RU2294645C2 - Method for production of iodinated products - Google Patents

Abstract

FIELD: food processing industry, in particular production of dairy-based iodinated probiotic products.

SUBSTANCE: claimed method includes preparation of nutrient medium, introducing of potassium iodide solution, heat treatment, cooling, inoculate adding, culturing, cooling, bottling and packing. As nutrient medium clarified curd whey is used, and as inoculate ferment of propionic-acid bacterium strain P. freudenreichii subsp. Shermanii or ferment of bifidobacterium strain bifidodobacterium longum B 379 M. Potassium iodide solution is introduced into nutrition medium before inoculate adding in amount of 57-67 mug/ml when propionic-acid bacteria are used as ferment; and in amount of 52-57 mug/ml when bifidobacteria are used as ferment. After cultivation process whey is separated to produce of bacterial cell suspension.

EFFECT: product with increased iodine content, higher biological value and antimutagenic activity, and increased iodine digestion.

7 tbl, 2 ex, 1 dwg

Description

The present invention relates to biotechnology and can be used in the production of iodinated probiotic milk-based products.

A known method for the production of iodized milk protein - casein (see TU 9229-001-48363077-99 NPP "Medbiofarm"), which provides for the iodination of tyrosine contained in casein under the influence of iodine monochloride with the formation of iodotyrosine and diiodotyrosine.

The disadvantage of this method is that iodine deficiency is caused not only by insufficient intake of iodine from the outside, but also by disturbances in the functioning of various organs of the digestive system, which negatively affects the digestibility of iodine by the body. Therefore, even regular use of iodine casein cannot completely solve the problem of hyperthyroidism.

Closest to the technical nature of the present invention is a method for the production of iodized kefir, which includes preparing the medium, heat treatment, cooling, introducing an iodine source - iodine casein, fermentation, stirring, fermentation, mixing, cooling, bottling, packaging (see TU 9206-005- 48363077-00 MRNTS RAMS, NPP Medbiofarm, Food and Processing Industry Department of the Department of Agriculture and Food of the Kaluga Region).

But the product obtained with this production method does not contain probiotic microorganisms that can survive in the gastrointestinal tract, which significantly reduces its biological value.

The inventors solved the problem of the possibility of using the enzyme system of probiotic microorganisms for iodination in order to produce products that not only contain iodine, but also have a positive effect on the condition of the gastrointestinal tract. From literary sources it is known that the iodination reaction can occur during the breakdown of proteins. Enzymes secreted by microorganisms are able to break down the proteins of the nutrient medium into amino acids, including tyrosine and histidine, etc. The latter are able to form strong compounds with iodine. The iodination reaction can also occur during the synthesis of proteins by microorganisms.

Also, an analysis of literature data showed that thyroid dysfunction occurs in some diseases of the gastrointestinal tract, for example, with insufficient absorption of iodine in the small intestine. Therefore, research was aimed at eliminating the very cause of iodine deficiency.

The technical result of the invention is to increase the digestibility of iodine by the body, maintaining a high iodine content in the product during storage, as well as increasing the biological value and antimutagenic activity of the finished products.

The specified technical result is achieved in that in a method for the production of iodized products, which includes preparing a nutrient medium, heat treatment, cooling, introducing an iodine and inoculum source, mixing, fermenting, cooling, bottling, packaging, according to the invention, a solution of potassium iodide in the amount of 52-67 μg / ml, and as an inoculum, the starter culture on pure cultures of probiotic microorganisms.

In addition, the specified technical result is achieved by the fact that as the inoculum use fermentation of propionic acid bacteria strain P. Shermanii freudenreichii subsp. shermanii.

In addition, the specified technical result is achieved by the fact that as an inoculum use the starter culture of bifidobacteria strain bifidobacterium longum 379 M.

Distinctive features of the claimed invention are the new conditions for the implementation of the fermentation process, namely the introduction of potassium iodide solution into the nutrient medium and fermentation with starter culture on pure cultures of probiotic microorganisms.

The use of probiotic microorganisms, which are regulators of the intestinal microbiocenosis, on the activity of which the state of the whole organism depends, will help to establish the mechanism of iodine penetration into the thyroid gland, and will ensure the intake of a better digestible organic form of iodine.

