RU2142234C1 - Method for producing age-specified bifidopreparations - Google Patents

Abstract

FIELD: biotechnology, in particular, production of eubiotic preparations. SUBSTANCE: method involves preparing ferment by cultivating bifidobacteria of Bifidibacterium bifidum, Bifidobacterium longum, Bifidobactrium infantis or Bifidobacterium adolescentis kinds in nutritive medium; introducing protective medium into it with following liophylic drying or preparing cultured milk product in liquid state by introducing sown dose of obtained ferment into pasteurized milk in an amount not exceeding 5% of volume of nutritive medium; providing repeated cultivation of mentioned kinds of bifidobacteria at temperature of 38 C plus and minus 1 C till bunching; cooling and packing product. To produce ferment or cultured milk product in liquid state, each kind of bifidobacteria is separately cultivated till obtaining of biomasses titrated to at least 10⁹ CFU/ml and joined together prior to drying of ferment or prior to packing of biomass of cultivated milk product of bifidobacteria to obtain product in the form of mixture of mentioned kinds of bifidobacteria. To produce preparation for babies of up to 3-5 years, bifidobacteria of Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve or Bifidobacterium infantis kinds are used, and for teenagers or grownups - bifidobacteria of Bifidobacretium bifidum, Bifidobacterium longum and Bifidobacterium adolescentis kinds are used. Ratio of biomass of separate kinds of bifidobacteria in preparation is set in accordance with ratio of these bifidobacteria in intestines of healthy individual of definite age, obtained on the base of statistic data and regression analysis. EFFECT: improved medicinal and prophylactic properties of bifidopreparations, wider range of usage, improved compatibility with individual intestines normoflora and simplified production method by using optimum kinds of bifidobacteria in preparation. 4 cl, 1 dwg, 2 tbl, 6 ex

Description

 The invention relates to medicine, food industry and biotechnology and relates to a technology for the treatment of prophylactic eubiotics containing live bifidobacteria.

 Violation of the qualitative and quantitative composition of normoflora, called dysbiosis, leads to diseases of the digestive system, hematopoiesis system, immune system, development of malnutrition, anemia. According to the Ministry of Health, 90% of the population of Russia to one degree or another suffer from dysbiosis. The wide spread of dysbiosis is one of the most important factors determining the currently observed increase in the frequency and severity of acute and chronic diseases.

 Dysbacteriosis is treated with the use of specific eubiotics, which use live microorganisms - representatives of the normal microflora of the human intestine. Intestinal flora is a symbiosis of various microorganisms, the most significant of which are bifidobacteria and lactobacilli. Bifidobacteria, constituting from 80 to 95% of the intestinal normoflora, play a crucial role in the life of the human body, inhibiting the growth of pathogenic, putrefactive and gas-forming microorganisms, participating in the metabolism and synthesis of vitamins, stimulating the immune system.

 Given the above, the most effective drugs for the treatment and prevention of dysbacteriosis are bifid-containing drugs.

A method of obtaining a dry bifidumbakterina comprising administering an inoculum culture of bifidobacteria one species - Bifidobacterium bifidum in a nutrient medium containing pancreatic hydrolyzate of milk, peptone, sodium chloride, lactose, cystine hydrochloride, and agar, followed by cultivation at a temperature of culture (38 ± 1) ^o C before receiving the clot, the introduction of a protective medium containing sucrose from 0.45 to 0.9% and gelatin from 0.05 to 0.1%, packaging and freeze drying of the finished product (Pharmaceutical article FS 42-132BC-94). The dose of dry bifidumbacterin contains 1 • 10 ⁷ living cells. The drug is used in children from the first days of life, as well as in adults. There are no contraindications to the use of bifidumbacterin, there are no side effects.

 However, clinical practice has shown low efficacy of monopreparations in the treatment of deep disorders of the intestinal microbiocenosis, when atypical forms of several types of symbiotic pathogens appear [1]. Dry bifidumbacterin contains freeze-dried bacteria of only one species of B. bifidum, which are in a state of suspended animation and have reduced adhesive activity, as a result of which dry bifidumbacterin does not have sufficient clinical efficacy.

A known method for producing a complex preparation "bificolforte" containing a freeze-dried mixture of co-grown bifidobacteria and Escherichia coli strain M 17. The method includes the introduction of seed doses of cultures of bifidobacteria and Escherichia coli in a ratio of 1: 1 in a nutrient medium, followed by growing crops at a temperature ( 38 ± 1) ^o C until a clot is obtained, application of a protective medium containing sucrose from 0.45 to 0.9% and gelatin from 0.05 to 0.1%, packaging and freeze drying of the finished product (Temporary Pharmaceutical Article VFS 42-65BC-86).

 However, when using the drug "Bifikol-forte", negative consequences of its effect on the human body can be observed. This is due to the variability of E. coli during its cultivation and freeze-drying of the drug, which can lead to the appearance of pathogenic forms of this type of bacteria. According to some leading microecologists of the world, it is permissible to use only inactivated strains of E. coli in the preparations [2].

