LT4900B - Symbiotic mixture of five bifidobacteria and foodstuff containing the same - Google Patents

Abstract

The invention is in the field of food products and covers a vast assortment of dairy products, made of fresh or fermented milk, as well as non-fermented food products, like beer or mayonnaise, containing a vital symbiotic mix of bifida bacteria distributed in the human intestine. The mix is made of Bifidobacterium bifidum AS 1 kind (deposited in International Museum of Microorganisms in Latvia, collection number P569) and AS 6 kind (deposited in the same place, collection number P573), Bifidobacterium breve AS 4 kind (deposited in the same place, collection number P572), Bifidobacterium adolescentis AS 3 kind (deposited in the same place, collection number P571), and Bifidobacterium longum AS 2 kind (deposited in the same place, collection number P570), at an even mass ratio with the concentration in the food products 10<6> - 10<7> V/ml. Such food products are beneficial to people of various ages in improving the immune system; they act against the negative effects of pathogenic micro flora in the human intestine. The invention presents the manufacturing process of food products with this symbiotic mix of bifida bacteria.

Description

pathogenic microflora which are found in the human intestine to be affected by various harmful effects.

The present invention relates to a process for the production of foodstuffs containing this symbiotic mixture of bifldobacteria.

The present invention is from the field of microbiology which provides a symbiotic mixture of bifidobacteria consisting of five viable bifibobacteria which can be used in the preparation of a variety of food products containing this mixture. The present invention further provides food products containing the mixture and a process for their preparation.

There is a very close relationship between human health and its gut microflora. The microflora of a healthy person is dominated by bifidobacteria, which perform various physiological functions, the most important of which are protective and synthesizing. They are also involved in the final stage of the digestive process (Dalias G. Hoover, Food

Technology, June 1993, 120-124).

There are five main types of bifidobacteria known to be found in the body of people living in Europe. These include Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve and Bifidobacterium infantis. The first three adult intestinal flora make up the major part of bifidobacteria (40 to 75%). These bifidobacteria are disappearing in the human body due to the current high levels of sterilized food consumed by mankind and various stresses, ecological conditions, illness, antibiotic treatment, radiation therapy or chemotherapy. Decrease in bifidobacteria is one of the signs of dysbacteriosis. Normal intestinal microbiocenosis can be maintained by the use of biologically valuable products with bifidobacteria capable of inhabiting the intestine, preventing the development of putrefactive and pathogenic microflora (U.S. Patent No. 4,298,819). As a result, humanity has been consuming a variety of foods containing the aforementioned bifidobacteria for a number of years to repair dysbacteriosis in the body. (U.S. Patents No. 4,298,819,

4645667, 4806368; EP - 0667107, FR - 2560046).

There are known fermented milk products containing bifidobacteria that are made from fermented whole milk or skimmed milk. The following bifidobacteria are used as milk yeast for fermentation of milk: B. adolescentis strain VKPM B-1987 (ex SU I. 863639), B. adolescentis strain MC-42 B-1987 (VNIIGenetika college) (LT patent No. 3955). ), (B. longum strain VKPM B-2000 (ex. SU aut. I. No. 849779), B. bifidum strain VKPM B-3300 (ex. SU aut. I. No. 1347221).

The main disadvantage of these products is that different products provide different therapeutic effects to different human beings, since only a certain type of intestinal bifidobacteria or their combinations dominates in each individual human organism. This deficiency is partially eliminated by the use of a symbiotic yeast of two bifidobacteria consisting of B. bifidum strain VKPM B-3300 and B. longum strain VKPM B-2000 (formerly SU No. 1817185). The use of 2 bifidobacterial yeasts is described to shorten the tanning time and increase the amount of active bifidobacteria to 10 ¹ ° units / ml.

Also known are fermented milk products containing three major types of bifidobacteria, B. bifidum (strain VKPM B-3300), B. longum (strain VKPM B-2000) and B. adolescentis (strain VKPM B-1987) the concentration in the medium of the fermented product is not less than 10 ⁸ units / ml. (LT Patent No. 3972).