The research results showed that when a solution of potassium iodide is added to a medium containing tyrosine, histidine, the iodine substitution reaction occurs in the benzene ring of aromatic amino acids.

A significant difference of the claimed invention is a new approach to the development of iodinated products through the use of the enzyme system of probiotic microorganisms.

Experimental studies of the invention were carried out in the laboratories of the departments "Technology of dairy products. Commodity research and examination of goods" VSGTU, "Microbiology" Moscow State University. M.V. Lomonosov, in the problem laboratory of VSTU.

For experimental studies, curd whey was used, in which the nutrients necessary for the growth of microorganisms were added. A potassium iodide solution was added to the resulting medium, fermented with pure cultures of probiotic microorganisms. The following samples were prepared:

Sample No. 1 - nutrient medium fermented with pure cultures of bifidobacteria - Control 1,

Sample No. 2 - a nutrient medium fermented with pure cultures of bifidobacteria with the addition of a solution of potassium iodide in the amount of 52 μg / ml,

Sample No. 3 - a nutrient medium fermented with pure cultures of bifidobacteria with the addition of a solution of potassium iodide in an amount of 57 μg / ml,

Sample No. 4 - a nutrient medium fermented with pure cultures of bifidobacteria with the addition of a solution of potassium iodide in an amount of 67 μg / ml,

Sample No. 5 - nutrient medium fermented with pure cultures of propionic acid bacteria - Control 2,

Sample No. 6 - nutrient medium fermented with pure cultures of propionic acid bacteria with the addition of a solution of potassium iodide in an amount of 57 μg / ml,

Sample No. 7 - a nutrient medium fermented with pure cultures of propionic acid bacteria with the addition of a solution of potassium iodide in an amount of 67 μg / ml,

Sample No. 8 is a nutrient medium fermented with pure cultures of propionic acid bacteria with the addition of a solution of potassium iodide in an amount of 76 μg / ml.

Samples No. 1, 2, 3, 4 were made of starter culture on pure cultures of bifidobacteria, and samples No. 5, 6, 7, 8 were made of starter culture on pure cultures of propionic acid bacteria in an amount of 5% by weight of the nutrient medium.

The following parameters were determined in the obtained samples:

- Biomass build-up

- The content of viable cells of microorganisms

- iodine content

Studies were carried out according to generally accepted methods.

- Antimutagenic activity

Studies were performed using the Ames test.

- Histomorphological studies

Studies were performed by microscopy of stained preparations.

- Content of thyroid hormones

The studies were carried out by a three-phase enzyme-linked immunosorbent assay using standard kits IFA-TT ₄ , IFA-TT ₃ manufactured by NVO "Immunotech".

As a result of the studies, optimal doses of potassium iodide solution were selected that did not inhibit the growth of probiotic microorganisms, which amounted to 57-67 μg / ml for propionic acid bacteria (samples No. 6, No. 7) and 52-57 μg / ml for bifidobacteria (samples No. 2, Number 3).

The results are presented in tables 1, 2.

Table 1
The effect of potassium iodide on the growth of bifidobacteria Samples No. Duration of cultivation, hour four 8 eighteen 24 Sample No. 1 8 · 10 ⁵ 8 · 10 ⁷ 2 · 10 ⁹ 4 · 10 ¹¹ Sample No. 2 8 · 10 ⁵ 6 · 10 ⁷ 3 · 10 ⁹ 4 · 10 ¹¹ Sample No. 3 1 · 10 ⁵ 3 · 10 ⁷ 5 · 10 ⁸ 8 · 10 ¹⁰ Sample No. 4 8 · 10 ⁴ 7 · 10 ⁶ 9 · 10 ⁷ 7 · 10 ⁹ table 2
The effect of potassium iodide on the growth of propionic acid bacteria Dose of iodine, mcg / ml Duration of cultivation, hour 6 12 eighteen 24 Sample No. 5 8 · 10 ⁶ 9 · 10 ⁹ 5 · 10 ¹⁰ 7 · 10 ¹² Sample No. 6 3 · 10 ⁷ 1 · 10 ¹⁰ 6 · 10 ¹⁰ 7 · 10 ¹² Sample No. 7 9 · 10 ⁶ 9 · 10 ⁹ 1 · 10 ¹⁰ 8 · 10 ¹² Sample No. 8 1 · 10 ⁶ 4 · 10 ⁸ 9 · 10 ⁹ 1 · 10 ¹¹

