Relevance of application of irradiated starter cultures to production of fermented milk products 242 D O I: 1 0. 15 82 6/ ch im te ch .2 02 0. 7. 4. 19 Zhuravleva D. A., Kazakov A. V., Selezneva I. S., Baranova A. A. Chimica Techno Acta. 2020. Vol. 7, no. 4. P. 242–249. ISSN 2409–5613 Relevance of application of irradiated starter cultures to production of fermented milk products D. A. Zhuravlevaa, A. V. Kazakovb*, I. S. Seleznevaa, A. A. Baranovaa a Ural Federal University, 19 Mira St., Yekaterinburg, 620002, Russian Federation b Ural State University of Economics, 62/45 8 Marta St./ Narodnoy voli St., Yekaterinburg, 620144, Russian Federation *email: prof_kazakov@mail.ru Abstract. Nowadays yogurt, fermented milk products, enriched with biologically active substances, acquire increasingly important significance in people’s diets. The tra- ditional method for producing fermented milk products and yogurt is to ferment the milk using starter cultures. The purpose of this research was to figure out if it is possible to use suspensions of probiotic microbial cultures irradiated with ionizing radiation to produce yoghurt products. Liquid live suspensions of bifidobacteria and lactobacilli, as well as a mixture of bifidobacteria, lactobacilli, propionibacteria and lactic acid streptococci were taken as a research model. The goal was achieved by receiving the yoghurt products enriched with active secondary metabolites due to using the suspensions of lysed cells of different microorganisms. We studied physico-chemical and organoleptic properties of the received products after 1, 7 and 14 days of storage. Keywords: fermented milk products; probiotic microorganisms; secondary metabolites; ionizing radiation Received: 10.10.2020. Accepted: 27.12.2020. Published:30.12.2020. © Zhuravleva D. A., Kazakov A. V., Selezneva I. S., Baranova A. A., 2020 Introduction Currently, it is generally accept- ed to use probiotics to produce a variety of  dietary supplements, fermented food products and drinks, and medicaments. It is  enough to  mention that almost all fermented milk products are obtained using lactic acid and probiotic microor- ganisms. All over the world, for decades this approach has been defined as  fully justified in  terms of  preserving and im- proving the health of people and animals. At the same time, most researchers, as well as wide public trained by them, undoubt- edly identify probiotic microorganisms with exceptionally useful ones, which do not cause any side effects and have no con- traindications to their unlimited use [1, 2]. Few people realize that production strains of bifidobacteria, lactobacilli, propionibac- teria and other probiotic microorganisms are selected from specific donors, such as astronauts, or from some environmental objects. It means that biomaterial, which represents just a part and is endowed with specific individual properties, applies to  the  entire community of  animal and human populations without any restric- tions or control. But, after all, it is known 243 that there is  a  huge variety of  races and strains of probiotic microorganisms, and every human or animal has got its unique microbial “landscape” and it is not really a correct measure to artificially plant some- one else’s heterogeneous microflora [3]. On the  other hand, it is  true that the benefits of liquid probiotics have been proven by numerous studies and experi- ments, or at least the apparent harm to hu- man and animal health from them has not been confirmed. The  purpose of  this research was to develop more physiological application of  production strains of  liquid probiotic microorganisms without any even hypo- thetical possibility of replacing their own beneficial microflora inherent in everyone. Experimental Liquid live suspensions of  bifidobac- teria and lactobacilli, as well as a mixture of  bifidobacteria, lactobacilli, propioni- bacteria and lactic acid streptococci were taken as  a  research model. A  positive impact of liquid probiotics on the organ- ism is  diverse. Improvement of  all parts of  the  gastrointestinal and urogenital tracts, skin surface, and mucous mem- branes of the upper respiratory tract; nor- malization of digestive functions; immu- nostimulating and anti-allergic effects — it is not a complete list of health-improving properties of the abovementioned probiot- ics in various age groups of the population, and also of  domestic and farm animals, confirmed by us with a dozen patents for inventions [4]. We have attempted to  convert liquid probiotics into more physiological forms with the elimination of any possible alien influence on the organisms of humans and animals, while increasing the nutritional value, as we have confirmed in our earlier works, and maintaining antimicrobial ac- tivity against opportunistic and pathogenic microflora [5]. This was achieved by lysis live cellular probiotic cultures of microor- ganisms through the irradiation and with subsequent application of  the  obtained cell-free suspensions as  starter cultures to produce sour-milk yogurt-like products. The  processing of  the  probiotic cultures with ionizing radiation was carried out on the  linear electron accelerator model UELR-10-10C2 in  the  Innovative-intro- duction Center of Radiation Sterilization of  the  Physical-Technological Institute (Ural Federal University, Yekaterinburg). There were used liquid probiotics, re- ceived at the production equipment, since in  almost all