Influence of the use of spore-forming bacteria with probiotic properties on the quality and safety of food

. The paper discusses the prospects for the use of probiotic drugs as an alternative to growth-stimulating antibiotics, which, according to current scientific research, have shown themselves as drugs that allow getting livestock and poultry products that are safe in all respects. The results of the conducted studies allow drawing conclusions about the biological safety of probiotics in terms of its residual amount in the tissues of slaughter animals, which excludes a negative effect on the consumer's body.


Introduction
According to the clauses of the Law of the Republic of Kazakhstan dated July 21, 2007 No. 301-ІІІ "On food safety", the supply of animal and poultry products that are safe in all respects to consumers is the main task of the producers in the Republic of Kazakhstan (RK).
The goals of state regulation in the field of food safety are to ensure food safety for human life and health, the environment, and also to protect the legitimate interests of consumers, the environment and national security.
Nowadays, all over the world, the main task of scientists is to prevent the destructive effect of technogenic transformations that occur during the production of livestock products on the human body.The importance of solving this problem is that the pollution of soil, water and air with chemical elements and physical processes affects their composition and becomes one of the reasons for the imbalance of the normal intestinal microflora, metabolic processes and, as a consequence, a decrease in the immune status of the body of not only animals, but also humans [1,2].
Changes in the ecological microbial systems in the digestive tract and the occurrence of dysbacteriosis are also facilitated by the transfer of animal husbandry to industrial technologies for keeping and feeding.The use of probiotics should be considered as part of the rational potential for obtaining high quality livestock products, both bacterially and chemically safe [3 -7].
Outbreaks of salmonellosis and colibacillosis in the industrial poultry industry became one of the reasons for choosing preventive measures without the use of antibiotics, although the proof of the regulatory effect of lactic acid bacteria on the intestinal microflora has already been established by I.I.Mechnikov (1903Mechnikov ( -1905)).
Nowadays, spore-forming bacteria, mainly from the genus Bacillus, are increasingly used to obtain drugs with probiotic properties.The results of experimental studies of scientists allow observing positive dynamics in increasing resistance, viability, weight gain in animals when they are used [8 -10].
At this time, there is no clear data on the mechanism of the probiotic action of sporeforming microorganisms.The question of the long-term consequences of the response of the immune system of the animal organism when using spore-forming bacteria as probiotic preparations has not been studied.The ability of microorganisms to produce harmful metabolites has not been studied too [11 -15].
Another factor that requires careful study of the effects of spore probiotics is the production of drugs from genetically modified strains of bacteria.For example, Vetom 1.1 contains a bacterial biomass of the recombinant strain Bacillus subtilis VKPM B 7092 modified with a plasmid that synthesizes human leukocyte interferon alpha-2, and the effects of genetically modified organisms on humans through the products they consume can manifest themselves only after 1-3 generations.
Research objectives: to study the ability of spore-forming bacteria with probiotic properties to accumulate in tissues and the timing of their excretion from the body of animals in order to confirm the safety of the obtained products of animal origin.
The lack of study of the issue of studying the cumulative properties of probiotics and biological safety in terms of the residual amount in the tissues of the body of animal drugs based on spore-forming bacteria set us the task of conducting experimental work to determine the safety of the probiotic Vetom 1.1 in the body of pigs.