Next, the iodine concentration in iodized concentrates was studied. The results indicate that sample 2 contains about 77% iodine, and sample 7 contained iodine 85%. A higher iodine content in the bacterial concentrate of propionic acid bacteria was noted, which is explained by the fact that peroxidase is formed during their metabolism, which increases the reaction rate and promotes more complete iodination. The rest of the iodine interacts with soluble serum peptides and remains in the culture fluid. The research results are presented in table 3.

Table 3
Iodine content in iodinated bacterial concentrates Samples No. The iodine content, mcg / ml Sample No. 2 40.15 Sample No. 3 42.8 Sample No. 4 43 Sample No. 6 46.8 Sample No. 7 57.4 Sample No. 8 55.6

Thus, based on the foregoing, it was found that the optimal amount of potassium iodide solution administered is 52-67 μg / ml.

Next, the shelf life of iodinated bacterial concentrates was studied. The results are presented in the drawing. When studying the shelf life, it was found that by the end of 3 months of storage, the iodine content in sample No. 2 decreased by 10.2%, and in sample No. 7 - by 9.9%. Minor changes in the amount of iodine can be explained by the fact that iodine in bacterial concentrates is in a bound state, which prevents its volatilization. This allows you to maintain a high iodine content in the product during storage.

Bacteria as sources of antimutagens are of undoubted interest as preventive food additives for the activation of natural repair systems and for the creation of a new type of medicine with antimutagenic properties.

Therefore, in further studies, the antimutagenic activity of iodinated bacterial concentrates was studied. The research results are presented in table 4.

Table 4
Determination of antimutagenic activity of bifidobacteria and propionic acid bacteria Type of microorganisms; Wednesday Cultivation time, hour The average number of revertants per cup Inhibition% Bifidobacteria: Cultivation medium 48 885 twenty Culture fluid 48 710 thirty Cells 48 134 62 Propionic acid bacteria: Cultivation medium 48 885 twenty Culture fluid 48 628 35 Cells 48 70 71.6

As a result of experimental studies, it was found that the culture fluid and cells of propionic acid bacteria and bifidobacteria have antimutagenic activity against mutagenesis induced by 4-nitroquinoline-N-oxide. The strongest antimutagenic effect was found in the culture fluid and cells of propionic acid bacteria, reaching 35% and 71.6% inhibition, respectively. Perhaps this is due to the fact that propionic acid bacteria synthesize significant amounts of antioxidant enzymes: superoxide dismutase, peroxidase and catalase. The simultaneous presence of these enzymes allows the cell to remove superoxide and peroxide radicals formed in oxidative reactions.

To confirm the effectiveness of the method for the production of iodinated products based on bacterial concentrates of bifidobacteria and propionic acid bacteria, an experimental test of the latter was performed on rats of the Wistar strain. The degree of digestibility of iodine was judged by the content of thyroid hormones in the blood serum and histomorphological studies of the thyroid glands of Wistar rats.

In experiments, administration of mercazolil to animals at a dose of 25 mg / kg for 14 days led to hypothyroidism. The data obtained are presented in table 5.

Table 5
The effect of iodinated concentrates on the level of thyroid hormones in the blood serum of Wistar rats with experimental mercazolyl hypothyroidism No. Group of animals Dose The level of thyroid hormones, nmol / l T ₄ total T ₄ St. T ₃ one Intact - 118.6 25.6 1.8 2 Merkazolil 25 mcg 61.5 15.92 0.95 3 Merkazolil + Yodbifioniks 50 mcg 133.3 24.86 1,59 four Merkazolil + Iodpropionics 50 mcg 117.3 27.1 1.93 5 Merkazolil + "KI 200" 50 mcg 104.3 24.75 1.38

As can be seen from table 5, the use of iodized concentrates for 14 days helps to restore the levels of thyroxine and triiodothyronine in the blood serum to the level of those of intact animals with mercazolyl hypothyroidism. The introduction of iodized concentrates is more effective than the introduction of "KI 200". The presence of this effect, apparently, can be explained by the fact that the enzyme system of microorganisms contributes to a more complete assimilation of iodine by the animal organism.