A known method of obtaining a comprehensive preparation "Bifiform", manufactured by Ferrosan, which includes introducing into a nutrient medium and cultivating a mixture of bifidobacteria and enterococci with subsequent freeze drying of the obtained biomass. In the finished product contains bacterial cells of each of these cultures 10 ⁷ CFU in 1 g of the drug, packaged in capsules [3].

 However, this drug in its composition has one type of bifidobacteria, which does not adequately correspond to the physiological and age-related parameters of the human body, in the biocenoses of the mucous membranes of which several types of bifidobacteria are simultaneously present, the quantitative content of which is 10-30 times higher than enterococci.

A known method for producing a complex fermented milk product, comprising introducing into sterile milk growth stimulants and a sowing dose of industrial yeast in an amount of 5-20 wt.%, Which is in the growth stage and contains eubiotics of the species Lactobacillus acidophilus, Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobact Bifidobacterium infantis. Cultivation is carried out to a bacterial count of more than 10 ⁸ cells per ml, after which the product is cooled and packaged in a container (Application EPO N0154614, IPC A 23 C 9/123, publ. 11.09.85).

A known method for producing a therapeutic and prophylactic preparation of the fermented milk product "Nomoflor", including normalization, homogenization, heat treatment of the raw milk raw material, cooling to the fermentation temperature, deoxygenation, introduction of the ferment containing bifidobacteria, fermentation, cooling, packaging. As a starter culture, a mixture of Bifidobacterium longum B-379 M, Bifidobacterium bifidum 791 and kefir fungi in a ratio of 1: 1: 0.5, respectively (Patent RF N2100936, IPC ⁶ A 23 C 9/127, publ. 10.01.98), is used.

 However, drugs prepared by the two above methods have insufficient therapeutic and prophylactic properties, because they do not take into account the age of patients using these drugs and physiological requirements.

The closest analogue (prototype) is a method for producing fermented milk bifidobiological product "Bifi-life", which includes the introduction of industrial starter culture on the basis of a consortium of strains of bifidobacterium species Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Bifidobacteriumeris 1 adis 1 and Bifidobacteriumenteris 1 : 1: 1, in a nutrient medium based on skim milk and reconstituted culturing the consortium of bacteria at a temperature of (38 ± 1) ^o C for 5 hours to obtain a clot biotitrom September ¹⁰ CFU / ml, followed by cooling of the product with its F ovkoy or freeze-drying (Patent RF N2109054, IPC C 12 N 1/20, A 23 C 9/12, A 61 K 35/74, publ. 20.04.98).

 However, due to the fact that different types of bifidobacteria in milk grow at different rates, the highest of which is species B. adolescentis, the species B. longum grows slightly more slowly, while the rest have a much lower growth rate, then bifidobacteria of type B prevail in the resulting product. adolescentis, which dramatically reduces the therapeutic properties of this product. In addition, the drug prepared according to this technology does not take into account the age of the patient using this drug, because It contains strains of bifidobacteria contained in the body of people of all ages (from newborns to the elderly), as a result of which it does not meet the physiological requirements. Moreover, the use of such a number of strains of bifidobacteria in one drug significantly complicates the technology for its preparation.

 The objective of the invention is the creation of such a technology for the preparation of bifid preparations, which would take into account the age of patients and contain a combination of several types of bifidobacteria: B. bifidum, B. longum, B. breve, or B. infantis, or B. adolescentis in a percentage ratio corresponding to the species the composition of the normoflora of the intestines of a healthy person of a certain age.

This problem is solved by the fact that in the method for producing age-specific bifid preparations, including the preparation of starter culture by cultivating bifidobacteria of the species Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium infantis, or Bifidobacterium nutrient medium with adolescent fermented milk product in liquid form by adding to the pasteurized milk a sowing dose of the obtained yeast in an amount of not more than 5% of the volume of the nutrient medium and re-cultivating the formation of these types of bifidobacteria at a temperature of (38 ± 1) ^o C until a clot is obtained, cooling of the product with its subsequent packing, according to the invention, when preparing the starter culture and fermented milk product in liquid form, each type of bifidobacteria is cultivated separately to obtain biomass with a titer of at least 10 ⁹ CFU / ml and combine before drying the starter culture or before packing the biomass of the fermented milk product of bifidobacteria to obtain the product in the form of a mixture of these types of bifidobacteria, and upon receipt of preparations for children after birth up to the age of 3-5 years, use bifidobacteria of the species Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve or Bifidobacterium infantis, and when receiving the drug for older children and adults, use bifidobacteria of the species Bifidobacterium bifidum, Bifidobacterobiumerium bumidum longum and of certain types of bifidobacteria in the preparation is established corresponding to their species ratio in the intestines of a healthy person of a certain age, obtained on the basis of a statistical data array processed by methods of mathematical statistics and regression analysis.

