Analyst, 2003, 128, 1048. [2] S.M. Ballantyne and M. Thompson, Analyst, 2004, 129, 219. [3] S. Sheikh, C. Blaszykowski and M. Thompson, RSC Advances, 2016, 6, 38037. [4] J.B. Chen, M.A.D. Neves and M. Thompson, Sensing BioSensing Research 2016, 11, 107. [5] L. Románszki, M. Tatarko, M. Jiao, Z. Keresztes T. Hianik and M. Thompson, Sensors. Act. B, submitted. [6] E. Chan and M. Thompson, to be published.



Label-free nanosensors for agro-environmental and food safety

Nóra Adányi1, István Szendrő2 and András Székács3

1 Food Science Research Institute, NARIC, Budapest, Hungary 2 MicroVacuum Ltd., Budapest, Hungary

3 Agro-Environmental Research Institute, NARIC, Budapest, Hungary

Agro-environmental and food safety have become a central concern within the European

Union, as all over the world. Consumers worry about food-borne pathogens, chemicals,

including mycotoxins and residues of pesticides and veterinary drugs. Due to the efficient

action of the RASFF – Rapid Alert System for Food and Feed – system operating in the EU,

numerous alerts or warnings occurred concerning the occurrence of toxic substances in

spices, foods and grains. For the quick and reliable detection of dangerous substances

new types immunosensors based on Optical Waveguide Lightmode Spectroscopy (OWLS)

detection were investigated.

In the lecture results obtained by OWLS based label-free immmunosensors will be

presented in the determination of different chemical contaminants in environmental or

food samples. Sensoric analytical methods were investigated for the determination of

mycotoxins (zearalenone, aflatoxin, ochratoxin and deoxynivalenol), a shellfish toxin

(ocadaic acid), an allergenic compound (histamine), a pesticide residue (trifluralin) and a

bioindicator protein for monitoring pollution with endocrine disrupting chemicals

(vitellogenin). High specificity/selectivity of the sensitised surface coupled with high

sensitivity of OWLS detection gives the possibility to develop immunosensors in most

cases with definitely lower limits of detection than those in traditionally used

immunoassays in direct/competitive formats. To study the detection possibilities of these

analytes, the corresponding antigens/antibodies were covalently attached on the amino-

modified sensor surface with glutaraldehyde or with EDC/NHS chemistry. The OWLS

technique as a label-free immunosensor has been successfully applied for the detection

of these target analytes, and the sensitivity could be successfully increased by Au

nanoparticles applied for the immobilisation to enhance the sensor surface.

Different types of environmental and food samples (e.g. surface water, grains, fish, tissues

of the European fire-bellied toad (Bombina bombina), mussel, fruit and vegetable juice,

spice paprika powder) were analysed using the newly developed methods, and a high

correlation was observed between the spike levels and the detected values using the

immunosensors and reference methods.

Acknowledgements This research was supported by the NVKP-16-1-2016-0049 project.



Detection of plasmin activity at biomimetic surfaces using electrochemical, acoustics and AFM methods

Tibor Hianik1, Marek Tatarko1, Alexandra Poturnayova1, Loránd Romanszki2,

Zsófia Keresztes2, Eric S. Muckley3, Ilia N. Ivanov3, Michael Thompson4 1 Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics

and Informatics, Comenius University in Bratislava, Slovakia 2 Functional Interfaces Research Group, RCNS, Budapest, Hungary

3 Center for Nanophase Materials Sciences, Oak ORNL, Oak Ridge, USA 4 Department of Chemistry, University of Toronto, Toronto, Canada

Plasmin is considered to be the major indigenous protease, which is transferred from blood

plasma to milk. It is mainly in a casein-bound state at nanomolar level, but its presence can have

a pronounced effect on long shelf-life milk products and also on the quality of certain cheese

formations. Accurate measurement of plasmin activity can be a significant factor in the quality

control of dairy products. We report a comparative analysis of plasmin detection by means of

electrochemical and acoustics biosensors. The topography of the biomimetic surfaces after

cleavage by plasmin has also been analyzed using atomic force microscopy (AFM) method. The

electrochemical biosensor was based on ferrocene-modified peptide substrate (Fc-P) specific to

plasmin. Fc-P substrate is enzymatically cleaved by plasmin and the short fragment of peptide

contained Fc is released causing decrease of redox current measured by cyclic voltammetry. The

biosensor was highly selective (limit of detection (LOD) =0.56±0.03 nM) demonstrating lower

reaction to other proteins such as thrombin. The concentration of plasmin has been determined

also by a photometric analysis based on UV-vis spectroscopy (LOD=3.68±0.04 nM) [1]. The

acoustics biosensor has been based on the monitoring of resonant frequency (fs) and motional

resistance (Rm) of thickness shear mode transducer (TSM) onto which the specific peptide

substrate has been immobilized. Addition of plasmin resulted in cleavage of the short fragments

of the peptides causing increase in the resonance frequency and decrease of motional resistance.

Plasmin was detected in the range of concentrations 1–20 nM in which affects the quality of milk.

The LOD value (0.65 nM) was similar to those of electrochemical method of plasmin detection.

The peptide substrate exhibited negligible response against to similar protease trypsin. The

topography of the sensing surface and their changes after cleavage by plasmin has been

monitored by AFM [2]. The electrochemical and acoustics sensors were also validated in

pretreated milk. Future improvements of the biosensor sensitivity will be discussed.

Acknowledgements. Supported by European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 690898. References: [1] G. Castillo et al., Electroanalysis, 2015, 27, 789.



Ensuring quality from dairy farm to finished product

Norbert Andruška LEVICKÉ MLIEKÁRNE a.s., Levice, Slovakia

There are three key factors in the dairy industry, which must be considered, such as economy, quality and long-term livestock. In the running world one should stop a bit, and consciously consider the options in order to ensure the appropriate yield. Our practical experiences have shown, that sometimes less is more. Considering these, we can achieve the maximum added value in both livestock breeding and milk processing plants. This aspect is not valid only specifically to the dairy industry only, but in a broader sense is true to the agriculture also and plays an important role for both producers and processing units as well.

