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Maria Sklodowska Curie Actions - Research and Innovation Staff Exchange

H2020-MSCA-RISE-2015 - FORMILK

International Workshop

“Recent applications of high-resolution ultrasonic spectroscopy for monitoring of

hydrolytic activities in milks"

Dublin, November 24, 2017.

Programme and Abstracts

Dublin, November 24, 2017

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“Recent applications of high-resolution ultrasonic spectroscopy for monitoring

of hydrolytic activities in milks"

Dublin, Ireland

November 24, 2017

Programme and Abstracts

Organized by School of Chemistry, University College Dublin, Belfield 4, Dublin, Ireland

(https://www.ucd.ie/chem/)

in the framework of

Marie Sklodowska-Curie Actions (MSCA) Research and Innovation Staff Exchange (RISE)

H2020-MSCA-RISE-2015

FORMILK, Project No. 690898 http://www.formilk.fmph.uniba.sk

http://www.ucd.ie/chem/

http://www.formilk.fmph.uniba.sk/

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The aim of the workshop is to provide a venue for discussions and training on application of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities of enzymes in milks in academic research and industry.

Workshop is organized in the framework of the project FORMILK funded by the European Commission under the programme H2020-MCSA-RISE-2015.

The workshop is open for PhD. students, young and senior researchers working in the areas of bioanalysis, biophysics, electrochemistry, analytical chemistry and related disciplines.

ORGANIZING COMMITTEE

Vitaly Buckin – Chairman, UCD, Dublin

Mark Dizon-Workshop Manager, UCD, Dublin

Rian Lynch, UCD, Dublin

Georgios Papoutsidakis, UCD, Dublin

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Maria Sklodowska Curie Actions - Research and Innovation Staff Exchange

H2020-MSCA-RISE-2015 - FORMILK


"Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks"

November 24, 2017

E0.01, Science East, University College Dublin, Belfield, Dublin 4

Program

9:00–09:30 Registration

09:30–09:40 Welcome and Introduction

09:40 – 12:00 Oral Presentations

09:40 – 10:15 V. Buckin (University College Dublin, Ireland): Ultrasonic monitoring of

hydrolytic reactions in milks.

10:15 – 10:50 M. Thompson (University of Toronto, Canada): Ultra-high frequency

acoustic wave detection in bioanalytical chemistry

10:50 – 11:05 M. Dizon (University College Dublin, Ireland): Monitoring of hydrolysis of

whey proteins with high-resolution ultrasonic spectroscopy. Degree of polymerization and

molar mass.

11:05 – 11:15 Coffee break

11:15 – 11:30 R. Lynch (University College Dublin, Ireland): Ultrasonic characterization

of performance of neutral and acid β-galacosidases in milks and in simulated conditions of

the gastro-intestinal tract of humans.

11:30 – 11:45 J. Byrne (Crosscare Ltd., Ireland): Application of high-resolution

ultrasonic spectroscopy for optimization of lactase formulations for infants. Effects of activity

of lactases on osmolarity of infant milks.

11:45 – 12:00 R. Sarok (Hungarian Dairy Research Institute Ltd.): Determination of

aflatoxin M1 in milk and milk samples using ELISA.

12:00 – 13:00 Lunch

13:00 – 14:00 Poster Presentations

14:00 – 16:00 HR-US Demonstration and Training in Real-time Ultrasonic

Monitoring of Hydrolytic Reactions in Milks

16:00 – 17:00 Discussion

17:00 – 18:00 Closing Reception

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Abstract for the

Oral Presentation

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International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.

Ultrasonic Monitoring of Hydrolytic Reactions in Milks

Vitaly Buckin

School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland

The rapidly growing field of enzyme-based technologies is dependent on analytical tools available for

non-destructive real-time assessment of various aspects of performance of enzymes in complex systems such

as emulsions, suspensions and gels, where most of the existing analytical techniques are limited in their

applicability, especially if the media is opaque, or if the reactants and the products do not possess an optical

activity. High-resolution ultrasonic spectroscopy (HR-US) is one of the novel technologies for real-time non-

invasive monitoring of enzymatic reactions. The technique is based on measurements of parameters of high

frequency sound (ultrasound) waves propagating through analyzed samples.