Going from the principle of optimal integrity of wildlife, it is inappropriate to produce fixed mono or bi-type bifidobacteria products over a long period of time, as this can cause adverse changes in the microbial status and gut flora of millions of people. The consequences of these changes can be poorly predicted in practice, as they may occur many years after the product has been used. The product with live bifidobacteria has a shelf life of 2 to 7 days, depending on the type of packaging (some products, such as yogurt, can be used for longer). It follows that the product is produced by national companies in each region, as the export and import of the product can be restricted not only by the short time the product is sold, but also by customs formalities and various controls.

It is an object of the present invention to provide a symbiotic mixture of viable bifidobacteria consisting of five strains of bifidobacteria commonly found in the human body, which can be added to a wide variety of foods such as various fermented milk products, various fermented milk products and non-fermented foods. types of beer and mayonnaise, etc.) and provide a method of making these products.

The object is achieved by providing a symbiotic mixture of viable bifidobacteria consisting of 5 strains of bifidobacteria such as Bifidobacterium bifidum

AS 1 strains (deposited at the Latvian International Museum of Microorganisms, collection P569) and AS 6 strains (deposited at Ibid, collection P57-3),

Bifidobacterium breve AS 4 strains (deposited ibid., Collection No. P572), Bifidobacterium adolescentis AS 3 strains (deposited ibid., Collection No. P571) and Bifidobacterium longum AS 2 strains (deposited ibid., Collection No. P570) which form a mixture taken in equal parts. All these bifidobacterial strains are stored and propagated by AB Bacterial Preparations in Kaunas. Their analysis is performed according to the methodologies described in Bergey's Manual of Systematic Bacteriology, V. 2, 1986, 1424-1430.

The bifidobacterial symbiotic mixture is obtained by mixing the above five viable bifidobacterial strains which are close in their cultural and physiological-biochemical properties. The Bifidobacterial Symbiotic Mixture is characterized by the fact that it has a common complex of properties that are specific to individual strains of bifidobacteria, allowing the mixture to enter the human intestine faster and, due to its broad specific activity, normalize the intestinal microflora faster. Bacteriology, V. 2, 1986, 1424-1430.

A symbiotic mixture of five viable bifidobacteria: B. bifidum

The cultural and morphological characteristics of AS1 strain, B. longum AS 2 strain, B. adolescentis AS 3 strain, B. breve AS 4 strain, B. bifidum AS 6 strain are shown in Table 1.

Table 1

Quality indicators Requirements according to normative-technical documentation Results of the analysis Description Puri, a brownish mass Compliant Solubility No> 5 minutes 3 min PH 5.7 ± 1.3 6.32 Residual humidity No> 3.5% 2.07% Identity Morphological properties: in aram method in tea - whether * non-porous rods with protrusions and ends at the ends or grain sticks with ends at the ends. Compliant

Cultural characteristics: Thiolicol terroes grow in the form of nails, grains (80%) and crumbs (20%); does not grow on rigid surfaces under aerobic conditions. Biochemical Properties: Catalytic Reaction Not Available. does not contain urease, does not liquefy gelatin, fermentes glucose, lactose, sucrose; does not ferment mannitol, maltose, salicin, xylose, arabinose, cellobiose, mannose, melocytosis, raffinose, sorbitol, rhamnose, trehalose. Antibiotic sensitivity: sensitive to penicillin, cefzol. Compliant Corresponds to "Api - System 20A Compliant for gentamicin. By-product contamination microflora No microflora present There is no Toxicity Must be nontoxic Non-toxic Specific activity (viable cells) quantity 1 ml) No <5x10 ' 11.35x10 " Acid formation Not <90 ° T 131.6 ^O T Antagonistic activity test strains S. flexneri 170, S. flexeksi 337, S. sonnei 5063 with respect to In mixed cultures, the cell cultures survived in the test cultures are not more than 2% compared to control cultures. Test cultures did not grow in mixed crop crops

The industrial cultivation of Bifidobacteria in milk medium is quite difficult as it requires strict anaerobic conditions, which requires expensive hardware and a high level of cultivation techniques. Bifidobacteria do not grow in pure cow's milk and need growth stimulants. In the present invention, three growth media for bifldobacteria were used:

-1. corn-lactose;

- 2. thioglycol; and

- 3. Blaurok's medium.

