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Ukrainian Journal of Ecology
Ukrainian Journal of Ecology, 2020, 10(3), 83-87, doi: 10.15421/2020_137
ORIGINAL ARTICLE
Enhanced cultivation technology for lacto- and bifidobacteria
A.P. Paliy1*, S.A. Gujvinska1, K.O. Rodionova2, N.V. Alekseeva3, O.V. Ponomarenko4, M.S. Alrawashdeh5, T.A. Yeletskaya6, G.V. Ponomarenko4, V.Yu. Kushnir7, A.P. Palii8
1 National Scientific Center Institute of Experimental and Clinical Veterinary Medicine, 83 Pushkinska
St, Kharkiv, 61023, Ukraine 2Luhansk National Agrarian University, 68, Slobojanska Str, Starobilsk, 92703, Ukraine 3Dnipro State Agrarian and Economic University, 25 Sergii Yefremov St, Dnipro, 49600, Ukraine 4Kharkiv State Zooveterinary Academy, 1 Akademicheskaya St, urban settlement Malaya Danilivka,
Dergachivsky district, Kharkov region, 62341, Ukraine 5University Mutah, P.O Box. 7, AL Karak, 61710, Jordan 6Institute of Animals Science of the National Academy of Agricultural Sciences, 1A Givotnovodov St, Kharkiv,
61026, Ukraine
7Odessa State Agrarian University, 13 Panteleimonovskaya St, Odessa, 65012, Ukraine 8Kharkiv National Technical University of Agriculture named after Petro Vasylenko, 44 Alchevskih St, Kharkiv, 61002, Ukraine
*E-mail: paliy.dok@gmail.com
Received: 04.06.2020. Accepted: 12.07.2020
The aim of this work was to improve a cultivation technology of lactobacteria and bifidobacteria based on the optimal growth substrate, which ensures a preservation of basic biological properties of microorganisms. Seven variants of experimental nutrient media were prepared for cultivation of lactic acid bacteria. The growth properties of experimental nutrient media were studied using following strains: Lactobacillus plantarum 7, L. casei 27, L. plantarum 7-317, Bffidobacterium adolescents 17 and B. adolescents 17-316. The number of living microbial cells was determined by serial dilution methods. The composition of nutrient media for cultivation of lactobacteria, bifidobacteria and technological parameters of their cultivation were determined by the temperature of 37 ± 0.5°C. Acidity of a nutrient medium has pH value ranges from 6.5 to 7.5. A nutrient medium for cultivation of lactobacteria is proposed. Composition of new nutrient medium: lactose - 10.0-15.0%, sucrose - 5.0-10.0%, cystine - 0.01%, skim milk - up to 100.0%. It was established that the optimal nutrient medium for bifidobacteria accumulation composed of the following components (ratio of components, mass %): peptone - 7.0 ± 0.01, yeast autolysate - 4.0 ± 0.01, glucose - 1,5 ± 0.02, sucrose - 1.5 ± 0.01, cystine - 0.03 ± 0.01, ammonium citrate - 0.4 ± 0.03, potassium phosphate monosubstituted - 0.4 ± 0.02, MgSO4 - 0.04 ± 0.002, MnSO4 - 0.007 ± 0.002, sodium phosphate disubstituted - 0.4 ± 0.02, microbiological agar - 2.5 ± 0.04, sodium citrate - 0.8 ± 0.05, distilled water - up to 100%.