Upon receipt of the drug for children after birth and 3-5 years of age, the following ratio of biomass of certain types of bifidobacteria in the preparation is established, wt.%:
Bifidobacterium longum - 38.0-52.0
Bifidobacterium bifidum - 30.0-42.0
Bifidobacterium breve or Bifidobacterium infantis - the rest is up to 100%
Upon receipt of the drug for older children and adults, the following ratio of biomass of certain types of bifidobacteria in the preparation is established, wt.%:
Bifidobacterium longum - 32.0-42.0
Bifidobacterium bifidum - 29.0-39.0
Bifidobacterium adolescentis - the rest is up to 100%
Upon receipt of the starter culture medium contains reduced skim milk, has a dry residue of 12-13%, pancreatic milk hydrolyzate, baker's yeast autolysate and ascorbic acid in the following ratio, wt.%:
pancreatic hydrolyzate of milk - 4.5-5.0%,
baker's yeast autolysate - 0.5-1.5%,
ascorbic acid - 0.003-0.005%,
reduced skim milk with a dry residue of 12-13% - up to 100%
A higher% milk solids content in the nutrient medium of 12–13% instead of the recommended 8–9% will make it possible to increase the survival time of bifidobacteria during storage of preparations and fermented milk products. In addition, a high% solids content of milk makes it possible to increase the biological value of starter cultures, since the general balance of milk substances is characterized by an anti-sclerotic focus and contains deficient arachidonic acid and a protein-lesetin complex.

 To create effective formulations of medicinal bifid preparations and medical and dietary fermented milk products, the dependence obtained by mathematical processing of clinical trial data on the species composition of intestinal bifidoflora depending on the age of the patients is used.

 In children of the first year of life, 85-95% of the total microbiocenosis of the colon is bifidoflora - the content of bifidobacteria in 1 g of feces reaches tens of billions (lg10). Bacteroids are unusual for children in the first half of life. The quantitative content of lactobacilli, enterococci, lactic streptococci and Escherichia coli (aerobic microflora) does not exceed 10%. Adult eubiosis is also characterized by the dominance of bifidobacteria (80-90% or lg8-10) in the total composition of normoflora. Lactobacilli, lactic streptococci, enterococci and Escherichia coli are excreted in approximately equal amounts (log6.6-7). Thus, among representatives of the normal intestinal microflora, representatives of the genus Bifidobacterium, which includes 11 species of bifidobacteria, dominate. It is bifidobacteria that play a decisive role in the regulation and stability of the biocenosis of the gastrointestinal tract and mucous membranes.

 Carrying out a physiological role useful for the body, bifidoflora increases its resistance to diseases. When creating bifid-containing preparations from strains of various types, an understanding of the mechanisms of their action on the body and knowledge of the biology of the bacteria of the Bifidobacterium genus themselves are required.

 Below are the steps for constructing age-specific bifidopreparations.

 Stage 1. The method of mathematical processing of statistical data regarding the species composition and quantitative content of eubiotics in the human body, followed by their use in the design of drugs.

 When studying the species structure of bifidobacteria isolated from infants and adults (see Table 1), it was found [4] that B. bifidum and B. longum are characteristic of newborns and infants who are breast-fed, the detection frequency which ranges from 40-60%. Species B.adolescentis has never been detected in breast-fed infants. This species is found only in premature babies who are breast-fed and suffering from purulent-septic diseases. In such children, the detection rate of B. bifidum, which is the most physiological in the child’s body, is reduced. Three types of bifidobacteria were distinguished in the intestinal biocenosis of healthy adults: B. bifidum, B. longum, and B. adolescentis. All types of bacteria were found in association with each other, as a rule, it was B. bifidum and B. longum in combination with other strains [4].

 Clinical studies [4-9] found that the species composition of bifidoflora is not the same and undergoes changes depending on age, the nature of nutrition and a number of other physiological factors. Bifidoflora of a healthy infant fed by mother's milk is represented by the species: B. bifidum - from 35 to 39%, B.longum - about 42-45%, B.breve - from 14 to 17%, B.infantis - up to 12% and B.adolescentis - up to 1.5% or not detected at all [5,7,8,9]. In breast-fed children, the detection rate in the intestine of B. adolescentis reaches from 22% to 48% with a decrease and even crowding out of other types of bifidobacteria. Some researchers consider this condition as a harbinger of developing dysbiosis [9]. By the age of 7 years, the species B.breve and B.infantis disappear from the intestines of the child. In older children and adults, mainly B. bifidum, B. longum, and B. adolescentis are found in the intestines; the latter after 35 years begins to prevail, reaching 60–85% in the elderly [8.9].