Materials and methods
In order to study the role of the probiotic Vetom 1.1 based on spore-forming bacteria in elucidating the mechanism of its action on the organism of young pigs, the object of the study was the pig population of the livestock complex (LC) Rubikom LLP of the Pavlodar region of the Republic of Kazakhstan.
LC Rubikom LLP is one of the largest enterprises in the Pavlodar region with a livestock of more than 50 thousand pigs.
The tests were carried out on a fattening livestock of 6-7-month-old pigs in the amount of 60 heads, which were given the Vetom 1.1 drug with feed at a dose of 50 mg/kg of live weight per head once every two days for 14 days of observation.After stopping the drug intake, pigs were slaughtered according to the following scheme: -10 heads -on the second day after stopping the drug intake; -10 heads -on the seventh day after stopping the drug intake; -10 heads -on the fourteenth day after stopping the drug intake.
In each period of the study, 10 heads of control animals were slaughtered, which were not given the drug.
The residual amount of the probiotic drug Vetom 1.1 and the degree of its safety for food consumers were determined in muscle tissue, heart, liver, spleen, and kidneys of slaughtered animals.
Vetom 1.1 is produced in Koltsovo, Novosibirsk region of the Russian Federation on the basis of The Scientific and Production Company "Research Center".According to the manufacturer's instructions, this drug "…contains immobilized dried spore biomass of bacteria of the recombinant strain Bacillus subtilis VKPM B 7092 modified with a plasmid that synthesizes human leukocyte interferon a-2 and auxiliary substances -sugar or powdered sugar and starch, which do not contain GMOs. 1 gram of Vetom 1.1 contains at least 1x106 CFU (colony-forming units) of live bacterial spores...".
Microbiological studies to determine the residual amount of probiotic Vetom 1.1 in the tissues and organs of the slaughtered animals were carried out taking into account the population level of bacteria of the Salmonella, Proteus, Staphylococcus, and Escherichia coli groups.
The technique for determining the cumulative properties of a probiotic based on sporeforming bacteria in the body of pigs was similar to the technique of operating procedures of the instruction "Methodological guidelines for the determination of residual amounts of antibiotics in animal products (Russian Standard 29.06.1984No. 3049-84)".

Determination of the concentration of spore probiotics in tissues and organs
To determine the concentrations of spore probiotics in tissues, a sample of 10.0 +/-0.1 g of muscle tissue, kidneys, liver, lung and other organs of animals, cut from the middle part of the sample, is removed, weighed and homogenized by grinding in sterile mortars with quartz sand or in a special mixer.A certain volume of isotonic sodium chloride solution (1 ml per 1 g of tissue mass) is added to the ground material and mixed well.Prepared samples are placed in a thermostat for extraction of probiotics for 90.0 +/-1 min at 37°C +/-0.1°C,periodically shaking the contents of the cones, after which, to inactivate possible inhibitory substances and better desorption of the probiotic, the mixture is heated in a water bath at a temperature of 60.0 +/-1.0°C for 20.0 +/-0.1 min.
The samples are centrifuged at 3000 rpm.within 20 +/-1 min.The supernatant is the first dilution and is ready to be injected into the wells.The second dilution is prepared in chemical test tubes: 1.0 +/-0.1 ml of the supernatant is added to 1.0 +/-0.1 ml of buffer (isotonic sodium chloride solution).The concentration of the test material is determined by the dilution of the standard.
The prepared solutions of each concentration of the standard and the test material are added to the wells in an amount of 0.1 +/-0.001ml of at least two Petri dishes.After adding the extracts and probiotic standard to the wells with the determined concentration, the dishes are placed in a thermostat for 20 +/-2 hours at 37 +/-1.0°C.
The next day, the zones of growth retardation of the test culture are measured and the probiotic content in the test products is calculated.

Accounting for results
Plotting standard curves and calculating the concentration of probiotics.
In order to construct the standard curve, working solutions with concentrations of probiotic Bac.subtilis VKPM B7092: 0.000001; 0.00001; 0.0001; 0.001; 0.01; 0.1 CFU/ml prepared according to scheme No. 1 are used (table 1).The arithmetic mean of the diameters of the growth inhibition zones of test microbes for each concentration of the working solution of the standard from two parallel dishes are calculated and plotted as points on the abscissa axis.From these points, as well as from points on the ordinate axis, corresponding to the concentrations of working solutions 0.000001; 0.00001; 0.0001; 0.001; 0.01; 0.1 CFU/ml, vertical and horizontal lines are drawn.The line that connects the intersection points of the specified lines is a standard curve.

Calculation of probiotic concentration
To calculate the concentration of the probiotic in the studied substrate, the average diameters of the corresponding zones are plotted along the abscissa axis of the same grid.From the obtained points, the perpendiculars are lowered until they intersect with the standard curve.From the intersection points, the perpendiculars to the ordinate axis are restored, on which the concentration indices of the standard solutions are plotted.According to the dilution of the test substrate, the concentration of the probiotic in it is considered, multiplying the obtained value by the degree of dilution.
The point of intersection of a parallel straight line with the ordinate axis indicates the activity of the probiotic in the extract of the test sample -"C".If the dilution of the extract was studied, then the obtained indicator of its activity is multiplied by the indicator of the dilution of the extract.
The calculation of the content of residual amounts of probiotics in 1 g of the studied product is carried out according to the formula: X = C x 1.5 where: X -probiotic activity in 1 g of product; C -probiotic activity in 1 ml of test sample extract; 1.5 -dilution of the sample with a buffer different from the dilution of the standard homogenate.
In the case when pure solutions of the standard probiotic were used to construct the standard curve, the calculation is carried out according to the same formula, changing only the degree of dilution of the sample with the buffer by 3, namely: X=C x 3.In this case, it is indicated in the answer that the standard curve is built using pure probiotic solutions.
All studies to determine the residual amount of probiotic Vetom 1.1 in the body (organs and muscle tissue) of the fattening pig herd were carried out according to this method.
Statistical processing of the data obtained was carried out using the Student's difference method described by V.A. Chistyakov.