Key words: Cultivation technology; Animals; Biological studies
Introduction
Livestock farms implement drugs with probiotic cultures in schemes of prevention and treatment of animals within connection with the increasing needs of population in high-quality and safe food products of animal origin. Today, lactic acid cultures are essential components for the production of probiotics, as their positive effects have been repeatedly proven (Bermudez-Brito et al., 2012; Yirga, 2015). Probiotics have a positive effect on intestinal flora of animals (Markowiak & Slizewska, 2018). It also reduces a risk of gastrointestinal disease in animals and increases their productivity (Chaucheyras-Durand & Durand, 2010). This statement refers to antibiotics, which are used for therapeutic purposes. An antibiotic resistance of many microorganisms is found to antibiotics (Hossain et al., 2017; Hadzevych et al., 2019). Biotechnological advancement has made it possible to create a complex probiotic additives consisting of several different strains and types of microorganisms (Zhu et al., 2018). This trend becomes very promising, since these strains complement each other and exhibit a more effective synergistic, prophylactic and metabolic effect in comparison with monotherapy (Gadde et al., 2017). Current requirements of the European regulatory legislation in the field of probiotics provide the need for comprehensive studies of biological activity of individual probiotic cultures and their combinations. These studies has to be implemented for creating probiotic additives based on monocultures of lactobacteria and bifidobacteria or their various combinations (Alayande et al., 2020). Study of biological properties of lactobacteria and bifidobacteria, as well as other microorganisms, requires an appropriate skill in long-term storage and cultivation of cultures (Gujvinska et al., 2018). This is necessary both for a maintaining collections of lactic acid bacteria in a highly active state and for a manufacture and storage of probiotic drugs (Herawati et al., 2020). Conducted biological studies have allowed to isolate a significant spectrum of bacteria, such as: Lactobacillus spp., Bffidobacterium spp. and Lactococcus spp. These isolates can be used in the development of probiotic drugs for farm animals (Chiang et al., 2015; Gujvinska & Paliy, 2018b; Paliy et al., 2020). Great attention in the biotechnological process of creating therapeutic and prophylactic probiotics is given to two main factors. These factors are an achievement of a maximum level of biomass yield of viable bacterial cells and a biologically active substances synthesized by them (Parvez et al., 2006). It is important to consider adaptability under production conditions and stability of strains during cultivation, as well as taking into
account its probiotic properties preservation during selection (Ozen & Dinleyici, 2015). These indicators determine the productivity, competitiveness and profitability of technological process (Gujvinska & Paliy, 2018a).
Lactic acid bacteria and bifidobacteria are introduced into the composition of probiotics, dietary supplements and functional foods. They are widely used in the prevention and treatment of many animal dysfunctions (Patel et al., 2012; Celiberto et al., 2017). In recent years, attention is paid to the use of lactic acid bacteria growth promoters for increase the effectiveness of probiotics (Uzunova-Doneva & Donev, 2005). Separate data are presented regarding the effect of lactulose, inulin and aerosil on the growth of lactobacteria. However, there is no evidence about an effect of phytocompositions on the biological properties of lactic acid bacteria. A study of these issues is relevant because it reveals an another page of biotechnology of an important group of microorganisms known as lactobacteria and bifidobacteria (Ben Salah et al., 2012; Chiu et al., 2014; Wang et al., 2015).
These circumstances call for finding a new composition of nutrient medium based on components from domestic raw materials. This will reduce a cost of the nutrient medium and increase its elective properties, accelerate the growth of lactobacilli and bifidobacteria in general, and the accumulation of their bacterial mass in particular.
The aim of this work was to improve a cultivation technology of lactobacteria and bifidobacteria based on the optimal growth substrate.
Materials and Methods
A work for improvement of cultivation technology of lactobacteria and bifidobacteria based on optimal growth substrate was performed in the Laboratory of Veterinary Sanitation and Parasitology of the National Scientific Center "Institute of Experimental and Clinical Veterinary Medicine". Seven variants of experimental nutrient media were prepared for cultivation of lactobacteria and bifidobacteria for the research study. Lactose, sucrose, cystine, and skim milk were used to design the medium for lactobacteria. Peptone, yeast autolysate, glucose, sucrose, cystine, ammonium citrate, potassium phosphate monosubstituted, magnesium sulfate, manganese sulfate, sodium phosphate disubstituted, sodium citrate, microbiological agar, distilled water were used in a medium for cultivation of bifidobacteria. The MRS medium was used as a control of growth properties of probiotic cultures (Nwamaioha & Ibrahim, 2018).
Selection and optimization of nutrient media were carried out according to the following criteria: pH of a medium, temperature, a number of microbial cells after incubation (Yang et al., 2018). Cultures of lactobacteria (Lactobacillus plantarum 7, L. casei 27, L p/antarum 7-317) and bifidobacteria (Bifidobacterium adolescentis 17, BB. adolescentis 17-316) have been a subject of the research study. The abovementioned cultures are isolated, selected and stored in the Laboratory of Veterinary Sanitation and Parasitology of the NSC "IECVM". The number of live microbial cells assessed the growth properties of bifidobacteria. The number of live microbial cells was determined by the method of serial dilutions of a resulting suspension in saline. Subsequently, bacterial cultures were inoculated on nutrient media in a volume 0.1 cm3 of the 10-6 dilution (Süle et al., 2014; Senz et al., 2015). The optimal conditions (pH, temperature) of cultivation of lactic acid bacteria were determined (Kneifel, 2000).
