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Foods and Raw Materials, 2019, vol. 7, no. 1
E-ISSN 2310-9599 ISSN 2308-4057
Research Article Open Access
DOI: http://doi.org/10.21603/2308-4057-2019-1-161-167 Available online at http:jfrm.ru
Changes in physico-chemical properties of milk under ultraviolet
radiation
Vladimir D. Kharitonov1* , Natalia E. Sherstneva1 , Dmitriy V. Kharitonov1, Elena A. Yurova1 , and Vladimir P. Kurchenko2
1 All-Russian Dairy Research Institute, Moscow, Russia
b Belarusian State University, Minsk, Belarus
* e-mail: gnu-vnimi@yandex.ru
Received October 10, 2018; Accepted in revised form November 26, 2018; Published June 08, 2019
Abstract: The use of ultraviolet radiation in the treatment of milk and other liquid foods is a very promising field of study since it reduces their bacterial load. It is rarely used to increase the vitamin D3 content and modify the protein and fatty acid composition of milk. The paper describes how different parameters of ultraviolet radiation influence such characteristics of raw and pasteurized milk as the mass fraction of total protein, nonprotein nitrogen content, active and titratable acidity, general bacterial load (QMA&OAMO), fatty acid composition, and vitamin D3 content. Low-pressure gas-discharge lamps were used to treat a 400 ^m moving layer of milk with ultraviolet radiation. The radiation time, its doses, and the milk flow rate changed in the ranges of 5-25 min, 5.1-102 mJ/cm2, and 0.044530.13359 m3/s, respectively. We identified optimal radiation ranges that lead to both a lower microorganism content and a higher vitamin D3 content. Our study also determined specific correlations in the mutual changes of the given parameters. The treatment ranges did not produce any significant changes in other physico-chemical properties of milk. We also found that vitamin D3 was synthesized in raw and pasteurized milk in a similar way. Moreover, there was an insignificant decrease in the vitamin D3 content in milk treated with ultraviolet radiation during storage for up to 48 hours. On the whole, the results indicate that the treatment of milk with ultraviolet radiation in the dosage range from 5.1 to 102 mJ/cm2 has a complex effect on the total bacterial load (QMA&OAMO) and vitamin D3 content, whereas it has almost no effect on the protein and fatty acid composition.
Keywords: Ultraviolet radiation, milk, protein, fatty acids, vitamin D3, (QMA&OAMO) CFU/cm3
Please cite this article in press as:. Kharitonov V.D., Sherstneva N.E., Kharitonov D.V., Yurova E.A., and Kurchenko V.P. Changes in physico-chemical properties of milk under ultraviolet radiation. Foods and Raw Materials, 2019, vol. 7, no. 1, pp. 161-167. DOI: http://doi.org/10.21603/2308-4057-2019-1-161-167.
INTRODUCTION
The assessment of the ultraviolet radiation (UVR) effect on the physico-chemical and vitamin composition, as well as the bacterial load of dairy and other food products, is a promising field of research as it permits directional regulation of their properties.
Numerous studies are currently conducted in this area. However, the data are still insufficient to accurately assess all the aspects and mechanisms of the ultraviolet radiation effect on food and dairy products during their processing. There is therefore a need for further research in this area.
Milk is an important source of nutrition. It contains proteins, milk fat, minerals, and different vitamins. The main components of milk vary according to the breed of cows, feeding, and livestock management. These changes
mostly affect the content of fat-soluble vitamins, in particular vitamin D3. In European countries, such as Denmark, the consumption of milk, cheese, and other dairy products accounts for about 12% of the total intake of vitamin D3 [1, 2]. Dairy products with a low content of vitamin D3 cannot serve as its natural source. Insufficient intake of vitamin D3 increases the risk of developing hypertension, autoimmune diseases, diabetes, rickets, and cancer [1].