Research results
In the course of microbiological studies to determine the residual amount of probiotic Vetom 1.1 in the tissues and organs of slaughter animals, the results were obtained, which are reflected in tables 2-4.
The content level of the probiotic Vetom 1.1 on the second day after stopping the drug intake is shown in Table 2.
According to the obtained results, a growth retardation of test microbes is observed on average in the range of 4.4-5.3mm.In samples from the control groups of animals, which were not tested by the action of the probiotic, zones of growth inhibition of test microbes were not observed (P <0.01).
The results of the study of the residual amount of probiotic Vetom 1.1 in the body of piglets on the seventh day after stopping the drug intake are presented in Table 3.
The results of the study showed that the growth retardation of test microbes is observed on average in the range of 6.1-6.8 mm.This is 1.7-1.5 mm more than in the piglets of the first experimental group.In samples from the control groups of animals, which were not tested by the action of the probiotic, zones of inhibition of the growth of test microbes were also not observed (P <0.001).
The results obtained when determining the residual amount of the drug in the body of piglets on the fourteenth day after taking the drug are shown in Table 4.
The research results shown in Tables 5, 6 and 7 indicate that the average activity of the probiotic Vetom 1.1 is highest on the seventh day after stopping the drug intake.This indicator indicates that the tested probiotic drug based on spore-forming bacteria has a cumulative capacity (P <0.01).
Table 8 and Figure 1 show the dynamics of changes in the average indicator of probiotic activity by the terms of use and in relation to the test cultures used.After determining the activity of Vetom 1.1 when exposed to strains of test cultures, we calculated the number of CFU of probiotic in the tested samples from organs and tissues of an experimental pig herd.Calculation data are shown in Table 9.According to the table, it can be concluded that the highest activity of the probiotic drug in the organs and tissues of experimental piglets of fattening livestock is observed on the seventh day after stopping the drug intake.
The data obtained allowed making calculations to determine the amount of probiotic Vetom 1.1 in the organs of the tested animals, the results of which are reflected in table 10.
The biomass concentration of bacteria of the recombinant Bacillus subtilis strain VKPM B 7092, which is the active principle in the body of animals, corresponds to 1*10 6 .

Conclusion
According to the results of our research, the residual amount of this probiotic drug did not exceed the permissible concentration.However, there are some cumulative properties of this drug.In the organs of pigs subjected to slaughter on the seventh day after taking the drug, there is an increase in its content by an average of 23%.The largest percentage of accumulation occurs in the spleen (94%) and muscle tissue (64%).A cumulative effect is also observed in the kidneys (6%).At the same time, a good percentage of probiotic excretion is observed in the lung tissue (87%) and in the liver (21%).
In the samples of pig organs from slaughter on the fourteenth day after the drug intake, its residual amount was minimal.
The results of the studies carried out on the residual amount of probiotics containing spore-forming bacteria in the tissues and organs of animals allow drawing conclusions about their biological safety for consumers of animal products after 15 days after stopping the intake of this drug by animals.

Table 2 .
The content of Vetom 1.1 in the piglets' organism on the second day after stopping the drug intake (n = 20).

Table 3 .
Vetom 1.1 content in piglets' organism on the seventh day after stopping the drug intake (n=20).

Table 5 .
Vetom 1.1 probiotic activity on the second day after stopping the drug intake, CFU.

Table 6 .
The activity of the probiotic Vetom 1.1 in 1 ml of the extract of the test sample on the seventh day after stopping the drug intake, CFU.

Table 7 .
The activity of the probiotic Vetom 1.1 in 1 ml of the extract of the test sample on the fourteenth day after stopping the drug intake, CFU

Table 9 .
Activity of probiotic Vetom 1.1 in 1 ml of test sample extract, CFU.