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
1
International Workshop
“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
2
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
3
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"
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
4
Abstract for the
Oral Presentation
5
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)
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
6
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.
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
7
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.
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
8
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
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
9
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.
10
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks",
Dublin, November 24, 2017.
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.
11
Abstract for the
Poster Presentation
12
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks",
Dublin, November 24, 2017.
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.
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
13
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.
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
14
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.
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.
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
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.
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
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.
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
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
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
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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
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
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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.
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
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Notes
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
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Notes
International workshop "Recent applications of high-resolution ultrasonic spectroscopy for monitoring of hydrolytic activities in milks", Dublin, November 24, 2017.
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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.