A megfelelő minőség biztosítása a tejtermelő farmoktól a késztermékig

Norbert Andruška LEVICKÉ MLIEKÁRNE a.s., Levice, Szlovákia

A tejiparban három kulcsfontosságú tényezőt kell figyelembe venni, mint a

gazdaságosság, minőség és a hosszú távú állatállomány. A mai rohanó világban, le kell

lassítani és tudatosan át kell gondolni a lehetőségeket, hogy a megfelelő hozamot

biztosítani tudjuk. Gyakorlati, tapasztalataink igazolták, hogy kevesebb többet jelent. Ezek

figyelembevételével érhetjük el a maximális hozzáadott értéket, mind az

állattenyésztésben, mind a tejfeldolgozó üzemekben. Ez a szemlélet nem specifikusan

csak a tejiparra, hanem tágabb értelemben a mezőgazdaságra is igaz és fontos szerepet

játszik a termelők és feldolgozók számára is.



Roles and possibilities of laboratory tests in dairy industry

Attila Hucker Hungarian Dairy Research Institute Ltd., Mosonmagyaróvár, Hungary

Various methods are available for the testing and analyzing of milk, milk products and operating environments. After a certain point, the selection of the methods requires a significant expertise, and proper laboratory and technological know-how (dairy industry) is also indispensable. The possibilities in test method choice is getting narrow and becomes more and more difficult, from self-checking, through legal compliance verification and justification, towards error detection and research supporting measurement methods. As functional foods gain ground, and also in the frame of FORMILK project, the measurement of protein and lactose content became important. The determination of both protein and lactose content is a basic measurement method in the dairy industry, but the measurement of the decomposition products resulted by enzyme activity or the measurement of the activity is still a research boundary area, while accurate measurement results provide valuable informations to professionals.

Laboratóriumi vizsgálatok lehetősége és szerepe a tejiparban

Hucker Attila Magyar Tejgazdasági Kísérleti Intézet Kft., Mosonmagyaróvár

Különböző módszerek álnak rendelkezésre a tejek, tejtermékek és az üzemi környezetek

vizsgálatára és elemzésére. A módszerek kiválasztása egy bizonyos pont után nagy

szakértelmet igényel, megfelelő laboratóriumi és technológiai (tejipari szakmai)

szakismeret is nélkülözhetetlen hozzá. A vizsgálati módszerek megválasztásának

lehetősége az önellenőrzéstől, a jogszabályi megfelelőség igazolásán keresztül, a

hibafeltárás, továbbá a kutatást támogató mérési módszerek irányába folyamatosan

nehezedik és szűkül. A projekthez köthetően és a funkcionális élelmiszerek térhódítása

miatt is előtérbe került többek közt a fehérje és a laktóz tartalom mérése is. Mind a

fehérje tartalom, mind a laktóz tartalom alapvető mérési módszer a tejiparban, de a

különböző enzimek hatására képződő bomlástermékeik, vagy az azt előidéző enzimek

aktivitásának mérése még inkább kutatási határterület, ugyanakkor a pontos mérési

eredmények fontos információval szolgálhatnak a szakemberek számára.



Determination of protein fractions by RP-HPLC

Réka Sarok Hungarian Dairy Research Institute Ltd., Mosonmagyaróvár, Hungary

Due to the economic and social importance of the dairy industry, milk products are given

special attention in terms of food quality and safety. The colloidal system of milk consists

essentially of different casein proteins and whey proteins. Both groups can be subdivided

into subgroups.

Caseins consist of the following groups: α-caseins (αs1 and αs2), β-caseins (β-CN) and κ-

caseins (κ-CN) Whey proteins, globular proteins, which are soluble at pH 4.6. 3: 1 by

weight of α-lactalbumin and β-lactoglobulin. The properties of micellar casein and whey

proteins largely determine the behavior of milk in technical processes. A solid knowledge

of the structure and structural changes in milk proteins is needed to design new

production technologies and to analyze dairy products. Knowledge of the fractions is also

important because reliable identification and quantitative determination of the main milk

proteins allows the determination of the fact of milk counterfeiting.

RP-HPLC technique was developed to determine the ratio of casein and whey protein as

two main groups of milk protein. As a stationary phase, silica-based Vydac (300 Å, 5 μm,

250 x 4.6 mm) C18 column was used. Mobile phase with gradient program A, B, C eluents

(A: acetonitrile, B: trifluoroacetic acid, C = high purity deionized water).

The individual casein and whey protein constituents differ greatly in their properties and

behavior so that the separation of all milk protein components during a gradient program

was only achieved with some compromise.

Figure 1 A mixture of recovered casein and whey protein. (1 = κ-casein, 2 = αs2-casein, 3 = αs1-casein, 4 = α-lactalbumin 5 = β-casein, 6 = β-lactoglobulin B, 7 = β-lactoglobulin A). Dissolve 0.0146 g of casein in 1 ml of buffer, add 2 ml of whey and 2 ml of 50% V / V acetonitrile. Gradient program: 0 min 26% A, 30 min 48% A, 35 min 48% A, 40 min 26% A. Elution flow rate 1 mL / min, detection wavelength 220 nm



Fehérjekfrakciók meghatározása RP-HPLC módszerrel

Sarok Réka Magyar Tejgazdasági Kísérleti Intézet Kft., Mosonmagyaróvár

A tejipar gazdasági és társadalmi jelentősége miatt az élelmiszerminőség és biztonság

területén a tejtermékek kitüntetett figyelmet kapnak. A tej kolloidrendszere

tulajdonságaikban alapvetően eltérő kazeinfehérjékből és savófehérjékből áll. Mindkét

csoport további alcsoportokra oszthatók.

A kazeinek a következő csoportokból állnak: α-kazeinek (αs1 és αs2), β-kazeinek (β-CN) és

κ-kazeinek (κ-CN) A savófehérjék, globuláris fehérjék, amelyek 4,6 pH-n is oldhatók. 3:1

tömegarányban α-laktalbuminból és β-laktoglobulinból állnak. A micelláris kazein és a

tejsavófehérjék tulajdonságai nagymértékben meghatározzák a tej technikai

folyamatokban tapasztalt viselkedését. Az új termelési technológiák tervezéséhez és a

tejtermékek elemzéséhez szükséges a tejfehérjék szerkezetének és szerkezeti

változásainak szilárd ismerete. A frakciók ismerete azért is fontos, mert a főbb tejfehérjék

megbízható azonosítása és mennyiségi meghatározása lehetővé teszi a tejhamisítás

tényének meghatározását.