Lactating cows have two primary sources of increased vitamin D3 content in milk. This vitamin can come with food, including vitamin-enriched supplements, or be produced endogenously under the impact of ultraviolet radiation on the cow's skin [3, 4]. Under ultraviolet light radiation, 7-dehydrocholesterol turns into precalciferols as a result of the prototropic rearrangement. Precalciferols serve to form the D vitamins (Fig. 1).
Copyright © 2019, Kharitonov et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.
7-dehydrocholesterol CH,
ch]
CK-CH;—CH;—CH;—CH-CM,
CH, r f
CH-CH;-CH;-CH;-CH-CKj
cholecalciferol (vitamin D3)
Fig. 1. Vitamin D3 biosynthesis under ultraviolet radiation.
The vitamin D3 content in milk and dairy products can be increased by its introduction at a particular stage of the technological process. This can also be achieved by ultraviolet treatment due to its directional effect on 7-dehydrocholesterol.
Various gas-discharge radiators are currently the most common sources of ultraviolet radiation. Mercury lamps are used because mercury in a gaseous state is activated at relatively low temperatures. Furthermore, the discharge in mercury vapor provides the largest number of intense lines in the ultraviolet spectrum. High- and low-pressure mercury lamps are used to ensure effective ultraviolet treatment of the material. They differ in their intensity depending on mercury vapor pressure. The advantage of low-pressure mercury lamps is that the largest share of radiation falls on the wavelength of X = 253.7 nm, which has a maximum bactericidal effect. Therefore, lamps of this type are mostly used to reduce the bacterial load of the product. In high-pressure mercury lamps, the spectral area of impact has a higher wavelength range, which makes them less suitable for the bacterial treatment.
Flash xenon or argon high-pressure lamps became quite widespread, along with mercury gas-discharge lamps. Compared to argon lamps, flash xenon lamps have better bactericidal activity, shorter exposure time, and higher safety. Their disadvantages include a shorter period of guaranteed action, as well as increased operating costs.
We should note a shortage of comparative studies into the use of ultraviolet radiators in the field of food biotechnology.
Depending on the modes, the UV treatment can have a different effect on the composition and properties of milk. The most important components of milk - proteins, fats, and vitamins - can absorb UVR throughout its range. The energy absorbed by them can change the physico-chemical properties of these organic molecules. Furthermore, UVR produces active forms of oxygen that can change the chemical composition and properties of the main components of milk as a result of free radical reactions. The active forms of oxygen cause the DNA damage in microorganisms and oxidation of specific protein groups. Therefore, UVR is successfully used to reduce the bacterial load of dairy raw materials [5, 6].
The changes occurring in bacteria under UVR are multistage, with DNA molecules being the final stage of their action [7].
In addition to the direct influence on DNA, increased UVR intensity can cause cell mutations. This phenomenon is accompanied by the formation of free radicals and peroxides with mutagenic properties. For example, [8] reports an increased number of mutational changes in microorganisms cultivated in nutrient media.
UVR has the greatest oppressive influence on microorganisms in the wavelength range of 205-315 nm [9].
The first practical application of UVR as bactericidal treatment was for disinfection of water [10, 11]. Positive results were also achieved in the treatment of other highly transparent liquid foods, such as sugar solutions, their derivatives, and juices [12-17].
Numerous studies of UVR influence on milk and dairy products showed that the treatment is complicated by their low transparency due to the screening ability of protein and fat. Furthermore, dairy products have a complex composition, and their components are closely related to each other [18-23].
Nevertheless, those studies served as a basis for industrial facilities designed to reduce the quantitative content of bacteria in milk [6, 10, 24, 25].
Ultraviolet treatment has no direct effect on milk proteins in a certain range of parameters [26-28]. At the same time, we can use ultraviolet light to change the structure of proteins and give them new properties by changing treatment modes. This is confirmed by Cho et al. which showed that UV treatment could affect the molecular structure of p-lactoglobulin, the main allergen in milk [29]. Similar results were obtained in another study that recorded shifts in the molecular structure of p-lactoglobulin. These results also indicate a possibility of regulating the peptide profile of milk proteins and using UVR to reduce milk allergenicity [8].