(1)
квантиль x, который используется для определения границ доверительного интервала, согласно таблице интеграла вероятности равен 1.64 [10].In the process of searching for promising probiotics, many researchers have found that drugs that are similar in composition to the physiological parameters of the intestine and are made on the basis of several types of bifidobacteria that complement each other in functional value have the optimal clinical effect. In the process of research, statistical processing of literature data [2, 4, 6–9] was carried out on the species composition of bifidoflora depending on the age of a person with a confidence level of 90%, under the assumption that the available sample reflects the general population distributed normally. This means that if the probability integral is equal to 0.9

(1)
the quantile x, which is used to determine the boundaries of the confidence interval, according to the table of the probability integral is 1.64 [10].

(2)
где n - число данных, принятых для расчета;
2) вычислялись среднеквадратические отклонения для каждого случая согласно

(3)
3) вычислялись границы доверительных интервалов случайных погрешностей, в котором x = 1.64, согласно (1)

(4)
Далее по статистически обработанным данным для каждого из видов бифидобактерий B. bifidum, B.longum, B.adolescentis, B.breve и B.infantis проводились вычисления значений функции f{% (P), возраст (x)}, т.е. выполнялась операция интерполяции методом Ньютона, согласно [11]:
1) При двух известных значениях P₀=f(x₀) и P₁=f(x₀+h) получается интерполяционный многочлен первой степени, описывающий наклонную прямую

(5)
2) Соответствующее приближенное значение интеграла дает суммарное изменение % содержания определенного вида бифидобактерий в биоценозе ЖКТ за промежуток h лет

(6)
3) При 3 известных значениях P₀=f(x₀), P₁=f(x₀)+h) и P₂=f(x₀+2h) получается интерполяционный множитель 2 степени, описывающий параболу

(7)
4) Данные рассуждения можно обобщить на произвольное число равноотстоящих значений x - при 4 известных значениях получается многочлен

(8)
Таким образом составлялись интерполяционные многочлены, которые были обсчитаны методом Ньютона для нелинейных задач в Microsoft Exel при следующих параметрах поиска решения: 1) число итераций - 100, 2) автоматическое нормирование параметров, качественно различающихся по величине, 3) точность расчетов - 0.001.The operations for calculating the boundaries of confidence intervals in determining the average values of the percentage ratio of bifidobacteria in biocenoses were as follows:
1) calculated average values of indicators in a certain time interval according to

(2)
where n is the number of data accepted for the calculation;
2) the standard deviations were calculated for each case according to

(3)
3) the boundaries of confidence intervals of random errors were calculated, in which x = 1.64, according to (1)

(4)
Further, according to statistically processed data for each of the species of bifidobacteria B. bifidum, B.longum, B. adolescentis, B.breve and B.infantis, the values of the function f {% (P), age (x)} were calculated, i.e. the interpolation operation was performed by the Newton method, according to [11]:
1) For two known values of P ₀ = f (x ₀ ) and P ₁ = f (x ₀ + h), an interpolation polynomial of the first degree is obtained that describes the inclined line

(5)
2) The corresponding approximate value of the integral gives the total change in% of the content of a certain type of bifidobacteria in the gastrointestinal biocenosis over a period of h years

(6)
3) For 3 known values of P ₀ = f (x ₀ ), P ₁ = f (x ₀ ) + h) and P ₂ = f (x ₀ + 2h), an interpolation factor of 2 degrees is obtained that describes the parabola

(7)
4) These arguments can be generalized to an arbitrary number of equally spaced values of x - for 4 known values, we obtain a polynomial

(eight)
Thus, interpolation polynomials were compiled, which were calculated by the Newton method for nonlinear problems in Microsoft Exel with the following solution search parameters: 1) the number of iterations - 100, 2) automatic normalization of parameters qualitatively different in magnitude, 3) calculation accuracy - 0.001.

(10)
где

i - индекс возраста,
A_i...F_i - аппроксимированная частота обнаружения видов бифидобактерий B. bifidum, B.longum, B.adolescentis, В.breve и B.infantis,
a_i. . . f_i - среднестатистическое содержание соответствующих видов бифидобактерий в биоценозе кишечника
Таким образом были найдены данные о видовом соотношении B.bifidum, B.longum, B.adolescentis, B.breve и B.infantis для возрастов в 1, 7, 15, 30, 50 и 70 лет, которые приведены в табл. 2. Относительные погрешности расчетных данных составили 5%, 5%, 6%, 8% и 8% для соответствующих видов.Further, in order to smooth out random fluctuations, to more clearly represent the model and to trace the nature of the data change, we used the least-squares polynomial approximation of these data in accordance with the equation
p = b + c ₁ x + c ₂ x ² + c ₃ x ³ + ... + c ₆ x ⁶ (9)
where b, c ₁ , c ₂ ... c ₆ are constants
Approximation of interpolated data in Microsoft Exel. The data processed in this way reflect the frequency of detection of a certain type of bifidobacteria in the intestinal biocenosis in children and adults of different ages. Since all these species are found, as a rule, in association with each other, it is logical to assume that if we normalize the detection frequency of all types of bifidobacteria to 1 (or 100%), then the normalized detection frequency will reflect the average statistical ratio of different types of bifidobacteria in the intestinal biocenosis. Rationing conditions

(ten)
Where

i is the age index,
A _i ... F _i is the approximate detection frequency of bifidobacteria B. bifidum, B.longum, B.adolescentis, B.breve and B.infantis,
a _i . . . f _i - the average statistical content of the corresponding species of bifidobacteria in the intestinal biocenosis
Thus, data were found on the species ratio of B. bifidum, B. longum, B. adolescentis, B.breve, and B.infantis for ages 1, 7, 15, 30, 50, and 70 years, which are given in Table. 2. The relative errors of the calculated data were 5%, 5%, 6%, 8%, and 8% for the corresponding species.