A tejfehérje két fő csoportját alkotó kazein és savófehérjék arányának meghatározására

és minőségi elválasztására RP-HPLC technikát dolgoztunk ki. Vizsgálatinkhoz állófázisként

szilika alapú Vydac (300 Å, 5 μm, 250x4,6 mm) C18-as oszlopot használtunk.

Mozgófázisként gradiens programmal A, B, C eluenseket használtunk (A: acetonitril, B:

trifluor-ecetsav, C= nagytisztaságú ionmentes víz).

Az egyes kazein és savófehérje alkotók tulajdonságaikban és viselkedésükben nagyon

eltérőek, így az összes tejfehérje komponens elválasztását egy gradiens program

lefuttatása alatt csak némi kompromisszum árán sikerült megvalósítani.

1.ábra Kicsapott kazein és savófehérje keveréke. (1= κ-kazein, 2= αs2-kazein, 3= αs1-kazein, 4= α-laktalbumin 5= β-kazein,

6= β-laktoglobulin B, 7= β-laktoglobulin A). 0,0146 g kazein oldása 1 ml pufferben, hozzáadva 2 ml savó és 2 ml 50 V/V%

acetonitril. Gradiens program:0 min 26% A, 30 min 48% A, 35 min 48% A, 40 min 26% A. Az eluens áramlási sebessége

1 mL/perc, detektálási hullámhossz 220 nm



The role of protein breakdown during cheese production

Zoltán Zapletál Hungarian Dairy Research Institute Ltd., Mosonmagyaróvár, Hungary

Protein degradation is the decomposition process that is most likely to affect the taste of

the cheeses during ripening. Proteins, constituted from amino acids bound through

peptide bonds, are macromolecules with very diverse structure and function.. Naturally

occurring proteins are composed of 20 amino acids, many of the are so-called essential

amino acids. The most important protein of milk is casein, which is found in the form of a

stable colloidal solution in milk.

One of the most important point int he production of of rennet curded cheeses is the

curding process. Coagulation is the destabilization of the milk colloidal solution, the gel

formation resulted by the action of proteolytic enzyme (protease). The enzymes used for

the process: calf rennet, microbial vaccine, FPC vaccine. A new member of the new

generation of FPCs is Chr. Hansen Chy-Max® M vaccine enzyme, a enzyme produced by

fermentation, containing 100% pure chymosine (camel chymosine) with many

advantages.

Protein decomposition is most commonly the process of cheese ripening. The maturation

of cheeses is the result of many physical, chemical, microbiological, and biochemical

processes, during which the typical characteristic features of cheeses are formed. During

maturation, casein breaks down into smaller, simpler, water-soluble compounds. Free

amino acids determine the taste and aroma of cheese.

We have many maturing culture concepts. Within the DVS® Flavor Control ™ culture, our

latest offering is the DVS® CR-Full flavor culture range. Surface maturing culture our

family is the SWING® family of cultures, which contains aerobic mold, yeast and bacterial

cultures. Our latest, unique performance maturing culture is F-DVS Delight® culture,

which is primarily intended for the production of low-fat cheeses.



A fehérjebontás szerepe a sajtgyártás során

Zapletál Zoltán Magyar Tejgazdasági Kísérleti Intézet Kft., Mosonmagyaróvár

A fehérjebomlás a sajtok gyártása -, érlelése során lezajló, a sajtok ízét -, állományát

leginkább befolyásoló bomlási folyamat. A fehérjék aminosavakból, peptid kötéssel

felépülő, nagyon változatos szerkezetű és tulajdonságú makro vegyületek. A

természetben előforduló fehérjéket 20 féle aminosav alkotja, köztük több, un. esszenciális

aminosav. A tej legfontosabb fehérjéje a kazein, amely stabil kolloid oldat formájában

található a tejben.

Az oltós alvasztású, érlelt sajtok egyik legfontosabb művelete a tej oltós alvasztása. Az

alvadás a tej kolloid oldat destabilizációja, gél képződéssel, amelyhez fehérjebontó enzim

(proteáz) szükséges. A tej oltós alvasztásához használt oltóenzimek: borjúgyomor oltó,

mikrobiális oltó, FPC oltó. Az FPC oltók új generációjának egyedi képviselője, beszállító

partnerünk a Chr. Hansen Chy-Max® M oltóenzime, amely fermentációval előállított, 100

%-ban tiszta kimozint (teve kimozin molekulát) tartalmazó oltóenzim, számos előnnyel.

A fehérjebomlás leginkább a sajtok érése során lezajló folyamat. A sajtok érése fizikai -,

kémiai -, mikrobiológiai -, biokémiai folyamatok összessége, amely során kialakul a sajtok

jellemző állománya, - lyukazottsága, - íze. A fehérjebontást enzimek (peptidázok) végzik.

Az érés folyamán a kazein kisebb, egyszerűbb, vízben oldható vegyületekké bomlik. A

szabad aminosavak határozzák meg a sajt ízét és aromáját.

Számos érlelő kultúra koncepcióval rendelkezünk. A DVS® Flavor Control™ kultúra

családon belül a legújabb ajánlatunk a DVS® CR-Full flavor kultúraválaszték. Felületi érlelő

kultúra családunk a SWING® kultúracsalád, amely aerob penész -, élesztő -, baktérium

kultúrákat tartalmaz. Legújabb, egyedi teljesítményű érlelő kultúránk az F-DVS Delight®

kultúra, amelyet elsősorban csökkentett zsírtartalmú (light) sajtok gyártásához javaslunk.



Overview of test methods suitable for quantitative determination of lactose

Katalin Szabó

Hungarian Dairy Research Institute Ltd., Mosonmagyaróvár, Hungary

The presentation summarizes the analytical methods suitable for the determination of lactose content of milk and milk products. The quantitative occurrence of milk lactose in dairy products, the physiological effect of lactose and the lactose intolerance is also explained. The principles of various test methods are presented, including their advantages and disadvantages. The talk ends with the presentation of practical applications in the laboratory of the previously discussed tests.