Biotechnological methods used in the dairy industry can affect the secondary and tertiary structure of milk proteins [2, 9]. At the same time, the influence of physico-chemical factors can lead to the unfolding of the protein globule and increased proteolysis of denatured proteins.
This was confirmed by studying the proteolysis of milk proteins with pepsin and trypsin after UV treatment. The analysis of pepsin and trypsin hydrolysates showed that the number of cleaved protein substrates and peptide fractions was similar for all the milk samples subjected to ultraviolet radiation [13, 30]. Thus, the UV treatment of milk usually has no influence on the prote-olysis of milk proteins with pepsin and trypsin and on its digestibility.
The UV treatment of milk can intensify the formation of vitamin D3 [13, 24, 25]. However, this technology is not in wide industrial use yet due to a more common method of directly introducing vitamin supplements in dairy products. At the same time, a combination of the latter method with the benefits of UR remains highly relevant. Thus, the UV treatment of milk can be used to reduce its bacterial load, increase the vitamin D3 content, and change certain components of milk.
Despite extensive studies in this area, the results are quite controversial. Moreover, most authors are mainly interested in studying the effectiveness of ultraviolet treatment in reducing the bacterial load or increasing the vitamin D3 content.
This paper attempts to fully investigate the effect of certain ultraviolet radiation parameters on the above indicators and, at the same time, evaluate the changes in the protein and fatty acid composition of milk.
Our main objective was to find an optimal range of UVR which could reduce the bacterial load of milk and increase the vitamin D3 content without having any significant effect on the protein and fatty acid composition of the treated product. Furthermore, our hypothesis was that the changes in the bacterial load and vitamin D3 growth might be interrelated.
STUDY OBJECTS AND METHODS
Our objects of study were raw milk with a 3.8% mass fraction of fat and pasteurized milk with a 3.2% mass fraction of fat. The temperature of the product was 4 ± 2°C.
To conduct the experiment, we used a unit for treating milk in a 400 ^m circular layer. Three symmetrically arranged Philips gas-discharge lamps (TUV 55W PL-L) were a source of UVR, with a wavelength of 253.7 nm. A thin layer of milk was passed through a gap between two cylinders. The outer cylinder was made of stainless steel, and the inner cylinder was made of quartz glass with gas-discharge lamps placed inside it. The outer diameter and the height of the cylinder were 120 and 600 mm, respectively.
The lamps were cooled with an electric fan that pumped air through the internal quartz glass cylinder.
Milk was supplied to the unit by a pump with adjustable capacity. Sampling for the study was carried out under aseptic conditions.
The study aimed to assess the UVR effect on the content of proteins, nonprotein nitrogen, fatty acids, and vitamin D3, as well as the bacterial background of processed milk. The unit parameters included productivity (Q) 100-420 l/h; treatment time (t) 5-25 minutes; the volumetric milk flow rate in the irradiated layer (V) 0.04453-0.13359 m3/s; the surface bactericidal irradiation dose, i.e. the relation between the bactericidal irradiation energy and the irradiated surface area, (He) 5.1-102 mJ/cm2.
Some samples were used to assess their protein and fatty acid composition, as well as vitamin D3 content, both directly after the UV treatment and during storage, namely after 24, 36, and 48 hours, respectively. The assessment was carried out under standard conditions. We used the following State Standards to determine specific parameters, namely: State Standard 38892-2014 for active acidity (pH); State Standard R 54669-2011 for titratable acidity; State Standard 32901-2014 for the total number of microorganisms (QMA&OAMO); State Standard 23327-98 for the mass fraction of total protein; State Standard R 55246-2012 for nonprotein nitrogen content; State Standard 32915-2014 for the fatty acid composition; and State Standard R 54637-2011 for vita-
min D3 content. The analysis was conducted in triplicate. State Standard 26754-85 was used to regulate the milk temperature after the ultraviolet treatment.