(11)
Построенные таким образом линии тренда - графические зависимости приведены на фиг. 1 и представляют собой распределение видов бифидобактерий в биоценозе кишечника в зависимости от возраста.The final stage of the work was the construction of trend lines for each type of bifidobacteria using Microsoft Exel. The error values for these trend lines were calculated in accordance with the criterion R ² according to

(eleven)
The trend lines constructed in this way — graphical dependencies are shown in FIG. 1 and represent the distribution of species of bifidobacteria in the intestinal biocenosis depending on age.

 The obtained dependences make it possible to reasonably construct the formulation of eubiotics depending on the purpose and age of the patient. As can be seen from FIG. 1, the species composition of bifidoflora in adults and children (especially young children) varies significantly. Therefore, when designing complex bifid-containing preparations for children or adults, it is necessary to take into account age-related changes in the intestinal flora. Accordingly, the developed formulations of medicinal bifidobacteria and therapeutic dietary fermented milk products should differ and correspond to the species distribution of bifidobacteria in intestinal biocenoses depending on age.

 For example, for children under 3 years of age, it is desirable that therapeutic and prophylactic food additives contain bifidobacteria B.bifidum 36 ± 6%, B.longum 45 ± 7% and B.breve 12 ± 3% and B.infantis 7 ± 2%

 For dosage forms of bifid preparations, it is desirable that the ratio of B. bifidum and B. longum correspond to the species composition of the intestines of a healthy person aged 18-23 years. From a clinical and physiological point of view, a drug of this composition will be universal for adults and older children.

 Step 2. Technology for the preparation of age-specific bifid preparations using the graph shown in FIG. 1.

 Example 1

 For the preparation of a dry complex preparation for children from birth to young children (up to 3-5 years), the ratio of bifidobacteria species corresponds to the quantitative composition of the normoflora of a child under the age of 1 year. Using the graph in FIG. 1, the following types of bifidobacteria are established in the preparation and their quantitative ratio: B.longum - 49%, B.bifidum - 39%, B.breve - 12%.

Vials containing lyophilized dried bifidobacteria of the species B. bifidum, B. longum and B. breve are opened sterile, diluted with 5 milliliters of 0.9% sodium chloride solution. The resulting suspensions are transferred sterile (each separately) into test tubes with a sterile modified liver medium Blaurock and incubated at (38 ± 1) ^o C for 18-20 hours. Thus obtained cultures of bacteria of the first generation are separately introduced in an amount of 3-5% (from volume of nutrient medium) in glass bottles with a nutrient medium of the following composition:
pancreatic hydrolyzate of milk - 4.5-5.0%,
baker's yeast autolysate - 0.5-1.5%,
ascorbic acid - 0.003-0.005%,
reduced skim milk with a dry residue of 12-13% - up to 100%
Crop doses of each type of bifidobacteria are incubated at (38 ± 1) ^o C for 18-20 hours until a clot with a biotiter of at least 10 ⁹ CFU / ml and acidity of not more than 120-160 ^o T.

 The contents of the bottles of each type of bifidobacteria are sterilely mixed in the above proportions (B.longum - 49%, B.bifidum -39%, B.breve - 12%), adjust the pH to 7.0 with a 5% ammonia solution, add sucrose from 0.45 up to 0.9% and gelatin from 0.05 to 0.1%, dispensed in 2-3 ml volumes into 10 ml penflasks and subjected to freeze drying. Thus, a complex dry bifid preparation for children from birth to the age of 3-5 years is obtained.

 Example 2

 To prepare a dry complex preparation for children from birth to young children (up to 3-5 years), using the graph in FIG. 1, establish the following types of bifidobacteria and their quantitative ratio in the preparation: B. longum - 50%, B. bifidum - 41%, B.infantis - 9%.

Vials containing lyophilized dried bifidobacteria of the species B. bifidum, B. longum and B.infantis are opened sterile, diluted with 5 milliliters of a 0.9% sodium chloride solution. The resulting suspensions are transferred sterile (each separately) into test tubes with a sterile modified liver medium of Blaurock and incubated at (38 ± 1) ^o C for 18-20 hours. Thus obtained cultures of the first generation are separately introduced in an amount of 3-5% (by volume) nutrient medium) in glass bottles with a nutrient medium of the following composition:
pancreatic hydrolyzate of milk - 4.5-5.0%,
baker's yeast autolysate - 0.5-1.5%,
ascorbic acid - 0.003-0.005%,
reduced skim milk with a dry residue of 12-13% - up to 100%
Crop doses of each type of bifidobacteria are incubated at (38 ± 1) ^o C for 18-20 hours until a clot with a biotiter of at least 10 ⁹ CFU / ml and acidity of not more than 120-160 ^o T.