Thioglycol medium is most desirable because of its ease of preparation and use.

It contains all the ingredients needed for growth and reproduction of bifldobacteria.

The lyophilized five strains of bifldobacteria resuscitated in these media are multiplied to the required amount, then mixed and added to the required prepared food product.

The concentration of bifldobacteria in the food is 10 ⁶ -10 ⁷ U / ml. This concentration of bifidobacteria is sufficient for a positive effect on the human body and, in addition, does not cause an increased rate of fermentation of unleavened dairy products. Products with five strains of bifidobacteria have prophylactic healing properties, have a wide range of homeopathic effects on pathogenic microorganisms, in the case of dysbacteriosis restore the bifidobacterial flora of the digestive tract and strengthen the human immune system.

The patented symbiotic mixture of viable bifidobacteria of the present invention is used in the production of a wide range of non-fermented milk products such as natural pasteurized milk, skimmed pasteurized milk, pasteurized milk (0.5%, 1.0%, 2.0%, 2.5% and 3.2). % fat), pasteurized protein milk (1.0% and 2.0% fat), sour milk products such as kefir (1.0%, 2.5%, 3.2% fat), sour cream (25% and 30% fat) and yogurt, non-fermented foods such as all types of beer or mayonnaise.

The proposed method of production of non-fermented milk products is as follows: for the production of pasteurized milk, skimmed milk, protein milk or other raw materials, the fat content is normalized by separating the milk to the required fat content or by adding cream or fat milk to lean milk. The milk selected for the production of protein milk is normalized to the required fat content and then to the required dry matter content by the following methods. The protein content shall be increased by the addition of a calculated quantity of condensed skimmed milk, reconstituted skimmed milk or milk powder.

The milk, normalized to fat and solids, is then heated and cleaned by centrifugal cleaners. To improve the taste, all milk with a fat content of more than 2,5% is homogenised before or after pasteurization. The purified milk at 45-47 ° C is homogenized at a pressure of 10-15 MPa.

Further, normalized or natural, homogenized or non-homogenized milk is pasteurized at 74-78 ° C for 20 sec. The milk is cooled to 15-17 ° C, the bifidobacterial symbiotic mixture concentrate (1.0 l / ton) is added and the mixture is stirred for 5-10 minutes. All milk is then cooled to 6 ° C and packed in standard packages.

The resulting products are stored at a temperature of not more than 6-8 ° C for up to 72 hours. Depending on the packaging, the lead time may be extended.

Organoleptic characteristics of pasteurized milk containing bifidobacteria:

Appearance and consistency: a homogeneous fluid with no sediment or flakes.

Color: white or yellowish white; lean to bluish; frosted - creamy shade.

Taste and smell: characteristic of milk, with no foreign taste or smell; pronounced pasteurization spice of frothed milk; milk with dry or condensed product - a sweet spice. May have a faint additive spice.

The microbiological characteristics of the pasteurized milk are presented in Table 2.

table

Name of the indicator Normal Total number of bifidobacteria per ml of product, not less than 8ο ⁸ Total bacterial count of at least 1 ml of product per ml Noah ⁵ Coliform bacteria per ml product (must be absent) 0.1 Enteropathogenic bacteria 1 mi product (must be absent) 25th S. aureus 1 ml product (must be absent) 0.01

Physico-chemical characteristics of pasteurized milk are given in Table 3.

table

Kind of milk Fat%, at least how Dry materials %, no less how Acidity, T °, no more how Phosphatase Cleanliness degree, no lower groups how Density in kg / m ³ , not less than natural 3.5 - 18th - 1 1027 skinny - - 21st - 1 1030 1.0% fat content 1.0 21st - 1 1029 2.0% fat content 2.0 - 21st - 1 1027 2.5% fat content 2.5 - 21st - 1 1027 3.2% fat content 3.2 - 21st - 1 1027 pasteurized protein-rich milk 1.0% 1.0 12.6 21st 1 1037

fat pasteurized protein milk 2,0% fat 2.0 11.2 21st 1 1036

Sour milk products (kefir, yogurt, sour cream) are manufactured according to state standards, and 0.1 wt. Such products are extremely valuable because, in addition to viable bifidobacteria, they also contain the leaven of lactic acid cultures.

