JPH0541981A - Liquid composition containing live bacteria - Google Patents

Abstract

(57) [Summary] [Purpose] To enhance the survival of viable bifidobacteria and / or viable lactic acid bacteria contained in a liquid composition. [Composition] A viable cell-containing liquid composition containing at least viable cells of bifidobacteria and / or viable cells of lactic acid bacteria, and lactoperoxidase at a concentration of 10 ppm or more, wherein the viable cell-containing liquid composition contains lactoperoxidase. Decomposes hydrogen peroxide generated in the. [Effect] Since useful microorganisms can survive for a long period of time and only a small amount of a natural product is added, the manufacturing cost is low and the hygiene is safe.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liquid composition containing a viable bacterium of a useful microorganism characterized by comprising at least a viable bacterium of a useful microorganism and lactoperoxidase.

The microorganisms useful in the present invention are microorganisms that live in the intestinal tract of humans or animals or other digestive tracts and are physiologically useful for humans or animals (for example, bifidobacteria, lactic acid bacteria, etc.), and live bacteria survive. Is a useful microbe, which is a liquid composition containing a viable bacterium, a liquid or semi-solid food product containing a viable bacterium of a useful microbe utilizing the above-mentioned useful microbe (for example, yogurt, lactic acid bacterium drink, etc.), a drug, a feed , Pet food, or raw materials thereof.

[0003]

Fermented foods such as yogurt, cheese, pickles and natto are the most common compositions containing live microorganisms, but in recent years, live microorganisms of useful microorganisms have been directly ingested,
Foods, feeds, pet foods, etc., containing live bacteria of Bifidobacterium and / or lactic acid bacteria that live in the intestinal tract of humans and animals or other digestive tracts are widely marketed for the purpose of physiological effects. In these compositions,
It is desirable that the target microorganisms contain viable bacteria in a concentration as high as possible, that the viable bacteria can be maintained at a high concentration during the storage period, and that the viable bacteria can be ingested at a high concentration.

However, it has been extremely difficult to secure the viable cell count at a high concentration during the storage period in the conventional composition containing the viable cell, and the viable cell concentration necessary for obtaining a physiological effect is not so high. It was no exaggeration to say that the majority of cases could not be maintained. For example, it is said that it is desirable for yogurt containing viable Bifidobacteria to maintain a viable Bifidobacterium viable count of at least 10 million per ml, but in many cases the viable count of Bifidobacterium cannot be maintained during storage.

Although many factors that affect the maintenance of the viable cell count as described above are known, the influence of oxygen contained in the composition cannot be ignored. That is, it is known that oxygen is particularly harmful to anaerobic and facultative anaerobic bacteria. Therefore, in order to eliminate the influence of oxygen, it is necessary to prevent the dissolution of oxygen in a composition containing live bacteria as much as possible. However, it has been considered as one solution in the past. For example, yogurt containing live bacteria of Bifidobacterium and a container for dairy products are made of a material that blocks the permeation of oxygen to prevent the effect of oxygen on the contents, but the actual production of yogurt and dairy products. In the process, it is impossible to prevent the dissolution of oxygen to some extent, which adversely affects the survival of viable bacteria during the storage period.

One of the toxicities of this enzyme is due to hydrogen peroxide produced from oxygen by the NADH oxidase possessed by useful microorganisms as shown in Chemical Formula 1, and the produced hydrogen peroxide exhibits toxicity. That is, the useful microorganisms produce hydrogen peroxide by the enzyme possessed by themselves, which adversely affects their survival.

[0007]

[Chemical formula 1]

On the other hand, the following is known about lactoperoxidase. This enzyme is an enzyme that is present in human and animal milk and body fluids, and accounts for about 1% (weight, the same applies to the following unless otherwise specified) of whey protein in raw milk. Lactoperoxidase is contained in unheated milk while maintaining its activity, but it is inactivated by heating (eg, normal milk sterilization conditions), so this enzyme is completely inactivated in fermented dairy products, for example. is doing. This enzyme also decomposes hydrogen peroxide in the presence of oxygen receptors, but SC
When N ^- is used as an oxygen acceptor, its product is 0SCN
^- a indicates the bacteriostatic action against many microorganisms due to the high antimicrobial, is used as lactoperoxidase system. However, this enzyme does not make SCN ^- only a specific oxygen acceptor, but has the action of decomposing hydrogen peroxide in the presence of a non-specific oxygen acceptor as in Chemical Formula 2.