It was noted that the action of an iodized concentrate of propionic acid bacteria is more effective compared to an iodized concentrate of bifidobacteria, since the content of the bound thyroxine and triiodothyronine in the former is higher.

Perhaps this is due to the fact that peroxidase synthesized by propionic acid bacteria catalyzes the iodine addition reaction.

At the next stage, we studied the change in the morphological structure of the thyroid glands of animals of various experimental groups. The experimental results are shown in tables 6 and 7.

Table 6
The area of tissue components of the thyroid glands of rats (%), M ± m, n = 10 Group Follicular epithelium Parafollicular Endocrinocytes Colloid Vascular bed Connective tissue Follicular colloid index Intact 45.86 ± 0.27 14.15 ± 0.34 20.05 ± 0.26 12.96 ± 0.27 7.39 ± 0.21 2.29 ± 0.11 Merkazolil 49.56 ± 1.04 24.96 ± 0.91 8.96 ± 0.11 16.12 ± 0.93 3.25 ± 0.17 5.53 ± 0.38 Merkazolil + Yodbifioniks 46.02 ± 1.17 15.18 ± 0.47 20.62 ± 0.68 12.07 ± 0.81 6.11 ± 0.27 2.23 ± 0.24 Merkazolil + Iodpropionics 45.89 ± 0.25 15.12 ± 0.36 20.53 ± 0.45 12.02 ± 0.26 7.68 ± 0.15 2.24 ± 0.31 Merkazolil + Potassium iodide 200 47.56 ± 0.26 17.58 ± 0.41 18.26 ± 0.24 14.06 ± 0.29 5.87 ± 0.31 2.6 ± 0.19 Table 7
Morphometric indicators of the constituent components of rat thyroid follicles, (μm), M ± m, n = 10 Group Follicle Diameter (D) Thyrocyte Height (h) Thyrocyte core diameter (d) Intact 22.62 ± 0.28 6.58 ± 0.29 3.24 ± 0.68 Merkazolil 18.39 ± 0.49 7.51 ± 0.76 5.2 ± 0.64 Merkazolil + Yodbifioniks 22.02 ± 0.47 6.91 ± 0.11 3.84 ± 0.14 Merkazolil + Iodpropionics 22.59 ± 0.34 6.95 ± 0.58 3.45 ± 0.25 Merkazolil + Potassium iodide 200 20.87 ± 0.25 7.03 ± 0.33 4.23 ± 0.36

Thus, it was found that the thyreostatic effect of mercazolil at a dose of 25 mg / kg for 14 days leads to functional overstrain of the thyroid gland, which may be explained both by a direct effect on thyrocytes and indirectly through hypothalamic-pituitary regulation. The results of the studies revealed that the use of iodinated concentrates of bifidobacteria and propionic acid bacteria at a dose of 50 μg / mg for 14 days restores the functional dependence of the thyroid gland in animals to the level of intact animals.

From the foregoing, it follows that the use in the present method of starter culture on pure cultures of bifidobacteria and propionic acid bacteria promotes the formation of strong iodine-containing organic compounds that are well absorbed by the body, and the iodine contained in them is involved in the formation of thyroid hormones. In addition, the developed products have high biological value and antimutagenic activity.

Thus, in the course of experimental studies it was proved that it is the claimed combination of distinctive features of the invention that ensures the achievement of the above technical result. This allows us to conclude that the claimed technical solution meets the criteria of patentability "novelty" and "inventive" level.

In the inventive method, use the starter culture on pure cultures of bifidobacteria, manufactured according to TU 9229-002-02069473-2005, the starter culture on pure cultures of propionic acid bacteria, manufactured according to TU 9229-007-02069473-2005.