As ultrasonic waves propagate through most of materials the technique does not require optical

transparency. It does not need optical markers, secondary reactions, or other consumables. High resolution of

this technique allows measurements of small changes in concentrations of substrates and products in a course

of analyzed reactions. In addition, ultrasonic scattering effects in dispersions provide real time information on

the structural changes in the medium. As ultrasonic measurements characterize the properties of the bulk

medium, the unwanted effects of surfaces, often associated with reflectance spectroscopies and electrode

techniques, are excluded. HR-US measurements can be performed in a wide range of temperatures, (typically

-20 to 120Co) in large and small (droplet size) sample volumes, in static and flow-through regimes, in dilute

solutions and semisolid materials, thus, allowing monitoring of enzyme catalyzed reactions in a variety of media and environmental conditions.

The obtained ultrasonically reaction progress curves (concentration of reactant/product vs time),

measured precisely over the whole course of reactions, can be utilized in advanced analysis of hydrolytic

processes in milks, including monitoring of

the evolution of the degree of

polymerization and molar mass of the substrates, and the change of the osmolarity of milks during hydrolytic processes. The

broad dynamic (concentration) range of the

technique allows monitoring reverse

reactions, measurements of reactions

equilibrium constants and reactions

standard Gibbs energies1. The technique can be applied for quantitative assessment

of enzyme activity (ultrasonic activity

units) with native substrates directly in

milks. Recently described methodology of

the ultrasonic analysis2,3 offers a set of “ultrasonic tools” for quantitative real-time

characterization of the impact of various

factors on performance of enzymes in

complex mixtures, including reversible and

irreversible effects of temperature and

temperature profiling, variation of enzyme

activity during reactions caused by enzyme

deactivation and inhibition, effects of

concentrations of enzymes and of

substrates.

Real-time ultrasonic profiles of hydrolysis of lactose in infant milk. Concentration of β-galactosidic bonds hydrolyzed by β- galactosidase (Kluyveromyces lactis, G3665, Lactozyme 2600 L,

Novozyme Co.) in Cow&Gate First Infant milk at different temperatures calculated from the measured ultrasonic velocity

profile as described earlier2. The enzyme was added to milk at zero

time. The dashed line indicates 50% level of hydrolysis. Concentration of the enzyme was 3.42 UU/g. One ultrasonic activity

unit, UU, represents the amount of enzyme, which hydrolyses 1

μmol of β-galactosidic bonds in Cow&Gate First Infant milk per

1. P. Resa and V. Buckin Analytical Biochemistry 415 1–11(2011)

2. M. C. Altas, E. Kudryashov and V. Buckin Analytical Chemistry 88, 4714−4723 (2016) 3. V. Buckin, M. C. Altas, Catalysts, 7(11), 1-42 (2017)


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Ultra-high frequency acoustic wave detection in bioanalytical chemistry

Michael Thompson

Department 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 [1]. In the present paper, we discuss

applications of a much higher frequency device – the electromagnetic piezoelectric

acoustic wave sensor (EMPAS). This technology is based upon excitation of up to 1

GHz waves in quartz via an electromagnetic field. This is achieved in an electrode-

less fashion through use of a flat spiral coil positioned close to the quartz surface

whereby the device is excited by the secondary electric field associated with the coil

[2]. Generally the sensor is operated around the 50th harmonic of a formal 20 MHz

crystal.

The following applications of the technology will be described concisely – detection of

cocaine [3], endotoxin [4], breast [5] and ovarian cancer [6] biomarkers in serum. All

of these assays involve operation of the EMPAS device in tandem with surface anti-

fouling chemistry [7]. The latter is based on ultra-thin surface modifiers involving the

ethylene glycol moiety. Probes such as proteins or aptamers are linked to the sensor

surface together with the anti-fouling modifier.

Relevant to the theme of the workshop, preliminary research on the EMPAS study of

β-casein deposition and β-casein cleavage by plasmin will be described. With respect to

the former deposition experiments, adsorption on the hydrophobic (OTS) treated surface

was far more reproducible and facile than on the surface of bare quartz. In addition early

work on the detection of plasmin via casein cleavage was achieved at a low limit of

detection compared to more conventional acoustic wave protocols. Future research in

this regard will be discussed.