All studies were performed in triplicate. Statistical processing of the results was carried out using traditional methods of variation statistics using the "Microsoft Excel" and "Statistica 10" programs. A significant difference between obtained results was considered at p-value < 0.05.
Results and Discussion
A nutrient media with various sources of carbon and nitrogen were used for cultivation microorganisms. There are MRS control medium (glucose is a carbon source; enzyme peptone, meat broth, ammonium citrate are nitrogen sources) and seven experimental nutrient media for cultivation of lactobacilli (n=4) and bifidobacteria (n=3). These nutrient media differed by the type and concentration of carbon and nitrogen sources and mineral components.
A comparative analysis of well-known nutrient media for the cultivation of lactobacilli and bifidobacteria shows that successful cultivation depends on a quality and composition of a medium due to a type of protein base as well as a specificity of growth components (Sánchez et al., 2019). Thus, many researchers used glucose, lactose, and sucrose as carbohydrate sources to prepare nutrient media. In addition to this, various microelements significantly affect a development of lactic acid bacteria that are present in the environment. In this regard, sodium chloride, sodium acetate, disubstituted ammonium citrate, sodium citrate, sources of manganese, magnesium, phosphorus, iron and other elements are used (Colombo et al., 2014). This fact requires a careful and focused approach in the selection process of raw ingredients for nutrient media preparation (Hayek et al., 2019). Therefore, a nutrient medium for cultivation of lactobacilli was enriched by a various ratios of lactose, sucrose and cystine with addition of skimmed milk up to 100% (Table 1).
Table 1. Composition of nutrient medium for lactobacteria cultivation.
No Components Nutrient Media
No. 1 No. 2 No. 3
1 Lactose, g - 5.0 ± 0.01 10.0 ± 0.02
2 Sucrose, g - 15.0 ± 0.02 10.0 ± 0.01
3 Cystine, g 0.01 ± 0.005 0.01 ± 0.005 0.01 ± 0.005
4 Skimmed milk, cm3 up to 100 up to 100 up to 100
Bifidobacteria are very demanding on composition of nutrient media, especially on easily digestible nitrogenous compounds, amino acids, carbohydrates, unsaturated fatty acids, vitamins and mineral elements (Roy, 2001). The components were selected for the production of a nutrient medium for bifidobacteria cultivation in the following ratio (mass %): glucose - 1.0-1.5, lactose - 1.0-1.5, cystine - 0.01-0.03, ammonium citrate - 0.2-0.4; potassium phosphate monosubstituted - 0.2-0.4, magnesium sulfate heptahydrate - 0.02-0.04, manganese sulfate tetrahydrate - 0.005-0.007, sodium phosphate bisubstituted - 0.2-0.4, microbiological agar - 0.22.5, sodium citrate - 0.5-0.8, distilled water - the rest. Distilled water was added up to 100% after dissolving the ingredients and then boiled. The pH was adjusted to 6.2-6.6, and the nutrient medium was filtered through a paper filter. The medium was sterilized and tested for sterility by keeping in a thermostat at a temperature of 37°C for 3-5 days (Table 2).
Table 2. Composition of nutrient medium for bifidobacteria cultivation.
No Components Nutrient Media
No. 1 No. 2
1 Peptone, g 5.0 ± 0.01 6.0 ± 0.02
2 Yeast autolysate, g 2.0 ± 0.03 3.0 ± 0.01
3 Glucose, g 1.0 ± 0.01 1.2 ± 0.01
4 Sucrose, g 1.0 ± 0.02 1.2 ± 0.03
5 Cystine, g 0.01 ± 0.01 0.02 ± 0.01
6 Ammonium Citrate, g 0.2 ± 0.01 0.3 ± 0.02
7 Potassium phosphate monosubstituted, g 0.2 ± 0.03 0.3 ± 0.02
8 Magnesium sulfate, g 0.02 ± 0.001 0.03 ± 0.002
9 Manganese sulfate, g 0.005 ± 0.001 0.006 ± 0.003
10 Sodium phosphate disubstituted, g 0.2 ± 0.01 0.3 ± 0.01
11 Microbiological agar, g 0.2 ± 0.02 2.0 ± 0.01
12 Sodium Citrate, g 0.5 ± 0.01 0.7 ± 0.04
13 Distilled water, cm3 up to 100 up to 100
Thus, as a result of the studies the components of nutrient media were determined. They are sources of nitrogen, vitamins, carbohydrates, minerals, and the protein component for lactic acid bacteria.