The inventive method is as follows.

After preparation of the nutrient medium, it is deoxidized to a pH of the medium in the range (7.0-7.1). The finished medium is sterilized at a temperature of (120 ± 1) ° C for 30 minutes, then cooled to a temperature of (30-40) ° C. A solution of potassium iodide is introduced into the nutrient medium, fermented with ferment on pure cultures of probiotic microorganisms. The growth of microorganism cells is carried out at a temperature of (30-40) ° C for (24 ± 2) hours under conditions of periodic cultivation with a single neutralization of the culture fluid after 12 hours, maintaining the pH at the optimum level with a saturated sterile solution of sodium carbonate (Na ₂ CO ₃ ) . After the cultivation process is complete, serum is separated to obtain a bacterial cell suspension, which is cooled to a temperature of (4 ± 2) ° C, poured into sterile vials, sealed, and stored.

The method is illustrated by the following examples of specific performance.

Example 1

The medium for the growth of bifidobacteria is clarified curd whey with the addition of buffer salts, ascorbic acid and agar. The clarification of cottage cheese whey is carried out by heating to a temperature of 95 ° C and holding to more fully isolate proteins for 60 minutes. After this, the serum is clarified by filtration.

After the preparation of the nutrient medium, it is deoxidized to a pH of 7.0. The finished medium is sterilized at a temperature of 120 ° C for 30 minutes, then cooled to a temperature of 37 ° C. A 10% solution of potassium iodide in an amount of 52 μg / ml is added to the nutrient medium, fermented with ferment on pure bifidobacteria cultures. The growth of bifidobacteria cells is carried out at a temperature of 37 ° C for 24 hours under conditions of periodic cultivation with a single neutralization of the culture fluid after 12 hours, maintaining the pH at an optimal level with a saturated sterile solution of sodium carbonate (Na ₂ CO ₃ ). After the cultivation process is completed, serum is separated to obtain a bacterial cell suspension, which is cooled to a temperature of 4 ° C, poured into sterile vials, sealed with sterile rubber stoppers under aseptic conditions, and rolled up with aluminum caps on a semiautomatic device, glued on a label, and stored.

Example 2

The medium for the growth of propionic acid bacteria is clarified curd whey with the addition of buffer salts, ascorbic acid and agar. The clarification of curd whey is carried out by heating to a temperature of 95 ° C and maintain it for a more complete release of proteins for 60 minutes. After this, the serum is clarified by filtration.

After the preparation of the nutrient medium, it is deoxidized to a pH of 7.0. The finished medium is sterilized at a temperature of 120 ° C for 30 minutes, then cooled to a temperature of 30 ° C. A 10% solution of potassium iodide in an amount of 67 μg / ml is added to the nutrient medium, fermented with ferment on pure cultures of propionic acid bacteria. Propionic acid bacteria cells were grown at a temperature of 30 ° С for 24 h under periodic cultivation conditions with a single neutralization of the culture fluid after 12 hours, maintaining the pH at the optimum level with a saturated sterile solution of sodium carbonate (Na ₂ CO ₃ ). After the cultivation process is completed, serum is separated to obtain a bacterial cell suspension, which is cooled to a temperature of 4 ° C, poured into sterile vials, sealed with sterile rubber stoppers under aseptic conditions, and rolled up with aluminum caps on a semiautomatic device, glued on a label, and stored.

Claims (1)

A method for the production of iodized dairy products with the preparation of a nutrient medium, the introduction of a solution of potassium iodide, heat treatment, cooling, making an inoculum, cultivation, cooling, bottling, packaging, characterized in that as a nutrient medium use clarified curd whey, as an inoculum use starter culture of propionic acid bacteria strain P.freudenreichii subsp. shermanii or yeast of bifidobacteria of the strain Bifidobacterium longum B 379 M, a solution of potassium iodide is introduced into the nutrient medium before the inoculum in the amount of 57-67 μg / ml when used as a starter propionic acid bacteria and in the amount of 52-57 μg / ml when used as a starter bifidobacteria, and after the cultivation process is complete, serum is separated to obtain a bacterial cell suspension.