Tejcukor mennyiségi meghatározására alkalmas vizsgálati módszerek

szakmai áttekintése

Szabó Katalin Magyar Tejgazdasági Kísérleti Intézet Kft., Mosonmagyaróvár

Tej és tejtermékek laktóz tartalmának meghatározására alkalmas analitikai módszerek

áttekintése. A tejcukor mennyiségi előfordulása tejtermékekben, a tejcukor élettani

hatása, laktózintolerancia. A különféle vizsgálati módszerek elvének bemutatása, az adott

vizsgálatok előnyeinek és hátrányainak elemzése. A bemutatott vizsgálatok gyakorlati

alkalmazása a laboratóriumban.



Application of lactose decomposition in dairy practice

Gábor Szafner, Árpád Novák, Solveig Bukovics Hungarian Dairy Research Institute Ltd., Mosonmagyaróvár, Hungary

The lactose, which is present in cows’ milk in an average amount of 4.7 g/100 g milk, can be degraded to glucose and galactose by enzymatic processes. The resulting glucose and galactose is sweeter, easier to dissolve in water, and also easier to ferment, than lactose [1]. Compared to the sweetening power of sucrose (100%), de relative sweetening power of lactose is 22%, that of galactose is 42%, while of the glucose is 45% [2]. The aim of our research was to develop the production process and recipe of a high added value permeate (this forms during the ultrafiltering of milk) based product, the use of which from organoleptic and inner content side is favourable, compared to the base permeate, thus it could be suitable for its replacement in the baking and confectionery fields. In order to solve this issue, two main problems had to be solved: development of the right sweet taste character and decreasing the salty taste. The development of the right sweet taste character was reached by the partial hydrolysis of lactose (increasing the content of the much sweeter glucose and galactose), while the decrease of mineral (salty) taste was done by nanofiltration (partial desalination, the removal of mainly monovalent ions) and controlled electrodialysis. During the process of industrial adaptation, we have checked the physico-chemical, microbiological and organoleptic property of the produced products. References [1] P.F. Fox, Advanced dairy chemistry volume 3, 1997 77-84. [2] H. R. Moskovitz, Perception & Psychophysics, 1970, 5, 315-320.



Laktóz bontás alkalmazása a tejipari gyakorlatban

Szafner Gábor, Novák Árpád, Bukovics Solveig Magyar Tejgazdasági Kísérleti Intézet Kft., Mosonmagyaróvár

A tehéntejben átlagosan 4,7 g/100g mennyiségben megtalálható laktóz enzimatikus

folyamatok révén glükózra és galaktózra bontható. A keletkezett glükóz és galaktóz

édesebb, vízben könnyebben oldható, valamint könnyebben fermentálható, mint a laktóz

[1]. A répacukor (sucrose) édesítő erejéhez viszonyítva (100%) a laktóz relatív édesítő

ereje 22%, a galaktózé 42% és a glükózé 45% [2].

Kutatásunk célja volt egy tej ultraszűrése során keletkezett permeátum bázisú, nagy

hozzáadott értékkel rendelkező termék gyártási eljárásának és receptúrájának

kifejlesztése, amelynek felhasználása organoleptikus-, és beltartalmi szempontból

kedvezőbb a permeátum pornál, ezáltal elsősorban a sütőipari és édesipari területeken

alkalmas lehet annak helyettesítésére is.

A feladat megoldásához kettő fő problémát kellett megoldani amelyek: a megfelelő édes

íz karakter kialakítása valamint a sós íz csökkentése. A megfelelően édes íz karakter

kialakítását a laktóz részleges hidrolízisével (az édesebb glükóz és galaktóz tartalom

növelésével), az ásványos (sós) ízérzet csökkentését nanoszűréssel (részleges sótalanítás;

elsősorban monovalens ionok eltávolítása) valamint irányított mértékű elektrodialízis

alkalmazásával biztosítottuk.

Az ipari adaptálás során vizsgáltuk az előállított termékek fizikai-kémia, mikrobiológiai

valamint organoleptikus tulajdonságait.

References [1] P.F. Fox, Advanced dairy chemistry volume 3, 1997 77-84. [2] H. R. Moskovitz, Perception & Psychophysics, 1970, 5, 315-320.



Monitoring of plasmin regulation on immobilised milk protein using acoustic methods

Marek Tatarko, and Tibor Hianik Department of Nuclear Physics and Biophysics, Faculty of Mathematics,

Physics and Informatics, Comenius University in Bratislava, Slovakia

Plasmin is essential protease that has immense importance in the regulation of milk

protein composition. Its key role is proteolysis of casein, the most common protein group

in milk [1]. The casein degradation causes changes in the milk quality, such as gaining

bitterness or rougher texture and even can cause loss of flavor. Plasmin activity in milk is

regulated by plasmin protease system and this regulation presents a serious disadvantage

for conventional plasmin detection methods [2]. Thickness shear mode (TSM) is promising

acoustic method for detection of immobilized casein degradation caused by plasmin

activity. TSM capability to sensitively detect removed casein mass is effective way of

identification for any plasmin regulatory effects [3]. Concentration of activated plasmin

was inhibited by equivalent or fractional concentration of antiplasmin, specific plasmin

inhibitor. As an alternative protease, trypsin was used with corresponding antiplasmin

concentration as well. While in case of the trypsin, decrease in signal corresponded to

added concentration of antiplasmin, plasmin measurement responses were not precisely

correlating with protease-inhibitor ratio. Changes in plasmin activity were contributed to

additional regulation factors, that needed to be inspected. Two series for ten-day

monitoring of freshly activated plasmin using TSM proved these changes in activity are

influencing activity measurements. Initial observed increase in activity was contributed to

active urokinase-type activator, continuously cleaving plasmin precursor plasminogen

several hours after initial addition. This was later closely researched by 24-hour

monitoring of plasmin activity. After reaching peak, activity gradually decreased by the

second order, which was contributed to ongoing plasmin autolysis. These plasmin

regulation measurements proved that TSM is suitable acoustic biosensor for cleaving

casein and it is capable of detecting plasmin concentration influenced by inhibition,

activation and even autoregulation effects.