 The contents of the bottles of each type of bifidobacteria are sterilely mixed in the above proportions (B. longum - 50%, B. bifidum - 41%, B.infantis - 9%), adjust the pH to 7.0 using 5% ammonia solution, add sucrose from 0.45 up to 0.9% and gelatin from 0.05 to 0.1%, dispensed in 2-3 ml volumes into 10 ml penflasks and subjected to freeze drying. Thus, a second complex dry bifid preparation for children from birth to the age of 3-5 years is obtained.

 Example 3

 For the preparation of a dry complex preparation for adults and children over 5 years old, the ratio of different types of bifidobacteria corresponds to the quantitative composition of the normal flora of the intestines of a young healthy person 18-23 years old. It was during this period that the body had already completed its formation and was less susceptible to various diseases, both somatic and infectious. Using the graph in FIG. 1, the following types of bifidobacteria are established in the preparation and their quantitative ratio: B. longum - 41%, B. bifidum - 33%, B.adolescentis 26%.

Vials containing freeze-dried bifidobacteria of the species B. bifidum, B. longun and B. adolescentis are opened sterile, diluted with 5 milliliters of a 0.9% sodium chloride solution. The resulting suspensions are transferred sterile (each separately) into test tubes with a sterile modified liver medium of Blaurock and incubated at (38 ± 1) ^o C for 18-20 hours. Thus obtained cultures of the first generation are separately introduced in an amount of 3-5% (by volume) nutrient medium) in glass bottles with a nutrient medium of the following composition:
pancreatic hydrolyzate of milk - 4.5-5.0%,
baker's yeast autolysate - 0.5-1.5%,
ascorbic acid - 0.003-0.005%,
reduced skim milk with a dry residue of 12-13% - up to 100%
Crop doses of each type of bifidobacteria are incubated at (38 ± 1) ^o C for 18-20 hours until a clot with a biotiter of at least 10 ⁹ CFU / ml and acidity of not more than 120-160 ^o T.

 The contents of the bottles of each type of bifidobacteria are sterilely mixed in the above proportions (B.longum - 41%, B.bifidum - 33%, B.adolescentis - 26%), adjust the pH to 7.0 using a 5% ammonia solution, add sucrose from 0.45 up to 0.9% and gelatin from 0.05 to 0.1%, dispensed in 2-3 ml volumes into 10 ml penflasks and subjected to freeze drying. Thus, a complex dry bifid preparation for adults and children over 5 years of age is obtained.

 Example 4

 For the preparation of a complex fermented milk preparation in liquid form for children from birth to young children (up to 3-5 years), the ratio of different types of bifidobacteria corresponds to the quantitative composition of the normoflora of a child under the age of 1 year. Using the graph in FIG. 1, the following types of bifidobacteria are established in the preparation and their quantitative ratio: B. longum - 48%, B. bifidum - 37%, B.breve - 15%.

Vials containing freeze-dried bifidobacteria of the species B. bifidum, B. longum and B. breve are opened sterile, diluted with 5 milliliters of a 0.9% sodium chloride solution. The resulting suspensions are transferred sterile (each separately) into test tubes with a sterile modified liver medium Blaurock and incubated at (38 ± 1) ^o C for 18-20 hours. Thus obtained cultures of bacteria of the first generation are separately introduced in an amount of 3-5% (from volume of nutrient medium) in glass bottles with a nutrient medium of the following composition:
pancreatic hydrolyzate of milk - 4.5-5.0%,
baker's yeast autolysate - 0.5-1.5%,
ascorbic acid - 0.003-0.005%,
reduced skim milk with a dry residue of 12-13% - up to 100%
Crop doses of each type of bifidobacteria are incubated at (38 ± 1) ^o C for 18-20 hours until a clot with a biotiter of at least 10 ⁹ CFU / ml and acidity of not more than 120-160 ^o T (second generation).

Further, the container with agitators containing pasteurized (UHT), cooled to (38 ± 2) ^o C, and reduced skimmed milk, 5% of biomass introduced each species of bifidobacteria second generation (biomass in each separate container). The mixture in the containers is stirred for 15 minutes and the milk is fermented for 16-18 hours upon reaching the product acidity (80-110) ^o T and titer 10 ⁷ CFU / ml.

The contents of the containers of each type of bifidobacteria are sterilely mixed in one container in the above proportions (B. longum - 48%, B. bifidum - 37%, B. breve - 15%). The finished product is chilled to a temperature of (8 ± 2) ^o C and poured into consumer packaging.