cases there is  a  statistically significant difference in  the  quality and safety criteria of  any products, received using laboratory and industrial methods. The BR-30 bioreactor (Fig. 1) was used to produce liquid live probiotics. This reac- tor allows carrying out sterilization of ini- tial nutrient hydrolysate-milk medium in  automatic mode (temperatures 106– 108 °С and excess pressure 0.3–0.4 atm for 60 min); cooling till 38–40 °С and holding at this temperature of mixture of nutrient medium with preliminarily prepared liq- uid starter material for 24 hours, based on the following proportions: 3–5% of starter culture to the volume of seeded sterile en- vironment [6]. The following raw materials were used to  prepare unfermented products: cow milk “Irbitsky” 2.5% fat, pasteurized, ho- mogenized, standardized in  the  package volume of  1 l was manufactured by  JSC “Irbitsky milk factory” (Yekaterinburg, Russia) according to  the  Russian State Standard 31450–2013 [7]. It was bought 244 from supermarkets in Yekaterinburg, Rus- sia. The milk ingredients were marked on the package as the following: fat — 2.5%, protein — 3.0%, and carbohydrate — 4.7%. Biologically active additives “Euflorine-L” (liquid Lactobacillus), (liquid Bifidumbac- terin) (Fig. 2a,b); non-alcoholic drink “Eu- florine-plus” (protein hydrolysate — meta- bolic) (Fig. 2c) in 100 ml dark glass bottles were selected as probiotic starter culture for yoghurt products preparation. They were produced by LLC NPC “PRIORITY” (Yekaterinburg, Russia). Weighing was performed on the labora- tory analytic balance AND HR-60 of the I accuracy class. The  рН was measured according to  the  requirements of  State Standard 33776–2016 [8] using electronic рН-meter Kelilong PH-911. The inocula- tion process was carried out under a ster- ile condition of  microbiological laminar flow (II  class, В2 type BМB-II  — “Lam- inar-S-1.2”, Lamsystem Company, Rus- sia) (a thermostable incubator). The milk was fermented in  Yogurt maker Marta MT-1854 MARTA TRADE INC., United Kingdom, equipped with the  timer and thermometer. All experiments and sample analysis were carried out in two parallels. Results and discussion Comparative chemical analysis to de- termine the  qualitative and quantitative spectrum of  amino acids, containing in  whole cell and lysed by  irradiation of the respective microbial cultures, was carried out in the scientific-research labo- ratory of united laboratory complex at Ural State University of Economics using highly efficient liquid chromatograph Agilent 1260 Infinity II (Germany), equipped with multi-wave detector and analytic tube with reversed phase Agilent ZORBAX Eclipse AAA 4.6 * 150 mm 3.5-Micron. Gradient elution with two eluents was used during the research process. We used the phos- phate buffer based on Na2HPO4 with 0.5 М concentration as the first eluent, and a mixture of acetonitrile:methanol:water with 45:45:10 ratio was used as  the  sec- ond eluent. Elution speed was 2 ml/min, and temperature of  the  tube was main- tained at the level of 40 °С. Before start- Fig. 2. Models of starter cultures for fermented milk products: a — Euflorine-L, b — Euflorine-B, c — Euflorine-plus Fig. 1. The BR-30 bioreactor to produce liquid live probiotics 245 ing the measurements, the chromatograph was calibrated using the standard of amino acids produced by  Agilent Technologies company. In the calibration the following non-essential amino acids were used: Ala- nine, Aspartic and Glutamic acids, Ser- ine; conditionally essential  — Arginine, Cysteine, Glycine, Proline, Tyrosine, and essential amino acids, such as Histidine, Threonine, Valine, Methionine, Phenyla- lanine, Isoleucine, Leucine, Lysine. The  performed research showed that in  the  irradiated liquid probiotic micro- bial cultures, the total content of the above- mentioned amino acids, expressed in mg per 100 g of sample, is 1.6–2.2 times higher than in live whole-cell monocultures of bi- fidobacteria, lactobacilli and their mixtures in the 1:1 ratio. In the average values, cel- lular microbial cultures, destroyed by ra- diation, contained 1.8 times more amino acids than the  corresponding live whole cell cultures. Besides, studies were carried out to de- termine the number of live colony-forming microorganisms in  liquid probiotic cul- tures after various modes of their irradia- tion. During this, the NMAFAM parameter was determined (number of  mesophilic aerobic and facultative anaerobic micro- organisms) [9]. When determining this value, we used the scales VMK-622, auto- matic single-channel dispenser BIOHIT, рН-meter “Anion 7000”. It was found that before irradiation the content of probiotic microorganisms in  liquid live whole-cell microbial cul- tures was from 1∙108 to  1∙1010 CFU/cm3. After irradiation with the  dose 10 kGr the NMAFAM parameter was at the level of values from 1.6∙104 to 2.0∙102 CFU/cm3. At  the  same time, colony-forming mi- crobial units in the culture of lactobacilli were not determined at all (the parameter matched the value less than 1.0∙101 CFU/ cm3). At a higher radiation dose of 15 kGr the  NMAFAM parameter in  all studied cultures of microorganisms was less than 1.0∙101 CFU/cm3; it means that the micro- bial cells have been completely lysed in this case. The next stage of our work was to study the possibility of using biologically active additives of brands “Euflorine-B”, “Euflo- rine-L” and “Euflorine-plus”, produced by  LLC NPC “PRIORITY”, as  a  starter in  the  production of  yoghurt products using probiotic cultures lysed by ionizing radiation and their secondary metabolites, as well as living microorganisms. At  first it was identified that the  рН value of  the  original milk was 6.65 ± 0.75%. The milk was pasteurized at 85 °C for 15 min and subsequently cooled to 38– 40 °C (fermentation temperature), then 125 ml of it was poured into clean, numbered 150 ml containers. After that the milk was divided in models inoculated by the dose 15 kGr and non-irradiated models of start- er cultures, with a dose of 100 g/l of milk. Containers with fermented milk and with reference samples were closed with lids, put into a yogurt maker at 40 °С for 8 hours. The  control sample was prepared using the previous method without the addition as a starter culture to the pasteurized milk neither the living culture, nor the suspen- sion of lysed cells of microorganisms, their secondary metabolites. Analysis of  the  appearance, organo- leptic and physical parameters of quality of yoghurt products, made by adding ir- radiated, as well as live cultures of micro- organisms, was performed after 1, 7 and 14 days of storage. While determining the рН of the sam- ples after 8 hours of fermentation, it was established that only in case of milk fer- 246 mentation by  not irradiated Euflorine-L the value lowered to рН = 4.4, the texture of this product was dense, homogeneous (Fig. 3), while the other samples were less dense, and the рН value varied from 4.9 to 5.8. Consequently, the samples were again placed into the yogurt maker for anoth- er 3 h and suddenly cooled to 4 °C after the fermentation time was determined. Af- ter 6-hour cooling, gelation was observed in  all samples (Figs. 4a, 4b, 5a, 5b), al- though the рН values remained at the same level. T h e re f o re , m i l k f e r m e nt at i o n in the samples with not irradiated starters is carried out due to the activity of micro- organisms, as well as the activity of their metabolites; when using sterile starters, fer- mentation process is conditioned by solely the interaction of secondary metabolites and milk components. The control sample during fermenta- tion and the consequent cooling has not changed even for 24  hours, so it can be argued the fermentation due to microor- ganisms contained in milk, without adding the model starters, does not occur. During the  research there were de- termined physical and organoleptic indi- cators of yoghurt product samples qual- ity, received using irradiated starters and mixtures with living microorganisms and during 1, 7 and 14 days of storage (see Ta- ble 1). Determination of sensory indica- tors — appearance and consistency, taste and smell, color of received products was performed according to the requirements of State Standard 31981–2013 [10]. The sensory evaluation of yogurt prod- uct samples was provided by 5 volunteers expert panel members from professors and students in  the  department of  tech- nologies of  organic synthesis according Fig. 3. Yoghurt products with the addition of not irradiated (а) and irradiated (b) Euflorine-L Fig. 4. Fermented products with the addition of not irradiated (а) and irradiated (b) Euflorine-В 247 to scoring scale (0–5) for its appearance (color and syneresis), flavor (aroma and taste), consistency (firmness and texture) and overall acceptability. The samples were served to panelist cold at 4 °C by random order. Samples No. 2 and No. 6 have shown the  best organoleptic parameters during 7 days of storage; after 14 days of storage the taste characteristics of all the samples have been decreased. It was determined that with the increase in the storage period of all the samples, pH value decreased, titratable acidity (from 55 °Т to  145 °Т [11]) and whey separa- tion (syneresis) increased. This is well cor- related with the fact that during storage, the protein-lipid complex is destroyed due to acidification of the medium by the form- ing lactic acid, which results in compac- tion of the yoghurt product structure and separation of the whey [12]. Conclusions In conclusion, it should be noted that during the  performed research live cell probiotic cultures of the microorganisms were irradiated by different doses at the lin- ear electron accelerator. Microbiological analysis showed that microbe cells were completely lysed after radiation by the dose 15 kGr. Fermented products were received using irradiated mixtures instead of tra- ditional starters. It should be also noted Fig. 5. Yoghurt products, made of not irradiated (а) and irradiated (b) Euflorine — plus Table 1 Results of determination of рН, density and sensory indicators of fermented products Samples number Euflorine type рН Density, g/ml Evaluation of sensory indicators (1, 7 days) 1 day 7 days 14 days taste smell appearance 1 Euflorine-В 5.8 5.6 5.2 0.9780 3.0 3.4 3.6 2 Euflorine-В irrad. 4.9 4.7 4.3 0.9820 4.0 4.0 4.4 3 Euflorine-L 4.4 4.0 3.8 0.9860 3.6 3.8 3.8 4 Euflorine-L irrad. 5.3 5.0 4.8 0.9750 3.4 3.4 3.6 5 Euflorine-plus 5.1 4.7 4.6 0.9630 4.0 4.4 3.8 6 Euflorine-plus irrad. 5.4 4.9 4.5 0.9450 3.8 4.0 4.6 248 that irradiated Euflorin-B and Euflorin- plus are preferable for this purpose. They can improve the  texture, viscosity, and the  rheological charteritics of  yogurt ef- fectively, ensure the formation of a denser and more uniform structure and consist- ency of yogurt products. 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