The physico-chemical characteristics of kefir with bifidobacteria are presented in Table 4.

table

Kind of Kefir Fatness, less,% no Acidity, ° T Temperature, no higher, ° C Peroxidase 1.0% fat content 1.0 85-120 8th There is no 2.5% fat content 2.5 85-120 8th There is no 3.2% fat content 3.2 85-120 8th There is no skinny - 85-130 8th There is no

Organoleptic characteristics of kefir with bifidobacteria:

Consistency and appearance: homogeneous with uneven flakes; there may be bubbles, the surface may contain whey (no more

2 kefir volume%).

Color: milky white, slightly creamy.

Taste and smell: lactic, fresh, pungent, with no foreign taste or smell. The nutritional and energy value of 100 g of kefir with bifidobacteria is given in Table 4.

table

Kind of Kefir Protein, g Fat, g Carbs, g Energetic value, kcal 1.0% fat content 3.5 1.0 5.3 42 2.5% fat content 3.4 2.5 3.8 51 3.2% fat content 3.4 3.2 4.1 59 skinny 3.0 0.05 3.8 27.6

The physico-chemical characteristics of sour cream with bifidobacteria are shown in Table 5.

table

Sour cream species Fatness, less,% no Acidity, ° T Temperature, no higher, ° C Peroxidase 25% fat content 25th 60-100 8th There is no 30% fat content 30th 60-100 8th There is no

Organoleptic characteristics of sour cream with bifidobacteria (both fats):

Consistency and appearance: viscous, sufficiently dense, shiny.

Salva: White, yellowish in color, uniform in volume.

Taste and smell: lactic acid, characteristic of the pasteurized product.

The nutritional and energy value of 100 g of sour cream with bifidobacteria is given in Table 6 15.

table

Kind of sour cream Protein, g Fat, g Carbohydrates, g Energy value, kkal 25% fat content 26th 25th 2.9 247 30% fat content 26th 30th 2.8 291

The following examples illustrate the invention.

example

1. Preparation of Bifidobacterial culture medium

1.1. Preparation of maize-lactose medium

Composition: g /! peptone 10.0 lactose 10.0 corn extract solution in water 1: 6 50 ml sodium citrate 6.0 magnesium sulfate 0.12 potassium dihydrogen phosphate 2.0 sodium dihydrophosphate 1.0 cystine hydrochloride 0.15 or ascorbic acid 0.5 agar 1.0 distilled water what remains to 1000 ml pH 7.0-7.2.

Preparation:

Add 1 g of agar to 150-200 ml of distilled water and melt in a water bath until completely dissolved. to the remaining (800-850 ml) distilled water add 10 g of peptone, 50 ml of water-diluted corn extract, 6 g of sodium citrate, 0.12 g of magnesium sulphate, 2 g of potassium dihydrogen phosphate and 1 g of sodium dihydrogen phosphate. The resulting mixture is heated to 85-90 [deg.] C., then mixed with molten agar, 10 g of lactose and 0.15 g of cystine hydrochloride or 0.5 g of ascorbic acid are added. (Cystine is pre-dissolved in 20-30 ml of distilled water with 30-40%

NaOH solution until everything is dissolved).

The resulting solution was made up to 1 L with hot distilled water and adjusted to pH 7.0-7.2 with 30-40% NaOH (about 10 mL).

The medium is sterilized for 30 minutes in two liter containers at 0.05 mPa, twice for at least 8 hours. is not longer than 20 or less. break.