References [1] Surface-launched acoustic wave sensors: Chemical sensing and thin film characterization,

M. Thompson and D.C. Stone, Wiley-Interscience, New York (1997).

[2] A.C. Stevenson, H.M. Mehta, R.S. Sethi, L-E. Cheran, M. Thompson, I. Davis and

C.R. Lowe, Analyst, 2001, 126, 1619. [3] M.A.D.Neves, C. Blaszykowski, S. Bokhari and M. Thompson, Biosensors and

Bioelectronics, 2015, 72, 383. [4] S. Sheikh, C. Blaszykowski and M. Thompson, RSC Advances, 2016, 38037. [5] V. Crivianu-Gaita, M. Aamer, R.T. Posaratnanathan, A. Romaschin; and M.

Thompson, Biosensors and Bioelectronics, 2016, 78, 92. [6] J.B. Chen, M.A.D. Neves and M. Thompson, Sensing and BioSensing Research,

2016, 11, 107. [7] S. Sheikh, D. Y. Yang, C. Blaszykowski and M. Thompson, Chemical

Communications, 2012, 48, 1305.


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Monitoring of hydrolysis of whey proteins with high-resolution ultrasonic spectroscopy. Degree of polymerization and molar mass.

Mark Dizon, Margarida Caras-Altas, and Vitaly Buckin

School of Chemistry, University College Dublin, Belfield 4, Dublin, Ireland.

Whey protein represents about 20% of the protein content in bovine milk. These

proteins are known to be great source of a range of biologically active peptides [1].

Bioactive peptides are specific protein fragments which displays improved functional,

immunological and bioactive properties. Proteolytic enzymes (‘proteases’) are

commonly used as a method of enzymatic modification to produce these functional

peptides, and their properties depends on the degree and control to which the

proteins are hydrolysed [1,2]. Optimising the control of the processes of hydrolysis to

produce the peptide fragments with desired properties and bio-activity requires

efficient tools for real time monitoring of hydrolysis of peptide bonds under various

environmental conditions.

High-Resolution Ultrasonic Spectroscopy (HR-US) was employed for real-time non-

destructive monitoring of hydrolysis of whey proteins, β–lactoglobulin and bovine

serum albumin, by α–chymotrypsin and Proteinase K (specific and unspecific serine

proteases, respectively) at different pH. With this technology, novel methodology has

been developed for ultrasonic real-time measurements of the number of cuts of

peptides and of the degree of hydrolysis. The ultrasonic results were compared and

verified with discontinuous TNBS (2,4,6-trinitrobenzenesulfonic acid) measurements,

which is a traditional discrete method to obtain degree of hydrolysis of the reaction

[3,4]. The obtained ultrasonic profiles of the concentration of peptide bonds

hydrolysed were used for real-time analysis of the degree of polymerization and

molar mass of fragments of β–lactoglobulin produced during hydrolysis [4].

References

[1] M. Lucarini, Beverages, 2017, 3, 1–10.

[2] a) C.C. Udenigwe, A. Mohan, M. C. Udechukwu, S. R. C. K. Rajendran, RSC Advances, 2015, 5,

97400–97407; b) A. Zambrowiczs, M. Timmer, A. Palonwoski, G. Lubec, T.,Trziszka, Amino

Acids, 2013, 44, 315–320.

[3] a) S. M. Rutherfurd, Journal of AOAC International, 2010, 90(5), 1515–1522; b) J. Adler-Nissen,

Journal of Agricultural and Food Chemistry, 1979, 27(6), 1256–1262

[4] V. Buckin, and M. Caras-Altas, Catalysts, 2017, 7(11), 336.


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Ultrasonic characterization of the performance of neutral and acid β-galactosidases in milks and in simulated conditions of the

gastro-intestinal tract in humans Rian Lynch, Vitaly Buckin.

School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland Lactose intolerance is a condition characterised by the inability to digest and absorb

lactose, a disaccharide found in milk, leading to negative clinical symptoms including

abdominal discomfort, bloating, flatulence, etc. Approximately 75% of the world’s

population is affected by this intolerance, which arises due to a deficiency in the

enzyme lactase.1 Foods containing lactose are a natural source of calcium, vitamin

D and protein,2 therefore their removal from the diet may have a negative impact on

the health of an individual. Alternatively, treatment with lactase enzyme (β-

Galactosidase) supplements hydrolyses lactose in food into glucose and galactose

which are absorbable in the small intestine, reducing or eliminating the symptoms

associated with lactose intolerance. The effectiveness of these supplements in vivo

depends greatly on several factors including enzyme concentration, transit time, pH

profile and digestive proteases present in the gastrointestinal tract.3 In this work, the performance of a neutral lactase, utilised as a supplement in infant

milk for reducing of the level of lactose, was studied using HR-US spectroscopy.