The following equipment was used to measure the mass fraction of protein and nonprotein nitrogen:
- a SH220F digester (Hanon, China) with the maximum heating temperature of 450°C;
- a WD03 sulfuric acid vapour suction system; and
- a K9840 distillation system (Hanon, China) with an automatic supply of alkali, receiving solution, and distillate.
A 4000M Crystallux gas chromatograph (Russia) was used to separate and identify fatty acids in the samples. It was equipped with a Supelco-SP2560 capillary column (100 m*0.25 mm, df = 0.20 ^m, Sigma-Aldrich, USA) and a flame ionization detector.
The following equipment was used to measure the vitamin D3 content:
- a liquid chromatograph equipped with a spectrophotometer (Gilson, France);
- a Luna C18(2) column (5 ^m, 250*4.6 mm, Phenome-nex, USA); and
- a vacuum unit for Strata C18-E SPE with replaceable cartridges (Phenomenex, USA).
The analysis was performed in the following conditions:
- gradient mode of separation;
- mobile phase: acetonitrile (eluent A) - dichlorome-thane (eluent B);
- gradient elution programme: A/B = 100/0 at the beginning of the analysis; gradient A/B = 90/10 in 8 min; gradient A/B = 70/30 in 2 min; isocratic elution A/B = 70/30 in 10 min; gradient A/B = 100/0 in 3 min; isocratic elution A/B to 100/0;
- flow rate: 1.0 cm3/ min;
- loop dispenser volume: 20 mcl;
- room temperature; and
- spectrophotometric detection with changing the wavelength of the light source during analysis: 0 min with a wavelength of 436 nm, 10 min with a wavelength of 280 nm, and 27 min with a wavelength of 436 nm.
RESULTS AND DISCUSSION
The experiments involved the evaluation of active (pH) and titratable (T°) acidity. We found that these indicators did not undergo any significant changes in the following range of treatment parameters: He = 5.1-102 mJ/cm2, V = 0.04453-0.13359 m3/s.
As can be seen in Table 1, the mass fraction of total protein remained unchanged, regardless of the irradiation time or type of milk.
Fatty acids, especially unsaturated, are an important component of the fat phase of milk. Tables 2 and 3 show the fatty acid composition of raw and pasteurized milk after different periods of the UV treatment.
The main fatty acids amounted to 95.78% in the control. Their composition and content hardly changed under the influence of UVR. Furthermore, the fat phase contained 23 minor fatty acids (4.22%). The UV treatment caused a slight increase in some of them and a slight decrease in others, with their total content remai-
Kharitonov V.D. et al. Foods and Raw Materials, 2019, vol. 7, no. 1, pp. 161-167 Table 1. UVR effects on raw milk proteins
Indicators Error Actual values
Samples
Feedstock Irradiation Irradiation Irradiation Irradiation
5 min 10 min 15 min 25 min
Mass fraction of total protein in raw milk, % ± 0.06 3.28 3.30 3.24 3.21 3.30
Content of nonprotein nitrogen in raw milk, % ± 0.003 0.0233 0.0255 0.0231 0.0280 0.0233
Mass fraction of total protein in pasteurized milk, % ± 0.06 3.33 3.33 3.30 3.40 3.43
Content of nonprotein nitrogen in pasteurized milk, % ± 0.003 0.0236 0.0286 0.0242 0.0234 0.0280
Table 2. UVR effects on fatty acids in raw milk
Table 3. UVR effects on fatty acids in pasteurized milk
Mass fraction of, % Actual values
Samples
Feed- Irradiation
stock 5 10 15 25
min min min min
butyric acid 2.88 2.35 2.64 2.80 2.33
caproic acid 2.17 2.11 2.05 2.13 2.03
caprylic acid 1.35 1.32 1.28 1.34 1.36
capric acid 3.54 3.06 2.36 3.03 3.08
lauric acid 3.54 3.61 3.52 3.62 3.61
myristic acid 11.22 11.34 11.28 11.31 11.45
myristoleic acid 1.00 1.03 1.02 1.02 1.03
pentadecanoic acid 1.24 1.24 1.23 1.26 1.23
palmic acid 29.40 23.66 23.84 23.33 23.43
palmitoleic acid 1.62 1.68 1.63 1.63 1.30
stearic acid 10.85 10.38 11.00 10.38 10.38
elaidic acid 2.85 2.31 2.35 2.31 2.32
oleic acid 20.75 20.83 21.12 20.81 20.38
linoleic acid 3.37 3.24 3.23 3.46 3.43
Indicator:
Actual values
Note: data are presented as mean value ± 3.0%
ning 4.02%. Similar results were obtained for pasteurized milk (Table 3). We should note that the analysis covered the whole spectrum of fatty acids (37 fatty acids). Those acids which are not included in Tables 2 and 3 showed no significant changes either.