Acknowledgements. Supported by European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 690898. References [1] E. D. Bastian et al., International Dairy Journal, 1996, 6, 435. [2] B. M. Prado et al., International Dairy Journal, 2006, 16, 593. [3] I. Politis and J. H. White, Journal of Dairy Science, 1995, 78, 484.



Comparing plasmin activity measurements at the air/liquid and liquid/solid interfaces

Loránd Románszki1, Marek Tatarko2, Mengchi Jiao3, Zsófia Keresztes1, Tibor Hianik2, Michael Thompson4

1 Functional InterfacesResearch Group, IMEC, RCNS, Hungary 2 Faculty of Mathematics, Physics and Informatics, Comenius University in

Bratislava, Slovakia 3Nanjing University, People’s Republic of China

4 Department of Chemistry, University of Toronto, Canada

Detection of residual plasmin activity in milk represents a difficult challenge for the dairy

industry. Conventional methods are either too expensive or incapable of providing

enough data from UHT treated milk. As an alternative, we designed and carried out low

concentration plasmin measurements based on β-casein as enzyme substrate, adsorbed

as layers at either air/liquid or liquid/solid interfaces. In the first case, the measurement

principle was that surface tension change caused by cleavage and desorption of the

substrate could be directly correlated to the concentration of the enzyme. In the second

case, the initial rate of the frequency increase of a quartz crystal (electromagnetic

piezoelectric acoustic sensor, EMPAS), caused by desorption of the substrate, was

correlated to the concentration of the plasmin. The detection limit of plasmin was

estimated to be about 1 nM in the tensiometric approach, and as low as 32 pM with

EMPAS.

0 2 4 6 8 10 12 140.00

0.05

0.10

0.15

0.20

0

2

4

6

8

10

(-

min

) / (

m

ax -

min

)

cPL

/ nM

df

/dt

) / s

-2

Figure 1.: Calibration curves for cPL plasmin concentrations obtained by tensiometric measurements (squares, solid line) and EMPAS (triangles, dashed line), respectively

Acknowledgements This work was supported by the European Commission within the project Innovative technology for the detection of enzyme activity in milk (FORMILK) under grant agreement number 690898 /H2020-MSCA-RISE-2015 and the National Competitiveness and Excellence Program Hungary, (NVKP_16-1-2016-0007).



The application of HR-US spectroscopy for monitoring of hydrolysis of lactose in milks and development of enzyme formulations for lactose

intolerant infants.

Rian Lynch, Vitaly Buckin School of Chemistry, University College Dublin, Ireland.

Lactose intolerance is the most prevalent enzyme deficiency in humans and is

characterised by cramps, bloating and diarrhoea following the consumption of foods

containing lactose.[1] Often avoidance of the symptoms of lactose intolerance is as easy

as cutting dairy products from the diet. However, this represents the exclusion of an

increasingly available, cheap and high-quality source of nutrition from the diet. Dairy is a

primary source of calcium in the diet, so the removal of dairy from the diet may result in

negative health effects on the individual, in particular, osteoporosis.[2] Alternatively, β-

galactosidase enzyme supplements are available as tablets, or in droplet form for infants,

which hydrolyse lactose into glucose and galactose, thus preventing the negative

symptoms associated with lactose maldigestion. The applicability and the efficiency of

these supplements in-vivo depends on several conditions including temperature, enzyme

source, enzyme concentration and pH [3], which shall be assessed and optimised at the

development stage of the supplements. However, traditional analytical techniques are

limited in their application for real-time evaluation of these supplements under various

conditions.

In our work, we have employed a novel analytical technique, which overcomes these

limitations, High-Resolution Ultrasonic Spectroscopy (HR-US), for the real-time, non-

destructive, high-precision monitoring of lactose hydrolysis by β-galactosidase

supplements. Lactose hydrolysis was performed with both neutral and acid active β-

galactosidases, in a range of milks (including infant milks) and buffered solutions of

lactose. The effects of varying pH and enzyme concentrations have been evaluated in

both. The performance of our neutral lactases showed a pH optimum of pH ~6.5 while

our acid lactases had pH optimum of pH ~4.5. Our ultrasonic measurements also allowed

real-time monitoring of the change in osmolarity of milks over the course of reaction of

hydrolysis of lactose. Our results indicated that the recommended maximum limit for

osmolarity is often exceeded during enzymatic lactose hydrolysis in infant milks. Our

results provide a background for the application of HR-US technique in the manufacturing

of infant milks with reduced amounts of lactose. References [1] M.C Altas, E. Kudryashov, V. Buckin, Anal. Chem, 2016, 88, 4714–4723. [2] S. Rosenberg, J.J Body, Calcified Tissue International, 2016, 98, 1-17. [3] K. Xenos, S. Kyroudis, A. Anagnostidis, P. Papastathopoulos, Eur J Drug Metab Pharmacokinet, 1998, 23, 350-355.



Protein stabilised nanoparticle assisted detection of proteolytic activity studied with

photometric and HR-US spectroscopic methods

Tamás Szabó,1 László Trif2, Nikoletta Molnár2, Barbara Csupor1, Breda O’Driscoll3, Vitaly Buckin4, Zsófia Keresztes1

1Functional Interfaces Research Group, IMEC, RCNS, Hungary 2Functional Nanoparticles Research Group, RCNS, Hungary

3SONAS Ltd, Dublin, Ireland 4School of Chemistry, University College Dublin, Belfield, Dublin, Ireland

Differently prepared casein stabilized gold nanoparticles have been used to

evaluate activity of proteolytic enzyme. Hydrolysis of casein shell could be followed by the

change of gold plasmonic effect, as resulted by the aggregation of nanoparticles. Protein

cleavage creates difference in the surface charge of nanoparticles, the subsequent charge

dependent aggregation can be followed in time by means of spectrophotometric

methods.