Hydrolysis was carried out in the milk under several differing conditions of pH, which

allowed for approximation of the activity of the enzyme under physiological conditions

in the stomach during infant feeding. We have also used HR-US to study the activity of an acid lactase (β-Galactosidase

from Aspergillus Oryzae), utilised it tablet supplements, under various conditions of

pH representative of the adult gastric pH profile in buffered solutions. Information on

the performance of this enzyme across the pH range found in the stomach allows for

the determination of required dosage of enzyme to achieve appropriate lactose

hydrolysis within the pH “window” in which the enzyme is active under gastric

conditions. Acknowledgements: Funding for this work has been provided by the Irish Research Council Enterprise Partnership Scheme in collaboration with Crosscare Ltd.

References:

[1] Altas, M. C., Kudryashov, E. & Buckin, V. Anal. Chem. 88 (2016) 4714–4723.

[2] Huth, P. J., DiRienzo, D. B. & Miller, G. D. J. Dairy Sci. 89 (2006) 1207–1221.

[3] Xenos, K., Kyroudis, S., Anagnostidis, A., & Papastathopoulos, P. Eur J Drug Metab Pharmacokinet. 23(2)

(1998) 350-355


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Colief® Lactase Enzyme Infant Drops Product Origin and Application Development

Crosscare limited - John Byrne

Crosscare limited Ireland.

This presentation is a brief introduction to Crosscare Limited and its products.

Emphasis is placed on the liquid neutral lactase product Colief Infant Drops and its

application in the reduction of the lactose content in infant formula feeds. Techniques

applied in the measurement of lactose conversion achieved by the application of the

drops are briefly touched upon in the context of product application development and

adjustment. The current engagement with High-Resolution Ultrasonic Spectroscopy

(HR-US) is considered in the context of the academic research work currently being

carried out in the department of Chemistry and Chemical Biology, UCD, by Dr. Vitaly

Buckin and his team under the auspices of Crosscare Limited’s participation in the

Enterprise Partnership Scheme of the Irish Research Council in conjunction with the

H2020-MSCA-RISE-2015 - Formilk Project.

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International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks",


Determination of aflatoxin M1 in milk and milk samples using ELISA

R. Sarok1*, A. Poturnayova2,3, K. Szabo1, A. Hucker1, T. Hianik2

1 Hungarian Dairy Research Institute, Mosonmagyarovar, Hungary

2 Department of Nuclear Physics and Biophysics, FMFI UK, Bratislava, Slovakia 3 Institute of Biochemistry and Animal Genetics, Center of Biosciences SAS, Bratislava,

Slovakia * Corresponding author: [email protected]

Aflatoxins are carcinogenic, highly toxic metabolites of the mold fungus varieties

Aspergillus flavus and Aspergillus parasiticus. Aflatoxin M1 is produced as a metabolite

of aflatoxin B1. It is secreted with the milk after the feeding of aflatoxin B1 containing

feed to lactating cows. As aflatoxin M1 is relatively stable thoughout the pasteurizing

process, not only a comprehensive routine check of the raw materials to be processed is

required, but also of the final products. For aflatoxin M1 the limit has been fixed at 0.05

µg/l (50 ppt). Several methods have been developed for AFM1 determination in milk,

mainly based on high-performance liquid chromatography (HPLC), which

advantageously substituted the thin layer chromatography technique (TLC). Enzyme-

linked immuno-sorbent assays (ELISA) have become very popular for mycotoxins

analysis, raising the development of many commercially available kits, which are

essentially based on competitive assays. These ELISA techniques have been in some

cases successfully transferred to biosensor technology.

In our work we used ELISA for the quantitative analysis of aflatoxin M1. Using this

method, it is possible to detect aflatoxin M1 in milk, milk powder and cheese

quantitatively and with accuracy. The basis of the test is the antigen-antibody reaction.