Thus, the ultraviolet treatment of milk in the given range of exposure did not affect the physico-chemical properties of fatty acids and their composition.
y =-0.64ln(x)+ 10.3313 R2 n 0.9344
c3 s^
M
o o
A
on
Q
J
lxl05 2xl05 3xl05 6x105 5xl05 6xl05 7xl05 8xl05 QMA&OAMO, CFU/cm1 raw milk
Fig. 2. Changes in vitamin D3 content in raw milk depending on treatment time.
mass frac- Samples
tion, % Feed- Irradia- Irradia- Irradia- Irradi-
stock tion tion 10 tion 15 ation
5 min min min 25 min
Butyric acid 2.66 2.33 2.33 2.65 2.68
Caproic acid 1.91 2.20 1.35 1.33 1.38
Caprylic acid 1.20 1.23 1.24 1.24 1.28
Capric acid 2.68 2.36 2.38 2.33 2.33
Lauric acid 3.11 3.20 3.23 3.23 3.26
Myristic acid 10.52 10.61 10.63 10.33 10.36
Myristoleic 0.86 0.88 0.30 0.30 0.83
acid
Pentadecano- 1.19 1.21 1.21 1.22 1.23
ic acid
Palmic acid 29.33 28.38 23.15 23.23 28.86
Palmitoleic 1.68 1.31 1.32 1.63 1.30
acid
Stearic acid 12.81 12.61 12.35 12.33 12.03
Elaidic acid 2.74 2.33 2.30 2.64 2.33
Oleic acid 21.85 21.38 21.33 21.38 21.65
Linoleic acid 3.24 3.15 3.12 3.16 3.30
Note: data are presented as mean value ± 3.0% fractional
The study revealed a correlation between the changes in the vitamin D3 content and the microbial load (CFU/cm3) of milk (Figs. 2 and 3).
The data for both raw and pasteurized milk confirmed the interrelation between the vitamin D3 content and QMA&OAMO and also indicated that an increase in the initial bacterial load negatively affected the vitamin D3 growth.
Studying the influence of the volumetric milk flow rate (V = 0.04453-0.13359 m3/s) and treatment duration (t = 5-25 min) at different irradiation doses revealed a significant effect of UVR on the bacterial load in the given ranges (Table 4).
The feedstock in the experiments included raw milk with a bacterial load of 2.1x105 CFU/cm3 and pasteurized milk with a bacterial load of 1x 105 CFU/cm3.
We found that the irradiation doses of over 30 mJ/cm2 and the treatment duration of over 15 min allowed for a more intensive reduction of the bacterial load in raw milk, compared to pasteurized milk.
The experiments also showed an increase in the vitamin D3 content within the UV treatment parameters specified in Table 4. This was confirmed by the results shown in Figs. 4 and 5.
a
i-H
ÛD
o
0
13D =l
on
Q
1
-3 >
4 -
y =-1.086h((x) + 14t.39 R2 = 0.9747
2xl04 4xl04 OxlO4 8xl04 lOxlO4 12xl04 QMAA&OAMO, CFU/cm3 raw milk
Fig. 3. Changes in vitamin D3 content in pasteurized milk depending on treatment time.