 Example 5

 To prepare a complex fermented milk preparation for children from birth to young children (up to 3-5 years) in liquid form, using the graph in FIG. 1, establish the following types of bifidobacteria and their quantitative ratio in the preparation: B. longum - 52%, B. bifidum - 43%, B.infantis - 5%.

Vials containing lyophilized dried bifidobacteria of the species B. bifidum, B. longum and B.infantis are opened sterile, diluted with 5 milliliters of a 0.9% sodium chloride solution. The resulting suspensions are transferred sterile (each separately) into tubes with sterile modified liver fluid Blaurock and incubated at (38 ± 1) ^o C for 18-20 hours. Thus obtained cultures of the first generation are separately administered in an amount of 3-5% (by volume) nutrient medium) in glass bottles with a nutrient medium of the following composition:
pancreatic hydrolyzate of milk - 4.5-5.0%,
baker's yeast autolysate - 0.5-1.5%,
ascorbic acid - 0.003-0.005%,
reduced skim milk with a dry residue of 12-13% - up to 100%
Crop doses of each type of bifidobacteria are incubated at (38 ± 1) ^o C for 18-20 hours until a clot with a biotiter of at least 10 ⁹ CFU / ml and acidity of not more than 120-160 ^o T (second generation).

Further, the container with agitators containing pasteurized (UHT), cooled to (38 ± 2) ^o C, and reduced skimmed milk, 5% of biomass introduced each species of bifidobacteria second generation (biomass in each separate container). The mixture in the containers is stirred for 15 minutes and the milk is fermented for 16-18 hours upon reaching the product acidity (80-110) ^o T and titer 10 ⁷ CFU / ml.

The contents of the containers of each type of bifidobacteria are sterilely mixed in one container in the above proportions (B.longum - 52%, B.bifidum - 43%, B.breve - 5%). The finished product is chilled to a temperature of (8 ± 2) ^o C and poured into consumer packaging.

 Thus, a second complex fermented milk bifid preparation in liquid form is obtained for children from birth to the age of 3-5 years.

 Example 6

 For the preparation of a complex fermented milk preparation in liquid form for adults and children over the age of 5 years, the ratio of different types of bifidobacteria corresponds, as mentioned above, to the quantitative composition of the normal intestinal flora of a young healthy person 18-23 years old. Using the graph in FIG. 1, the following types of bifidobacteria are established in the preparation and their quantitative ratio: B.longum - 42%, B.bifidum - 39%, B.adolescentis 29%.

Vials containing freeze-dried bifidobacteria of the species B. bifidum, B. longum and B. adolescentis are opened sterile, diluted with 5 milliliters of 0.9% sodium chloride solution. The resulting suspensions are transferred sterile (each separately) into test tubes with a sterile modified liver medium of Blaurock and incubated at (38 ± 1) ^o C for 18-20 hours. Thus obtained cultures of the first generation are separately introduced in an amount of 3-5% (by volume) nutrient medium) in glass bottles with a nutrient medium of the following composition:
pancreatic hydrolyzate of milk - 4.5-5.0%,
baker's yeast autolysate - 0.5-1.5%,
ascorbic acid - 0.003-0.005%,
reduced skim milk with a dry residue of 12-13% - up to 100%
Crop doses of each type of bifidobacteria are incubated at (38 ± 1) ^o C for 18-20 hours until a clot with a biotiter of at least 10 ⁹ CFU / ml and acidity of not more than 120-160 ^o T (second generation).

Further, the container with agitators containing pasteurized (UHT), cooled to (38 ± 2) ^o C, and reduced skimmed milk, 5% of biomass introduced each species of bifidobacteria second generation (biomass in each separate container). The mixture in the containers is stirred for 15 minutes and the milk is fermented for 16-18 hours upon reaching the product acidity (80-110) ^o T and titer 10 ⁷ CFU / ml.

The contents of the containers of each type of bifidobacteria are sterilely mixed in one container in the above proportions (B. longmn - 42%, B. bifidum - 39%, B. adolescentis - 29%). Ready-made complex fermented milk bifidopreparation in liquid form for adults and children over 5 years old is cooled to a temperature of (8 ± 2) ^o C and poured into consumer containers.

 Thus, the proposed technology involves the use of a combination of several types of bifidobacteria (B. bifidum, B. longum, B.breve, or B. infantis, or B. adolescentis) in a percentage ratio corresponding to the species composition of the normal human intestinal flora. The combined use in the preparations of three strains of bifidobacteria in a certain percentage, corresponding to the age of the patient, provides a high therapeutic and prophylactic effect, increasing the compatibility of the drug with human intestinal normoflora. The proposed method is based on a simpler and more technologically advanced drug production scheme by reducing (optimizing) the number of bifidobacteria species used in the preparations.

Used scientific and technical information
1. Abramov N. A. Technological problems of providing eubiotics with the health needs of Russia. Abstracts of the All-Russian Conference "Dysbacteriosis and Eubiotics", M., 1996, p. 1.