The microtiter wells are coated with capture antibodies directed against anti-aflatoxin M1

antibodies. Aflatoxin M1 standards or sample solutions, aflatoxin M1 enzyme conjugate

and anti-aflatoxin M1 antibodies are added. Free aflatoxin M1 and aflatoxin M1 enzyme

conjugate compete for the aflatoxin M1 antibody binding sites (competitive enzyme

immunoassay). At the same time, the anti-aflatoxin M1 antibodies are also bound by the

immobilized capture antibodies. Any unbound enzyme conjugate is then removed in a

washing step. Substrate/chromogen is added to the wells, bound enzyme conjugate

converts the chromogen into a blue product. The addition of the stop solution leads to a

color change from blue to yellow. The measurement is made photometrically at 450 nm.

The absorbance is inversely proportional to the aflatoxin M1 concentration in the sample.

The ELISA allowed detectin of aflatoxin M1 in milk with detection limit of 0.005 µg/l (5

ppt)

This work was financially supported by European Union's Horizon 2020 research and

innovation programme under the Marie Sklodowska-Curie grant agreement No 690898.

mailto:[email protected]

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Abstract for the

Poster Presentation

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International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks",


Detection of microorganisms in food products by atomic force

microscopy

J. Nagy1*, Z. S. Steinerné 1, A. Poturnayova2,3, A. Hucker1, K. Szabo1, T. Hianik2

1 Hungarian Dairy Research Institute, Mosonmagyarovar, Hungary

2 Department of Nuclear Physics and Biophysics, FMFI UK, Bratislava, Slovakia 3 Institute of Biochemistry and Animal Genetics, Center of Biosciences SAS, Bratislava,

Slovakia

* Corresponding author: [email protected]

The enumeration of different lactic acid bacteria’s and „probiotics“ from several products

are not only as a manufacturer's requirement, but also as an authority and a market

requirement. The used level of microorganisms in foods or in drugs is normally high and

sample preparation is difficult. The current methods of detection of lactic acid bacteria’s

are focused on biochemical characteristic (plate method). Not any rapid method is

available. Although these methods have advantages each method also has limitations.

Another technology that is applicable in microbiological studies is atomic force

microscopy (AFM). AFM has been used to image a wide variety of biological materials,

like bacterial cells, and human chromosomes. The basic idea of AFM is to use a sharp tip

scanning over the surface of a sample while sensing the interaction between the tip and

the sample. The tip with a flexible cantilever or the sample is mounted on a piezoelectric

scanner which can move precisely in three dimensions. During the test, a laser diode

emits a laser beam onto the back of the cantilever over the tip. As the cantilever deflects

under load from the laser, the angular deflection of the reflected laser beam is detected

with a position-sensitive photodiode. Magnitude of the beam deflection changes in

response to the interaction force between the tip and the sample. The AFM system senses

these changes in position and can map surface topography or monitor the interaction

force between the tip and the sample. AFM was applied to investigate morphology and

characteristic parameters of the microorganisms. Using AFM we successfully detected

the Lactobacillus spp., Streptococcus spp., Bifidobacterium spp., immobilized at the

surface of glass, and determined their shape and dimensions.

This work was financially supported by European Union's Horizon 2020 research and

innovation programme under the Marie Sklodowska-Curie grant agreement No 690898.



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Study of the solutions of interpolymer complex forming synthetic polymer pairs by high resolution ultrasonic spectroscopy

Ákos Szabó,1 Zsófia Osváth,1 Mark Dizon,2 Breda O’Driscoll,3 Vitaly Buckin3

and Béla Iván1

1, Polymer Chemistry Research Group, Institute of Materials and Environmental

Chemistry, Research Centre for Natural Sciences of the Hungarian Academy of

Sciences, Magyar tudósok krt. 2., Budapest, H-1117, Hungary

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

3, SONAS Ltd, Dublin, Ireland

Solutions of synthetic polymer pairs, i.e. poly(acrylic acid) (PAA) and

poly(poly(ethylene glycol) methacrylate) (PPEGMA), were investigated by high

resolution ultrasonic spectroscopy (HRUS). Previous temperature-dependent

transmittance and light scattering measurements indicate that these macromolecules

form interpolymer complexes in their common solutions in water by

intermacromolecular H-bonds. The change in the sound velocity difference between

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

solution indicates that HRUS could be a relevant method to study these

supramolecular structures.