Hydrolytic resistance of casein shell can be tuned by microwave power enhanced

formation of stabilised nanoparticles. High microwave power results in very stable shell

on nanoparticle entities, where enzymatic cleavage cannot really be observed. In such

case aggregation effect cannot be detected by photometric methods. High resolution

ultrasonic spectrometry on the other hand offers the possibility to follow subtle changes

resulted by the cleavage products from the protein shell, which are creating a change in

the ultrasonic properties of the solution.

The method based on protein stabilised nanoparticle aggregation offers a good

general, although not specific evaluation of proteolytic activity. Peptide shell stabilisation

improves the selectivity of measurement, on the other hand the price and availability

creates drawbacks in general use. Acknowledgements This work was supported by the European Commission within the project Innovative technology for the detection of enzyme activity in milk (FORMILK) under grant agreement number 690898 /H2020-MSCA-RISE-2015 and the BIONANO_GINOP-2.3.2-15-2016-00017 project.

POSTER PRESENTATIONS

POSZTEREK



Poly(poly(ethylene glycol) methacrylate) based interpolymer complexes in aqueous solutions

Ákos Szabó,1 Mark Dizon,2 Zsófia Keresztes,3 Breda O’Driscoll,4 Vitaly Buckin4, Béla Iván1

1 Polymer Chemistry Research Group, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences of the Hungarian Academy of

Sciences, Hungary 2 School of Chemistry, University College Dublin, Belfield, Dublin, Ireland

3 Functional Interfaces Research Group, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences of the Hungarian Academy of

Sciences, Hungary 4 SONAS Ltd, Dublin, Ireland

Aqueous solutions of a synthetic polymer family, i.e. poly(poly(ethylene glycol)

methacrylate)s (PPEGMA), were investigated by various methods to study the change of

their thermoresponsive behaviour by the H-bonded interaction with poly acrylic acid

(PAA). Temperature-dependent transmittance and light scattering measurements indicate

that interpolymer complex formation occurs between PPEGMA and PAA in water by

intermacromolecular H-bonds [1]. This phenomenon can also be observed by high

resolution ultrasonic spectroscopy (HRUS) since the sound velocity difference between

PAA-containing solution and water changes after the addition of the same amount of

PPEGMA. The change of the hydrophilicity of the random copolymers of PEGMA and

methyl methacrylate (MMA) with the increasing MMA-content was also studied by these

methods.

20 30 40 50 60 70 800

20

40

60

80

100

PPEGMA

T / oC

Tra

nsm

itta

nce

%

PPEGMA + PAA

Figure 1.: Transmittance-temperature curves of aqueous poly(poly(ethylene glycol) methacrylate) (PPEGMA) solutions in the presence and absence of poly(acrylic acid) (PAA)



Poly(N-isopropyl acrylamide) interactions with proteins, investigated

by UV-Visible, NMR and Ultrasonic spectroscopy

Zsófia Osváth,1 András Láng2, Beáta Szabó3, Mark Dizon4, Vitaly Buckin4, Béla Iván1

1 Polymer Chemistry Research Group, IMEC, RCNS, Hungary. 2 Department of Organic Chemistry, Eötvös Loránd University, Hungary.

3 Research Group of Intrinsically Disordered Proteins, RCNS, Hungary. 4 School of Chemistry, University College Dublin, Ireland.

Poly(N-isopropylacrylamide) (PNIPAAm) is widely applied in medical science, its

interactions with different proteins have never been extensively studied so far. Our

experiments focused on the interactions between PNIPAAm and proteins. The proteins

selected as model systems that have good water solubility, have been studied earlier so

their structure is known and are sufficiently small that potential atomic-level interaction

studies could be conducted.

The 44-residue-long tβ4 is an actin binding small protein that belongs to the group of IDPs

and is disordered throughout its full sequence. NMR, UV-visible and Ultrasonic

spectroscopy studies showed that the critical solution temperature of PNIPAAm does not

change upon the addition of tß4 but the reversibility is incomplete over multiple thermal

cycles, indicating that the molecules interact with each other. The time-dependent

changes of the N-terminal half provided the atomic level proof of this interaction.

Figure 1.: Aqueous PNIPAAm solution temperature dependence phase



The synthesis of polymer-enzyme nanoparticles and their reaction with

beta-casein

Györgyi Szarka1, Márk Bisztrán2, György Kasza1, Tímea Stumphauser1, Mark Dizon3, Breda O'Driscoll5, Zsófia Keresztes4, Béla Iván1, Vitaly Buckin3

1Polymer Chemistry Research Group, RCNS Hungarian Academy of Sciences 2Eötvös Loránd University, Budapest, Hungary

3School of Chemistry, University College Dublin, Belfield, Dublin, Ireland 4 Research Group of Intrinsically Disordered Proteins, RCNS, HAS

5 SONAS Ltd, Dublin, Ireland

Nowadays, polymer-enzyme systems have significant interest, because such

conjugates can prevent the catalytic activity of the enzyme under extreme conditions (e.g.

high temperature or wide pH range).Poly(diethyl acrylamide-co-glycidyl methacrylate)

(p(DEAAm-co-GMA)) random copolymers were synthesized by free radical copolymerization

with different comonomer monomer/initiator ratios. The copolymers were characterized by

GPC, 1H NMR spectroscopy and DSC. The results show that the compositions of the obtained

copolymers correspond well to the theoretical ones and the average molecular weight can be

influenced by the initial monomer/initiator ratios. The thermoresponsive behaviour of the

produced p(DEAAm-co-GMA) copolymers were investigated by UV-vis spectroscopy. It was

found that the critical solution temperatures (CST) of the copolymers are lower than that of

the pDEAAm homopolymer, and the CST decreases linearly with the increasing GMA content

in copolymer under the investigated conditions.

Conjugates of α-chymotrypsin and the p(DEAAm-co-GMA) copolymers were

synthesized by the amine-oxirane reaction with different concentrations and feed ratios of

the reactants. The obtained enzyme-copolymer nanoparticles (NP) were examined by DLS.