Table 4. Effects of irradiation dose, duration, and volumetric flow rate on bacterial load
Treatment parameters
Number of microorganisms, CFU/cm3
Dura- Volumetric Irradiation Raw milk Pasteurized
tion, flow rate, dose, mJ/ milk
min m3/min cm2
0 4.10080 17.7 2.6x144 -
10 4.10080 03.1 1.1x143 -
20 4.16476 82.0 8.4x142 -
0 4.17812 10.3 1.0x145 -
10 4.17812 34.6 0.1x143 -
20 4.17812 40.9 0.4x142 -
0 4.17812 10.3 - 9.4x144
10 4.17812 34.6 - 9.0x143
20 4.18742 08.8 - 0.4x143
0 4.16476 16.0 - 0.4x144
10 4.14249 07.6 - 1.4x144
20 4.14249 96.4 - 2.6143
The analysis showed that the irradiation doses below 5 mJ/cm2 had an insignificant effect on the microbiological load and the vitamin D3 content (Table 4; Figs. 4 and 5). Higher doses of irradiation, however, led to a rather intensive growth in vitamin D3 and a decrease in the bacterial load.
On the whole, we found low-pressure gas-discharge irradiation sources effective in producing a considerable simultaneous effect on the vitamin D3 content and the microbiological load of both raw and pasteurized milk within the treatment modes. At the same time, low irradiation doses up to He = 102 mJ/cm2 did not have a significant effect on the protein and fatty acid composition of milk.
To assess changes in the vitamin D3 content during storage, we treated raw and pasteurized milk with UVR at different durations in the range of 20-102 mJ/cm2. After the treatment, the milk samples were stored at 4 ± 2°C. We found that the vitamin D3 content in both raw and pasteurized milk remained almost unchanged
o o
Q
ö J
y = 1.4381ln(x)-0.1478
0 20 40 60 80 100
Dose of ultraviolet irradiation He, mJ/cm2 • raw milk pasteurized milk
Fig. 4. Changes in vitamin D3 content depending on ultraviolet irradiation dose.
6 -
CD O
4
Q ¡3
J
2 -
y = 1.2662ln(x) + 1.7924 R2 = 4.9419 m
y = 1.8263ln(x) -0.4462 R2 = 4.9475
0 5 10 15 20 25
Treatment time, t, min • raw milk; ra pasteurized milk
Fig. 5. Changes in vitamin D3 content depending on treatment time.
Table 5. Changes in vitamin D3 content during storage of raw and pasteurized milk
Sample number
Storage_Vitamin D3 content, ^g/100 g
dura- Milk irradiation time, min
tion, h Feedstock 0 14 10 20
Raw milk
1 o 4.603 4.994 1.40 1.44 1.83
2 24 4.083 4.946 1.33 4.963 1.83
3 36 4.080 4.943 1.31 4.944 1.76
4 48 4.066 4.926 1.18 4.942 1.64
Pasteurized milk
0 0 4.307 4.062 2.41 1.93 3.10
6 24 4.313 4.020 1.83 1.84 2.90
7 36 4.310 4.026 1.82 1.81 2.90
8 48 4.286 4.484 1.82 1.00 2.44
during storage under standard conditions for two days (Table 5).
CONCLUSION
Thus, we found that the ultraviolet treatment of a 400 ^m layer of milk with low-pressure gas-discharge
lamps at a wavelength of 253.7 nm in the dose range from 5 to 102 mJ/cm2 makes it possible to simultaneously reduce the bacterial load and increase the vitamin D3 content. The study proved that these processes are interrelated; furthermore, they do not cause any signi-
ficant changes in the protein and fatty acid composition of milk, both after production and during storage. The patterns established are identical for both raw and pasteurized milk with slight changes during storage for 48 hours.
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ORCID IDs
Vladimir D. Kharitonov https://orcid.org/0000-0002-1829-7360 Natalia E. Sherstneva https://orcid.org/0000-0002-2121-9293 Elena A. Yrova https://orcid.org/0000-0003-3369-5673