[0009]

[Chemical formula 2]

[0010]

As described above, there has been a long-awaited demand for the development of an effective means for survival of viable bacteria of useful microorganisms. The present inventors, as a result of intensive research on a method for survival of viable bacteria of useful microorganisms (especially bifidobacteria) in a liquid composition containing viable bacteria of useful microorganisms such as bifidobacteria and / or lactic acid bacteria Hydrogen peroxide is produced from oxygen contained in a liquid composition containing useful microorganisms, and the hydrogen peroxide is harmful to the survival of useful microorganisms, and hydrogen peroxide is a viable bacterium of useful microorganisms. It has been found that live bacteria of useful microorganisms can be preserved without being killed for a long period of time by being removed from a liquid composition containing the.

[0011] An object of the present invention is to provide a liquid composition containing a viable bacterium of a useful microorganism that can survive for a long period of time, is inexpensive to manufacture, and is safe in terms of hygiene.

[0012]

The present invention which solves the above-mentioned problems is a liquid composition containing a viable bacterium of a useful microorganism characterized by comprising at least a viable bacterium of a useful microorganism and lactoperoxidase, Specifically, it is a viable cell-containing liquid composition containing at least viable cells of bifidobacteria and / or viable cells of lactic acid bacteria and lactoperoxidase at a concentration of 10 ppm or more.

The lactoperoxidase used in the present invention is usually industrially produced from unheated whey or skim milk as follows. Lactoperoxidase is
It is marketed as a reagent in a small amount, but not in a large amount. Of course, if the price is ignored, commercially available lactoperoxidase as a reagent can be used in the present invention.

Unheated whey or skim milk is passed through an ion exchange resin [for example, CM Sephadex C-50 (trademark, manufactured by Pharmacia Co.)] to adsorb lactoperoxidase on the ion exchange resin, and then water is added. After thorough washing, a buffer solution (for example, phosphate-saline buffer solution or the like) is passed through to elute the lactoperoxidase adsorbed on the ion exchange resin.
The eluate is concentrated and desalted at the same time using an ultraconcentrator and freeze-dried to obtain a powdery lactoperoxidase-containing material. The lactoperoxidase-containing product thus obtained usually contains 45 to 50% of lactoperoxidase measured by the following method. In addition, the measurement of the purity of lactoperoxidase in the following description,
All this way.

An aqueous solution of the sample was analyzed by gel filtration using Asahi Pack GFA-50 (trademark; manufactured by Asahi Kasei Corporation) by a gel filtration method (phosphate-saline buffer solution) to determine the area of the lactoperoxidase peak relative to the total peak area of the elution curve. Calculate the percentage,
The purity of lactoperoxidase was determined. In addition, a commercially available reagent (manufactured by Sigma, purity 100%) was used as a standard and analyzed under the same conditions to determine the elution position of lactoperoxidase. Further, the purity of lactoperoxidase obtained by the above method is determined by the Purpurrogallin method [Method in Enzymolog].
y), Volume 2, 769 pages, Academic Press (Acad
emic Press), 1955], and the activity ratio [(activity of lactoperoxidase-containing product / activity of commercially available reagent) × 100] compared with the activity of the above commercially available reagent compared to the activity of lactoperoxidase (per unit weight) It was a match.

The lactoperoxidase produced in this way is added to, mixed with, dispersed in, a liquid composition containing live cells,
When dissolved, the lactoperoxidase solution is subjected to aseptic filtration or heat sterilization at a temperature at which lactoperoxidase is not inactivated (for example, 70 ° C for 15 seconds) in order to prevent contamination by bacteria from lactoperoxidase. It is desirable to do.

The concentration of lactoperoxidase in the viable cell-containing liquid composition of the present invention is at least 10 ppm, as is clear from Test Example 3 described below, and if it is less than 10 ppm, a sufficient survival effect cannot be obtained. Therefore, the concentration of lactoperoxidase in the viable cell-containing liquid composition of the present invention is 10 ppm or more, and 200 ppm is the upper limit from the viewpoint of production cost and deterioration of flavor of the viable cell-containing liquid composition.