The enzyme contents and the enzyme activity were determined by UV spectroscopy. It can be

concluded that the activity of the NP is lower than that of the native enzyme, but less

sensitive to the pH changes. The enzymatic reaction of some polymer-enzyme NPs was

studied with beta-casein via High Resolution Ultrasound Spectroscopy (HRUS) and density

measurements. In the case of nanoparticles with higher polymer content, a significant side

reaction was observed during the HRUS measurement. This is possibly caused by the

formation of aggregates at the beginning of the reaction. The filtration of the NPs can

prevent the aggregation in the case of lower polymer contents, but the difference between

the HRUS and density measurements may indicate some other type of side reaction as well. Acknowledgement This work was supported by the European Commission within the project Innovative technology for the detection of enzyme activity in milk (FORMILK) under grant agreement number 690898 /H2020-MSCA-RISE-2015 and the BIONANO_GINOP-2.3.2-15-2016-00017 project.



Milk derived extracellular vesicles: physico-chemical characterization

Róbert Deák, Diána Kitka, Judith Mihály, Teréz Kiss,

András Wacha, Attila Bóta, Zoltán Varga Biological Nanochemistry Research Group,

Institute of Materials and Environmental Chemistry RCNS, Hungarian Academy of Sciences

H-1117 Budapest, Magyar tudósok körútja 2, Hungary http://bionano.ttk.mta.hu

Milk (particularly breast milk) functions not only as a nutrition source, but also delivers

immune modulatory factors for neonates. Extracellular vesicles (EV), lipid bilayer enclosed

structures (vesicles) as potent vehicles for intercellular communication, have been

identified in human breast milk and in bovine milk [1]. The isolation of milk-derived

extracellular vesicles (MEV) is still challenging since milk contains many colloidal

structures with comparable size to MEV, e.g. milk fat globules (0.1–15 μm) and casein

micelles (100–200 nm) [2].

The Biological Nanochemistry Research Group has a unique potential in the

characterization of bio-related nanosized systems providing structural information from

the atomic level to several micrometers. For example, the wealthy morphology of the

complex milk matrix may be visualized directly by freeze fracture combined with

transmission electron microscopy (FF-TEM). Size and size distribution of separated milk-

derived colloid particles can be determined by dynamic light scattering (DLS), and the

results can be correlated with small-angle X-ray scattering (SAXS). Quite recently, we

proposed a protocol to estimate the spectroscopic protein-to-lipid ratio, adaptable as a

fast and cheap method for EV preparation screening by Fourier-transform infrared (FTIR)

spectroscopy [3].

Isolation of cell-free MEV from breast milk was performed by two different methods,

namely size-exclusion chromatography (SEC) and ultracentrifugation (UC). The different

MEVs were characterized by DLS and FTIR techniques and the results were compared with

human blood derived EVs (red blood cell and plasma EVs, respectively).

References [1] M.J. van Herwijnen, M.I. Zonneveld, S. Goerdayal, E. N. Nolte-'t Hoen, et al., Mol Cell Proteomics. 2016, 15, 3412. [2] M.I. Zonneveld, A. Brisson, M.J. van Herwijnen, S.Tan, C.H.A. van de Lest, et al, J Extracellular Vesicle, 2014, 3, 24215. [3] J. Mihály, R. Deák, I.Cs. Szigyártó, A. Bóta, Z. Varga, Biochim Biophys Acta-Biomembranes, 2017, 1859, 459.



Ultrasonic Monitoring of Hydrolysis of bovine β-Casein by Trypsin

Georgios I. Papoutsidakis,1 Mark Dizon,1 Sopio Melikishvili,2 Tibor Hianik2, Vitaly Buckin,1

1 School of Chemistry and Chemical Biology, University College Dublin, , Ireland 2 Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynska,

Dolina F1, Bratislava 84248, Slovakia

Hydrolytic enzymes are commonly used in food and biopharmaceutical industries for

various applications including production of bioactive peptides that exhibit a range of

nutritional benefits [1]. In particular, milk protein β-casein serves as an accessible and

affordable source of the aforementioned peptides [2]. One of the key challenges in

control of the enzymatic processing of proteins is the lack of analytical techniques for

real-time, non-destructive monitoring of hydrolysis in bioreactors. High-Resolution

Ultrasonic Spectroscopy (HR-US) has already been applied for the real-time non-

destructive monitoring of hydrolysis of globular whey proteins, such as β-lactoglobulin

[3]. Our work explores the capability of HR-US for the real-time monitoring of hydrolysis

of ‘rheomorphic’ proteins,

normally arranged in a form

on nanoparticle aggregates

(micelles) of 10 to 50

molecules, an example of

which is bovine β-casein.

We have applied HR-US

technique for real-time

monitoring the process of

hydrolysis of bovine beta-

casein by trypsin under various enzyme/substrate ratios ([E/S]: 1/25, 1/100, 1/300, 1/500)

and various pH values (6.6, 7, 7.8). The ultrasonic profiles of hydrolysis were compared

with the discontinuous method TNBS (2, 4, 6-trinitrobenzenesulfonic acid) from which the

degree of hydrolysis and the reaction extent (number of peptide bonds hydrolysed per

one molecule of beta-casein) can be derived. The obtained results provided information

on the effects of different trypsin concentrations, enzyme/substrate ratios and pH on the

performance of this proteolytic enzyme.

References [1] D.P. Mohanty, S. Mohapatra, S. Misra and P.S. Sahu, Saudi J of Biol Sci, 2016, 23, 577. [2] D.A. Clare and H.E. Swaisgood, J Dairy Sci, 2000, 83, 1187. [3] V. Buckin, and M. Caras-Altas, Catalysts, 2017, 7, 336.

(c

)

(b)

Fig. 1: (a) Illustration of micelle of β-casein; (b) Illustration of single molecule of β-casein (left) and of molecule of β-lactoglobulin (globular whey protein in milk, right); (c) Illustration of hydrolysis of peptide bond by a protease enzyme.