Fig. 1: Model of the PPEGMA-PAA interpolymer complex formation

(black chain: PPEGMA, red chain: PAA)

References

[1] Á. Szabó, I. Szanka, Gy. Tolnai, Gy. Szarka and B. Iván, Polymer, 2017, 111, 61-

66.


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High resolution ultrasonic spectroscopy used for the study of thermal behavior of hyaluronan

Andrea Kargerová1, Adam Jugl1

1Brno University of Technology, Faculty of Chemistry, Materials Research Centre, Purkyňova

118, 612 00 Brno, Czech Republic.

[email protected]

Abstract

High resolution ultrasonic spectroscopy (HR-US) is a new technique for material analysis based on the measurements of parameters of ultrasonic waves propagating through samples. This technique is based on the measurements of velocity and attenuation on acoustical waves at high, ultrasonic frequencies propagating through materials. This technique allows direct and non-destructive measurements without formation of derivatives or changing state of sample. Advantage of this technique is the ability of ultrasonic waves to propagate through optically non-transparent materials. The analysed sample can be highly coloured or even opaque. In this contribution, a high-resolution ultrasonic spectroscopy technology (HR-US 102T) has been employed to study of thermal behavior and stability of hyaluronan aqueous solutions. The ultrasonic velocity was measured in hyaluronan solutions in water or in 0.15M NaCl and in the temperature range from 25 to 70°C for the hyaluronan molecular weights from 10 to 1750 kDa. The ultrasonic velocity increased linearly with concentration of hylauronan and decreased with temperature. The effect of molecular weight was negligible. The addition of NaCl changed only the numerical values of ultrasonic velocity while not changing the character of their dependence on temperature and concentration. Reversible changes of ultrasonic parameters were observed during several cycles of heating and cooling. The regular profile indicates that the structure of this solution changes with temperature, but all the changes are reversible, and the structure is not affected by the thermal history.

References

LAPCÍK, Lubomír, Lubomír LAPCÍK, Stefaan De SMEDT, Joseph DEMEESTER, Peter CHABRECEK.

Hyaluronan: Preparation, structure, properties, and applications. Chemical Reviews. 1998, vol. 98, issue 8, pp.

2663-2684.

BUCKIN, Vitaly, Cormac SMYTH: High-resolution ultrasonic resonator measurements for analysis of liquids,

Seminars in Food Anaysis. 1999, vol. 4, pp. 113-130.

OCHENDUSZKO, Agnieszka, Vitaly BUCKIN. Real-time monitoring of heat-induced aggregation of β-

lactoglobulin in aqueous solutions using high-resolution ultrasonic spectroscopy. International Journal of

Thermophysics. 2010, vol. 31, issue 1, pp. 113-130.

LOWRY, Karen M., Ellington M. BEAVERS, Joanne M. HOEFLING, Shiro MATSUOKA, Endre BALAZS,

Mary K. COWMAN a Shiro MATSUOKA. Thermal stability of sodium hyaluronate in aqueous solution.

Journal of Biomedical Materials Research. 1994, vol. 28, issue 10, pp. 75-78.

Acknowledgements

This study was supported by the COST action CM1101. The Materials Research Centre at the Faculty

of Chemistry, Brno University of Technology is supported by Project No. LO1211 from the Czech

Ministry of Education, National Sustainability Program I.




15

Bioavailability of dairy proteins after gastrointestinal digestion using CaCo2/HT-29 co-culture model

V. Buckin1, L. Giblin2 and A. R. Corrochano1, 2

1, Chemistry Department, University College Dublin, Ireland

2, Food Bioscience Department, Teagasc Food Research Centre, Moorepark, Ireland

Dairy proteins are prized for their nourishment and their bioactive components. The

aim of this study was to identify dairy peptides that are transported across the

intestinal barrier and are therefore available to provide a health benefit to

downstream target cells. Commercially available dairy proteins (whey protein isolate

(WPI), β-lactoglobulin and bovine serum albumin) and a whey-based sport product

were subjected to a static in vitro gastrointestinal digestion using Cost-Infogest

method. The intestinal barrier co-culture model, CaCo2/HT-29, was exposed to

digested samples at physiologically relevant concentrations. After a two hour

incubation, apical (intestinal lumen) and basolateral (containing absorbed peptides)

solutions were analysed for peptide content by Ultra-Performance Liquid

Chromatography/Electrospray Ionisation-High Resolution Tandem Mass

Spectrometry (UPLC/ESI-HR-MS/MS). The peptides were characterised using the

Proteome Discoverer 1.4 software against the Bos taurus database UniProt taxon ID

9913. The false discovery rate of peptide identification was set to FDR = 0.01.

Among the peptides identified, several known bioactive peptides were found in

basolateral solutions after WPI, β-lactoglobulin, bovine serum albumin and whey-

based sport product treatments. In conclusion, gut transit generates bioactive

peptides from dairy that are bioavailable across the intestinal barrier.