The reconstituted medium should be stored for up to 1 month at 20 ° C. Before use, heat the medium for 10 to 15 minutes in a boiling water bath and then cool to 37-38 ° C.

1.2. Preparation of thioglycolic medium

Composition: aZI enzymatic hydrolyzate of casein 15.0 yeast extract (vitamin preparation) 5.0 sodium chloride 2.5 glucose 5.0 thioglycolate sodium 0.5 cysteine dihydrochloride 0.75 agar 0.75 sodium carbonate 0.6-1.0. pH 6.8-7.2.

OXOID dry powder is used. The medium is prepared as follows: 1 liter of distilled water contains sodium thioglycoclate and agar. The mixture is heated until all is dissolved, then the remaining components are added and the pH adjusted to 6.8-7.2 with sodium carbonate. The reconstituted medium is dispensed into vials or tubes and sterilized at 121 ° C for 15 minutes. The prepared medium is kept in the refrigerator for up to 1 month.

1.3. Preparation of the nasty medium

Composition: a / To a decoction of the liver 1.01 sodium chloride 5.0 peptone 10.0 agar 0.75 lactose 10.0 L-cysteine hydrochloride 0.1. pH 7.2-7.4 Liver decoction oamvba:

Fresh non-frozen animal liver is removed, film is removed, skin is removed, and bile ducts are removed. The prepared liver is cut into pieces and weighed. 1 kg of liver is filled with 2 l of distilled water and boiled

1 hour. The strainer is then filtered through a paper filter into bottles, capped and sterilized by autoclaving at 112 ° C for 30 minutes under flowing steam.

The decoction of the liver can be stored for up to 1 year at a temperature not exceeding 20 ° C.

Media Preparation:

Add the prepared liver decoction, peptone, sodium chloride and agar in 5 volumes, heat until the agar is dissolved, adjust to pH 7.8-7.9, then boil for 20 minutes on low heat, leave to settle and filter. The resulting solution is diluted to 1 L with distilled water. Lactose and L-cysteine hydrochloride are then added, mixed thoroughly and dispensed into vials or tubes and sterilized at 112 ° C for 30 minutes.

The Blauroc medium thus obtained is suitable for use for 1 month.

example

Preparation of a viable bifidobacterial symbiotic mixture concentrate

2.1. Revitalization of Bifidobacterial strains:

Bifidobacterial strains are resuscitated using maize-lactose, thioglycose, or Blauroc medium.

add 4 ml of the lyophilized bifidobacteria to each vial (thoroughly wipe the alcohol stopper) with 4 ml of the culture medium. Mix well and transfer 1 ml of bacterial suspension into 10 ml of culture medium. Mix thoroughly and transfer 1 ml of the bacterial suspension into a successive tube containing 10 ml of culture medium and then 1 ml into a third tube with 10 ml of culture medium.

The tubes are kept in a thermostat at 37 ° C for 48-72 hours until the medium begins to cloud (vigorous growth).

This revives all five strains of bifidobacteria that can be stored at 2-8 at 25 ° C for up to 30 days, after adjusting the pH to 6.2-7.0. The pH is adjusted with 5% NaOH solution. 0.3 ml of 5% NaOH are added to 10 ml of the grown bifidobacterial culture. Stir well, leave at room temperature for several hours and then place in refrigerator for storage.

2.2. Preparation of viable bifidobacterial symbiotic mixture concentrate:

From 2.1. Bifidobacteria, an additive for milk, kefir, sour cream or other non-fermented products, prepared in the first stage (revitalized) of bifidobacteria.

1. Add 5 ml of reconstituted bifidobacterial medium to 100 ml of thioglycol culture medium. Cultured for 16-24 hours at 37 ° C.

2. Prepare the required amount of bifidobacteria from the grown bifidobacterial 'inoculum' (about 105 ml) by adding the medium to 2 l of a new nutritional swab to add 1 tonne of milk product to the 11 bifidobacterial mixture.