 2. Goncharova G.I., Dorofeychuk V.G., Smolyanskaya A.Z., Sokolova K.Ya. Microbial ecology of the intestine in normal and pathological conditions // f. Antibiotics and chemotherapy, vol. 34, No. 6, 1989.

 3. Reference Vidal. Medicines in Russia: A Handbook. M .: AstraPharmService, 1998, B-91.

 Liannaya A.M., Intizarov M.M., Donskikh E.E. Biological and environmental features of microbes of the genus Bifidobacterium.// Sat. under the editorship of Nikitin "Bifidobacteria and their use in the clinic, medicine, etc.", Moscow Scientific Research Institute of Power Engineering named after Gabrichevsky, Moscow: 1986.

 4. Jacobs J.P., Magrath D.J., Garret A.I. et al. J. Biol. Stand. 1981, N 3, p. 331.

 5. A. M. Liannaya, H.A. Silchenko, C.A. Lisunova et al. Biological and technological characteristics of bifidobacteria strains of the species B. breve and B. infantis. // Sat Problems of medical biotechnology and immunology of infectious diseases. Moscow NIIEM them. Gabrichevsky. M., 1996.

 6. L. N. Evtukhova, I. G. Skorikova and others. New strains of bifidobacteria, proposed for bacteriotherapy. // Standards, strains and methods for controlling bacterial and viral preparations. Moscow Research Institute of Vaccines and Serums I.I. Mechnikov. M., 1987.

 7. G.I. Goncharova, A.M. Lyanskaya, L.P. Semenova et al. Treatment and prevention of intestinal dysbiosis with bifidum preparations. // Sat Bacterial biological products. MNIIEM them. G.N. Gabrichevsky. M., 1989.

 8. E.P. Kozlova, N.A. Kurnosova, L.V. Feklisova. Theoretical and biomedical aspects of the use of various types of bifidobacteria as part of the means for the prevention and treatment of intestinal dysbiosis. // Sat under the editorship of Nikitin "Bifidobacteria and their use in the clinic, medicine, etc.", Moscow Scientific Research Institute of Power Engineering named after Gabrichevsky, Moscow: 1986.

 9. Zeldovich Ya.B., Myshkis A.D. Elements of applied mathematics. M .: Nauka, 1967, 645 p.

Claims (2)

1. A method of producing age-specific bifidobacteria, including the preparation of a starter culture by cultivating bifidobacteria of the species Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, or Bifidobacterium infantis, or Bifidobacterium adolescentis in a nutrient medium with a dry solution and then supplementing it with fresh and dry solution by introducing into the pasteurized milk a sowing dose of the obtained yeast in an amount of not more than 5% of the volume of the nutrient medium and re-culturing these types of bifidobact ry at a temperature (38 ± 1) ^o C until a clot, cooling of the product and its packaging, characterized in that in the preparation of sourdough and fermented milk product in liquid form each kind of bifidobacteria were cultured separately to obtain biomasses with a titer of not less than 10 ⁹ cfu / ml in the starter culture and with a titer of not more than 10 ⁷ CFU / ml in the fermented milk product and combined before drying the starter culture or before packing the biomass of the fermented milk product of bifidobacteria to obtain the product in the form of a mixture of these types of bifidobacteria, and upon receipt of preparations for after birth and up to the age of 3–5 years, bifidobacteria of the species Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve or Bifidobacterium infantis are used, and when receiving the drug for older children and adults, bifidobacteria of the species Bifidobacterium bifidum, Bifidobacteridum bifidobactent ratio bifidobacterium types of bifidobacteria in the preparation are determined according to their species ratio in the intestines of a healthy person of a certain age, obtained on the basis of a statistical array of data processed by mathematical methods ISTIC and regression analysis. 2. The method according to claim 1, characterized in that upon receipt of the drug for children after birth and the age of 3 to 5 years, the following ratio of biomass of certain types of bifidobacteria in the preparation is established, wt.%:
Bifidobacterium longum - 38.0 - 52.0
Bifidobacterium bifidum - 30.0 - 42.0
Bifidobacterium breve or Bifidobacterium infantis - Else up to 100%
3. The method according to claim 1, characterized in that upon receipt of the drug for older children and adults, the following ratio of biomass of individual types of bifidobacteria in the preparation is established, wt.%:
Bifidobacterium longum - 32.0 - 42.0
Bifidobacterium bifidum - 29.0 - 39.0
Bifidobacterium adolescentis - The rest is up to 100%
4. The method according to claim 1, characterized in that when receiving the starter culture medium contains reduced skim milk with a dry residue of 12 to 13%, pancreatic milk hydrolyzate, baker's yeast autolysate and ascorbic acid in the following ratio, wt.%:
Pancreatic milk hydrolyzate - 4.5 - 5.0
Baker's yeast autolysate - 0.5 - 1.5
Ascorbic acid - 0.003 - 0.005
Restored skim milk with a dry residue of 12 - 13% - Up to 100

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