The study of the growth properties of the nutrient medium for lactobacilli was the next stage of our work. Moreover, studies have shown that all samples of experimental nutrient media for cultivation of lactobacilli provided a higher level of accumulation of Lactobacillus spp. compared to the control nutrient medium (Table 3).
Table 3. Growth properties of culture media for the cultivation of Lactobacillus spp. (here and then n=3).
Lactobacillus strain
Lactobacillus plantarum 7
Lactobacillus casei 27
Lactobacillus plantarum 7-317
Nutrient medium Growth, CFU/cm3
No. 1 7.1 ± 0.23 x 107
No. 2 6.7 ± 0.23 x 108*
No. 3 8.8 ± 0.19 x 108*
No. 4 8.9 ± 0.17 x 1010*
control 6.3 ± 0.21 x 107
No. 1 7.3 ± 0.20 x 107*
No. 2 8.9 ± 0.24 x 107*
No. 3 10.7 ± 0.21 x 108*
No. 4 10.8 ± 0.19 x 109*
control 7.5 ± 0.23 x 106
No. 1 7.1 ± 0.19 x 107*
No. 2 7.7 ± 0.24 x 108*
No. 3 8.7 ± 0.18 x 108*
No. 4 9.4 ± 0.22 x 109*
control 6.9 ± 0.21 x 106
* р<0.05 compared to control
The most intensive growth of Lactobacillus spp. was observed on experimental nutrient media No. 3 and No. 4. Thus, the growth rate of Lactobacillus plantarum 7 was 8.8 ± 0.19 x 108 CFU/cm3 on experimental nutrient medium No. 3 and 8.9 ± 0.17 x 1010 on medium No. 4. This index was 6.3 ± 0.21 x 107 CFU/cm3 on a control nutrient medium. A Lactobaclllus casei 27culture also grew better on experimental nutrient media No. 3 and No. 4. A growth rate was 10.7 ± 0.21 x 108 CFU/cm3 and 10.8 ± 0.19 x 109 CFU/cm3 on listed media (7.5 ± 0.23 x 106 CFU/cm3 on control medium). A Lactobacillus plantarum 7-317 bacteria grew on developed nutrient media No. 3 and No. 4. Active accumulation of a significant number of viable cells was observed within the range of 8.7 ± 0.18 x 108 CFU/cm3 and 9.4 ± 0.22 x 109 CFU/cm3 respectively (6.9 ± 0.21 x 106 CFU/cm3 on control medium). It should be noted that experimental nutrient medium No. 4 is the best option for cultivation of L paantarum 7, L casei 27, L plantarum 7-317lactobacilli cultures compared to other experimental and control media. It should be noted that experimental nutrient medium No. 4 is the best option for cultivation of L. plantarum 7, L. casei 27, L. plantarum 7-317 lactobacilli cultures compared to other experimental and control media.
The next stage of our work was to test the growth properties of the developed media for bifidobacteria Bifidobacterium adolescent's 17and B. adolescentis 17-316 (Table 4).
Table 4. Growth properties of culture media for the cultivation of Bifidobacterium spp.
Nutrient medium Growth, CFU/cm3
Bifidobacterium adolescentis 17 Bifidobacterium adolescentis 17-316
No. 1 4.5 ± 0.22 x 106 5.5 ± 0.19 x 106
No. 2 5.7 ± 0.27 x 107* 5.7 ± 0.27 x 107*
No. 3 6.4 ± 0.23 x 108* 5.9 ± 0.23 x 108*
control 4.1 ± 0.19 x 106 5.2 ± 0.17 x 106
Studies have shown that nutrient medium No. 3 is optimal for the cultivation of microorganism B. adolescentis 17 and B. adolescentis 17-316. It provides a high yield of a bacterial mass of these bifidobacteria in the range of 6.4 ± 0.23 x 108 and 5.9
± 0.23 x 108 respectively. The next step in our work was to determine the optimal conditions for the cultivation of lactobacteria and bifidobacteria. The conditions under which microorganisms are cultivated have played a decisive role in their biological activity manifestation, including those used in the composition of probiotic preparations (Ram & Chander, 2003). The acidity and alkalinity of nutrient media are the most important properties of these media that influences on the life processes of microorganisms. It is expressed as the negative logarithm of hydrogen ions concentration (pH). The pH value influences on a dissociation of acids in the nutrient medium, solubility of nutrients and their transport into the cell, activity of enzymes etc. Thus, this factor determines the possibility of the growth of microorganisms on a nutrient medium that is optimal by its composition (Waddington et al., 2010). We have studied a growth of L plantarum 7, L casei 27, L plantarum 7-317, BB. adolescentis 17and BB. adolescentis 17-316strains at various pH and temperature. It was found that there is an inverse correlation between the pH value of nutrient medium and the number of lactic acid bacteria that grow on it. A high pH value accompanied by the presence of a small number of these bacteria (Table 5).