16

Application of High-Resolution Ultrasonic Spectroscopy for real- time monitoring of trypsin activity in β - casein solution

Sopio Melikishvili1, Mark Dizon2, Breda O’Driscoll3, Vitaly Buckin2, Tibor Hianik1

1Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynska

Dolina F1, Bratislava 842 48, Slovakia 2School of Chemistry and Chemical Biology, University College of Dublin, Belfield,

Dublin 4, Ireland 3 Sonas Technologies, Ailesbury Grove 42, Dublin 16, Ireland

Proteolysis, the enzymatic hydrolysis of a protein, plays an important role in various fields of bioscience and biotechnology. Technologically, there are broad applications of proteolysis in food processing [1]. For instance, the proteolytic activities in milk affect the texture and flavor of dairy products [2]. Therefore quantification of protein activity in milk could have important industrial impacts.

High-Resolution Ultrasonic Spectroscopy (HR-US) was applied for real-time non- destructive precision monitoring of trypsin-catalyzed hydrolysis of β – casein at varying temperature and enzyme concentration. Ultrasonic velocity and attenuation were measured using a HR-US 102PT ultrasonic spectrometer (Sonas Technologies Ltd., Ireland) equipped with the precision programmable temperature controller. The addition of enzyme into the β - casein solution was accompanied by the increase of velocity in the reaction mixture caused by cleavage of peptide bonds. 2,4,6- Trinitrobenzene Sulfonic Acid (TNBS) assay was employed in order to convert a change of ultrasonic velocity in the reaction mixture caused by hydrolysis of β-casein to the change in concentration of covalent bonds hydrolyzed. It was demonstrated, that temperature has the significant effect on the performance of enzyme, showing optimal activity at 45°C and negligible activity at 15°C. The obtained results have clearly demonstrated the capability of ultrasonic tools for nondestructive real-time assessment of the enzyme activity in complex formulations.

Acknowledgements: This work was supported by European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 690898.

References

[1] M.M. Vorob’ev, V. Vogel, G. Güler, W. Mäntele, Food Biophysics, 2011, 6, 519-526.

[2] N. Datta, H.C. Deeth, Diagnosing the cause of proteolysis in UHT milk, LWT Food Sci. Technol.,

2002, 36, 173–182.


17

Solubility and stability study using HR-US 102 of Beta-cyclodextrine and Gelatine as carrier systems for Curcumin

Maria Pavai,1 Mark Dizon2, Breda O'Driscoll3, Vitaly Buckin3 and

Zsofia Keresztes1

1, Research Centre of Natural Sciences, Hungarian Academy of Sciences, Hungary

2, UCD, Dublin, Ireland

3, SONAS Technologies Ltd., Dublin, Ireland

Curcuminoids are the major phenolic compounds that which are isolated from turmeric (Curcuma longa) and contain curcumin (the bioactive component of turmeric), demethoxycurcumin and bisdemethoxycurcumin. It can said, that the natural dye of curcumin is one of the strongest active ingredients in the curcuma root. Thanks to its impact on body health (antioxidant property; slows/kills the growth of tumour cells; lowers cholesterol level and reduces obesity; stimulates muscle regeneration after trauma; corrugate skin diseases; it is a healing herb for the liver, spleen, stomach, intestines, lungs and blood; can prevent and cure Alzheimer’s disease and can reduce inflammatory reactions) it has been intensively investigated [1]. 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 foods. Gelatine is pure protein and a natural foodstuff. It can be made from the skins of pigs and cows and are approved for human consumption by the veterinary authorities. They contain the collagen (the most important scleroprotein of the body). The basic unit comprises a protein chain of about 1050 amino acids. These intertwine in groups of three to form triple helix structures. Cross-linking between many of these triple helices produces collagen fibrils that have a three-dimensional network structure. And it’s these structures that form the connective tissue in skin and bone. Cyclodextrins, produced from starch by means of enzymatic conversion, are a family of compounds made up of sugar molecules bound together in a ring (cyclic oligosaccharides).They are used in food, pharmaceutical, drug delivery and chemical industries. The ability of gelatine and cyclodextrins to carry and stabilise curcumin has the potential to enable the delivery of these components into functional foods. In this study the potential of 1.25% β- cyclodextrin solution and 1.25% gelatine solution as a “solvent” or carrier for curcumin 95% Alfa Aesar curcumin were investigated using a HRUS 102 SS (High Resonance Ultrasonic Spectrometer) by measuring at 45°C the differential sound velocity and relative attenuation at different frequency ranges (frequency ranges between 2-15MHz).