(a

)



Liquid or semi-solid dairy products, hydrolysing substrate for the

anti-inflammatory hydrophobic curcumin

Mária Pávai1, Zsófia Keresztes1, Breda O'Driscoll2, Vitaly Buckin3

1 Research Centre of Natural Sciences, Hungarian Academy of Sciences, Hungary. 2 Sonas LTD., Ireland

3 School of Chemistry and Chemical Biology, University College Dublin, Ireland

The low solubility of the curcuminoids in aqueous solutions, their susceptibility to

degradation in aqueous environments and the low bioavailability compromise their

usefulness as a bioactive ingredient in functional, added values foods. Liquid or semi-solid

dairy products (such as yoghurt, buttermilk and sour-cream) contain milk proteins

(caseins and whey proteins), milk fat and the milk fat globule membrane. The presence of

proteins and lipids (including the phospholipids of the milk fat globule membrane) in

liquid or semi-solid dairy products make them an attractive substrate for curcumin, what

interacts with these proteins and lipids by moving into the hydrophobic groups on these

molecules. The the interaction of curcumin with the proteins and lipids from the liquid or

semi-solid dairy products improved its solubility and stability. Due to the ability of the

liquid and semi-solid dairy products to carry and stabilise curcumin it is possible to exist a

potential of it to enable the delivery of these components into functional foods. In this

study it is investigated the potential of yoghurt, what acts as a substrate for curcumin

(95% Alfa Aesar). The investigation was made at 5°C by the HRUS 102 SS (High Resonance

Ultrasonic Spectrometer) by measuring the differential sound velocity and relative

attenuation at different frequency ranges (2-10 MHz).

References [1] M. Pávai, J. Mihály, A. Paszternák, Food Analytical Methods, 2015, 2243-2249 [2] Shoba G, Joy D, Joseph T, Majeed M, Rajendran R, Srinivas PS. Planta Med. 1998, Volume 64, 4, 353-356



Novel ultrasonic technology for real-time monitoring of enzymatic

hydrolysis of whey proteins

Mark Dizon, Vitaly Buckin University College Dublin, Belfield, Dublin 4, Ireland

Enzymatic hydrolysis of proteins has been the most common protein modification method used by both research and industrial sector to manufacture peptides with desirable functional properties. These properties mainly depend on the degree and control to which the proteins are hydrolyzed. [1] Controlling and optimization of the hydrolytic processes requires efficient, non–destructive analytical techniques that allow real time analysis of enzymatic protein hydrolysis on its natural substrate under various hydrolytic conditions. Traditional methods are invasive and requires substrate labelling which can alter the enzymatic activity. Thus, novel analytical technology and approach are required to optimize the applicability of hydrolytic process.

High-Resolution Ultrasonic Spectroscopy (HR–US) is one of the promising technologies for direct, real-time and non-destructive monitoring of enzymatic activity on its natural substrate. HR–US employs high-frequency (MHz range) waves of compressions and decompressions (longitudinal deformations), which probe the elastic properties of materials determined by the intermolecular interactions and the micro-structural organization.[2] We have applied this technology to monitor in-real time the hydrolysis of whey proteins, β–lactoglobulin and bovine serum albumin (BSA), by individual and mixture of serine proteases, and to assess the effect of different reaction conditions on hydrolytic process. The ultrasonic reaction progress curve provided real-time precise characterization of number of peptide bonds hydrolyzed and the time profiles of degree of hydrolysis. This was verified with traditional and discontinuous TNBS (2,4,6-trinitrobenzene sulfonic acid) method, a ‘wet chemistry’ method to determine the degree of hydrolysis of protein. [3] The obtained ultrasonic profiles were further utilized in advanced analysis of hydrolytic process such as real-time reduction of average degree of polymerization and molar mass of fragments during the hydrolysis. [4]

References [1] O.L. Tavano, Journal of Molecular Catalysis B: Enzymatic, 2013, 90, 1. [2] V. Buckin, Journal of Sensors and Sensor Systems, 2018, 7, 207. [3] S. Rutherfurd, Journal of AOAC International, 2010, 93(5), 1515. [4] V. Buckin, M. C. Altas, Catalysts, 2017, 7 (11), 336.

Trypsin

Figure 1.: HR-US measurement cell set-up. (top left picture), Real-time monitoring of reduction of average

degree of polymerization, DP , and average molar mass,

MM , during hydrolysis of β–lactoglobulin, 162 amino

acids, (right picture).

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SAW and EIS biosensors for environmental particulate monitoring

Giuseppe Maruccio1, Maria Serena Chiriacò1, Sandro Spagnolo1,2 1 CNR NANOTEC – Institute of Nanotechnology c/o Campus Ecotekne, Italy.

2 Faculty of Mathematics, Physics and Informatics, Comenius University, Slovakia.

In this work we report a comparison between two optimized biosensing tools based on

innovative methods for environmental particulate monitoring. One of these is based on

surface acoustic waves (SAW) [1] and the other one on electrochemical impedance

spectroscopy (EIS) [2]. Both techniques allow the monitoring of events that occur on the

surface of sensitive areas of the biosensor, and have been used for the detection of

nanoparticles, similar in size to environmental particulate (aerosol). As a model system,

we chose polystyrene nanoparticles of three different dimensions (1 μm, 200 nm and 40

nm), which can mimic the different composition of PM1, very difficult to be detected with

standard gravimetric techniques [3] and very dangerous for health. In a deeper analysis,

obtained data were confirmed by SEM microscopy, and correlation with the percentage

of covered area by particles was performed. Finally, the two technologies have been

compared, evaluating the performance of the biosensors, and concluding that both

transduction methods are able to detect the presence of PM1 particulate; however, EIS

technique appears to show detection limits for 40 nm particles. SAW method is more

efficient in the detection of these particles, providing also in this case a linear response.

The results highlight also the suitability of the two methods to miniaturization, label-free

detection and integration in the so-called "Lab on a chip".

Figure 1.: a: SAW device with delay lines for detection of nanoparticles; b: EIS device; c: spectrum for the detection of nanoparticles of 200 nm; d: linear correlation between SAW signal and covered area of the device; e: EIS detection of 200 nm NP; f: linear correlation between Ret and covered area. References [1] O. Tigli, M.E. Zaghloul, Surface Acoustic Wave (SAW) Biosensors, in 53rd IEEE International Midwest Symposium on Circuits and Systems. 2010, IEEE, New York. p. 77-80. [2] M.S. Chiriacò et al., Talanta, 2015. 142, 57-63 [3] P. Kulkarni, P.A. Baron, and K. Willeke, Aerosol measurement: principles, techniques, and applications. John Wiley & Sons, 2011.

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