Table 5. Effect of nutrient medium Ph on the growth of lactic acid bacteria.
pH Growth, x 106 CFU/cm3
Control Experiment
5.5 13.7 ± 0.15 14.1 ± 0.14
6.0 14.4 ± 1.16 14.5 ± 1.10
6.5 23.2 ± 1.70 24.0 ± 1.16
7.0 24.3 ± 1.30 25.0 ± 1.14"
7.5 22.5 ± 1.10 23.0 ± 1.01*
8.5 10.1 ± 0.50 12.0 ± 4.10
As a result of statistical analysis, the most intensive growth of microorganisms was observed on nutrient medium at pH 6.5-7.5. Thus, the maximum accumulation of probiotic cultures was observed at pH 7.0 (control - 24.3 ± 1.30 x 106 CFU/cm3; experiment -25.0 ± 1.14 x 106 CFU/cm3), and the number of microbial cells was 14.1 ± 0.14 x 106 CFU/cm3 and 14.5 ± 1.10 x 106 CFU/cm3 at pH 5.5-6.0 respectively. The lowest concentration of microorganism 12.0 ± 4.10 x 106 CFU / cm3 was observed at pH 8.5. We also studied the optimal temperature conditions for the cultivation of lactic acid bacteria.
The cultivation of lactic acid bacteria was carried out at temperatures from 30°C to 50°C (Table 6). Studies have shown that the best growth of lactic acid bacteria was at a temperature of 37.0-40.0 ± 0.5°C. Growth of cultures was low and ranged from 9.00 ± 0.87 to 1.20 ± 0.05 x 106 CFU/cm3 at a temperature of 30.0, 45.0 and 50.0 ± 0.5°C. Consequently, an optimal pH value is 7.0, and incubation temperature of an inoculum is 37.0 ± 0.5°C at different cultivation modes of lactic acid bacteria. The results of these studies make it possible to improve the cultivation technology lactobacilli and bifidobacteria that based on the optimal growth substrate. This will allow to apply the obtained results for a manufacture of innovative probiotic preparations.
Table 6. Effect of temperature on the growth of lactic acid bacteria.
Temperature,°C Control Growth, x 106 CFU/cm3 Experiment
30.0 ± 0.5 14.5 ± 1.77 6.25 ± 0.15"
37.0 ± 0.5 24.7 ± 0.36 25.00 ± 0.34"
40.0 ± 0.5 23.5 ± 0.92 24.00 ± 0.87"
45.0 ± 0.5 8.05 ± 0.92 9.00 ± 0.87
50.0 ± 0.5 9.05 ± 0.06 1.20 ± 0.05
Conclusion
A nutrient medium for the cultivation of lactobacilli is proposed. A new nutrient medium has the following composition: lactose -10.0-15.0%, sucrose - 5.0-10.0%, cystine - 0.01%, skim milk - up to 100.0%. The nutrient medium optimal for the accumulation of bifidobacteria had the following composition of components (mass %): peptone - 7.0 ± 0.01, yeast autolysate - 4.0 ± 0.01, glucose - 1.5 ± 0.02, sucrose - 1.5 ± 0.01, cystine - 0.03 ± 0.01, ammonium citrate - 0.4 ± 0.03, potassium phosphate monosubstituted - 0.4 ± 0.02, magnesium sulfate - 0.04 ± 0.002, manganese sulfate - 0.007 ± 0.002, sodium phosphate disubstituted - 0.4 ± 0.02, microbiological agar - 2.5 ± 0.04, sodium citrate - 0.8 ± 0.05, and distilled water - up to 100%. We determined that the optimal temperature for cultivation of lactic acid bacteria is 37.0 ± 0.5°C, and the pH value of the nutrient medium is 6.5-7.5.
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Citation:
Paliy, A.P., Gujvinska, S.A., Rodionova, K.O., Alekseeva, N.V., Ponomarenko, O.V., Alrawashdeh, M.S., Yeletskaya, T.A., Ponomarenko, G.V., Kushnir, V.Yu., Palii, A.P. (2020). Enhanced cultivation technology for lacto- and bifidobacteria. Ukrainian Journal of Ecology, 10(3), 83-87.
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