References

[1] M. Pávai, J. Mihály, A. Paszternák, Food Analytical Methods, 2015, Volume 8, 9, 2243-2249


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Poly(N-isopropyl acrylamide) interactions with proteins by UV- visible, NMR and ultrasonic spectroscopy

Zsófia Osváth,1 András Láng,2 Beáta Szabó,3 Mark Dizon,4 Breda O’Driscoll,5

Vitaly Buckin4 and 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. Department of Organic Chemistry, Eötvös Loránd University, Hungary

3. Research Group of Intrinsically Disordered Proteins, Research Centre for Natural

Sciences of the Hungarian Academy of Sciences, Hungary

4. School of Chemistry, University College Dublin, Belfield, Dublin, Ireland

5. SONAS Ltd, Dublin, Ireland Poly(N-isopropylacrylamide) (PNIPAAm) is widely applied in medical science, its interactions with different proteins is not extensively studied so far. Our experiments focused on the interactions between PNIPAAm and tß4 protein. 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.

Fig. 1: The aqueous PNIPAAm solution phase transformation curve


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Ultrasonic Monitoring of Hydrolysis of β-Lactoglobulin by Digestive

Enzymes.

George Papoutsidakis,1 Mark Dizon,1 Aoife Scanlon,1 Vitaly Buckin,1

1, School of Chemistry and Chemical Biology, University College Dublin, Belfield,

Dublin 4, Ireland

Hydrolytic enzymes are commonly used in the industry for various applications including

production of bioactive peptides that exhibit a range of functions in the body [1]. In particular,

whey proteins serve as accessible and affordable sources of the aforementioned peptides [2].

Whey proteins can be cleaved at very specific residues by serine proteases and thus they are

useful enzymes for hydrolysis of this type of proteins. One of the key challenges in control of

the enzymatic processing of proteins, is the lack of analytical techniques available for real-time

non-destructive monitoring of hydrolysis in bioreactors. This work explores the capability of

High-Resolution Ultrasonic Spectroscopy (HR-US) for real-time monitoring of hydrolysis of

whey proteins and analysis of the effects of enzyme mixtures, and environmental conditions,

such as pH, temperature, buffer, on the performance of the proteolytic enzymes.

Fig. 1 . Illustration of hydrolysis of peptide bond by a protease enzyme.

HR-US was applied for assessing the hydrolysis of whey protein β-lactoglobulin by a mixture

of digestive enzymes (trypsin, α-chymotrypsin and pancreatin) under the effect of pH,

concentration of enzyme and type of buffer (phosphate, Simulated Intestinal Fluid).

It was found that the activity of the trypsin and α-chymotrypsin enzyme mixture in SIF buffer

is significantly higher than in phosphate buffer at the same pH. At initial stages of hydrolysis,

the mixture of trypsin and α-chymotrypsin showed significantly higher specific activity than

pancreatin. However, the extent of hydrolysis (number of peptide bonds hydrolyzed per one

molecule of β-lactoglobulin) at later stages is higher for pancreatin.

References

[1] Pihlanto, Anne, and Hannu Korhonen. "Bioactive peptides and proteins." Advances in food and nutrition

research 47 (2003): 175-276

[2] Ha, Ewan, and Michael B. Zemel. "Functional properties of whey, whey components, and

essential amino acids: mechanisms underlying health benefits for active people (review)." The

Journal of nutritional biochemistry 14.5 (2003): 251-258.


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Acknowledgements

This workshop was supported by European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 690898.

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