If there is 10 tonnes of milk product (milk, kefir, sour cream or other 5 non-fermented products), it is necessary to prepare, for example, 2 liters of suspension of each strain in thioglycol nutrient medium to produce a viable symbiotic mixture of 5 liters of bifidobacteria placed in containers of 10 tonnes of chilled milk, kefir, sour cream or other non-fermented product such as beer, mayonnaise, mix well (about 5 to 10 minutes), cool and pour into appropriate packaging for that product.

example

Method of Production of Mayonnaise Containing a Viable Symbiotic Mixture of Bifidobacteria Into 1 tonne of ready-to-use mayonnaise (reduced to 60% fat) is added 1 15 liters of a symbiotic mixture of bifidobacteria prepared according to Example 2. The resulting product is thoroughly mixed (6-7 minutes), packaged in a consumer container, cooled to 2-6 ° C, where it is kept for the shelf life. The energy value per 100 g is 388 kcal.

Example 4

Method of production of yogurt containing a viable mixture of bifidobacteria

One liter of ready-to-eat yogurt is added with 1 liter of a symbiotic bifidobacterial mixture prepared according to Example 2. The resulting product is thoroughly mixed (6-7 minutes), packaged in a consumer container, cooled to 2-6 ° C, where it is kept for the shelf life. 100 g of such yogurt contains: 0.7 g protein; fat -1.0 g; carbohydrates - 8.2 g.

example

Method of Production of Beer Containing a Viable Bifidobacterial Symbiotic Mixture 30 To 10 tonnes of ready-to-dispense fresh unpasteurized strong beer is added 10 l of a symbiotic bifidobacterial mixture prepared according to Example 2. The resulting product is thoroughly mixed (6-7 minutes), packed in consumer containers, cooled to 2-12 ° C, where it is kept for the shelf life.

Claims (8)

DEFINITION OF INVENTION 1. A symbiotic mixture of viable bifidobacteria, characterized in that it consists of five viable bifidobacteria, such as Bifidobacterium bifidum strain AS 1 (deposited in the Latvian International Organization for Microorganisms). 5 in Museum, Collection no. P569) and AS 6 strains (deposited ibid, collection no P573), Bifidobacterium breve AS 4 strains (deposited ibid, collection no P572), Bifidobacterium adolescentis AS 3 strains (deposited ibid, collection no P571) and Bifidobacterium longum AS 2 strains (deposited ibid., Collection No. P570) in the same weight ratio. 2. A fermented milk product containing bifidobacteria, characterized in that it contains 0.1% by weight of a viable bifidobacterial symbiotic mixture according to claim 1 in a concentration of 10 ^{6 to} 10 ⁷ U / ml. 15th 3. A non-fermented milk product according to claim 2, characterized in that it may contain a non-fermented milk product, such as natural pasteurized milk, skimmed pasteurized milk, 1%, 2%, 2, 5% or 3,2% fat milk or 1%, and 2% fat pasteurized protein milk containing a viable bifidobacterial symbiotic mixture according to claim 1. 4. A fermented milk product containing bifidobacteria, characterized in that it contains 0.1% by weight of a viable bifidobacterial symbiotic mixture according to claim 1 in a concentration of 10 ^{6 to} 10 ⁷ U / ml. 25th 5. A fermented milk product according to claim 4, characterized in that it may contain a fermented milk product such as 1%, 2.5%, 3.2% fat kefir or 25% and 30% fat cream or various types of yogurt containing viable yoghurt. a bifidobacterial symbiotic mixture according to claim 1. 30th An unfermented food containing a bifidobacterium, characterized in that it contains 0.1% by weight of a viable bifidobacterial symbiotic mixture according to Claim 1 at a concentration of 10 ⁶ -10 ⁷ U / ml in the product. 7. An unfermented food product according to claim 6, characterized in that it may contain various types of mayonnaise or beer. 8. A process for the preparation of a food product according to claims 2 to 7, comprising the step of preparing and chilling the food product according to a defined state standard, wherein 0.1 wt.% Of viable bifidobacteria are added to the prepared and chilled food at 15-17 ° C. of the symbiotic mixture of claim 1, mix well for 6-7 minutes, and store the resulting product at 6-8 ° C.