The production method of the liquid composition containing the viable bacteria of the present invention is as follows. 1) Method for producing bifidobacteria yogurt containing lactoperoxidase Milk is homogenized, sterilized, cooled to about 45 ° C, bifidobacteria and lactic acid bacterium culture are added as a starter, and a lactoperoxidase-containing aqueous solution is sterilized with a membrane filter. And add. Fill the desired container, 37 ~
Ferment in a constant temperature room at 39 ° C for 4 to 5 hours, and then cool.

2) Method for producing lactoperoxidase-containing bifidobacteria milk: Milk is homogenized, sterilized, cooled to about 10 ° C., bifidobacteria and lactic acid bacteria culture are added as a starter, and a lactoperoxidase-containing aqueous solution is further added at 70 ° C. Sterilize for 15 seconds, add, and fill into desired container.

In the liquid composition containing live cells of the present invention produced as described above, the effect of improving the storage stability of live cells of lactoperoxidase is exhibited.

Next, the present invention will be described in detail by showing test examples. (Test Example 1) This test was carried out in order to examine the preservative effect of the production of hydrogen peroxide in yogurt containing live cells and the addition of lactoperoxidase.

1) Preparation of Sample 10 kg of raw milk having a milk fat content of 3.3% and a non-fat milk solid content of 9.0% was homogenized and sterilized at 90 ° C. for 10 minutes. Cool to about 42 ℃ and use Streptococcus thermophilus [St
reptococcus (abbreviated as Str. below) thermophilus],
Lactobacillus (Lactobacillus (below
Bulgaricus] and Bifidobacterium (hereinafter abbreviated as B.) lo
ngum] milk culture 100g, 100g and 50g respectively
Was added. Further, 100 ml of a 1% aqueous solution of commercially available lactoperoxidase (manufactured by Sigma, lot No. L-2005) was passed through a 0.45 µ microfilter to remove bacteria and added. The mixture was thoroughly stirred, filled in a 100 ml polyethylene cup, sealed, fermented for 3.5 hours in a constant temperature room of 40 to 41 ° C., transferred to a refrigerator after completion of fermentation, and stored at 5 to 8 ° C. A sample prepared by the same method as above without adding lactoperoxidase was used as a control. The microorganisms used are all commercially available yogurt cultures or readily available from ATCC.

2) Test method The number of lactic acid bacteria and acidity contained in each sample are officially determined (1948
Dec. 27, Ministry of Health and Welfare Ordinance No. 52 "Ministerial Ordinance Concerning Ingredient Standards for Milk and Dairy Products") to determine the number of Bifidobacterium by Teraguchi et al. (Food Sanitation Magazine, Vol. 23, page 39, 1982) The hydrogen peroxide concentration was measured by ORITECTOR (trademark, manufactured by Oriental Electric Co., Ltd.) 1 day, 7 days, 14 days, and 21 days after production, and the changes in viable cell count and acidity of each microorganism were compared and tested. ..

3) Test Results The results of this test are shown in Table 1. As is clear from Table 1, it was confirmed that the addition of lactoperoxidase markedly suppressed the decrease in the survival number of Bifidobacterium (B. longum) during storage. In addition, when lactoperoxidase was added, no production of hydrogen peroxide was observed during storage of the sample, but when lactoperoxidase was not added, significant production of hydrogen peroxide was observed. It should be noted that almost the same result was obtained even when the test was performed by changing the test conditions (for example, the microorganism used, lactoperoxidase, etc.).

[0025]

[Table 1]

Test Example 2 This test was carried out to examine the preservative effect of hydrogen peroxide production and addition of lactoperoxidase in a milk drink containing viable bacteria of Bifidobacteria and Acidophilus.

1) Preparation of sample 100 kg of raw material milk adjusted to a milk fat content of 3.0% and a non-fat milk solid content of 8.9% was homogenized by a conventional method, sterilized at 130 ° C for 2 seconds, and then heated to about 4-5 ° C. Cooled. To this raw milk, 1 kg each of Bifidobacterium breve (B. breve) and L. acidophilus milk culture was added, mixed uniformly and prepared by the same method as in Reference Example 1. Oxidase (purity 50%)
4 kg of 1% aqueous solution of was sterilized by a plate sterilizer at 70 ° C. for 15 seconds and added. Mix evenly, fill a polyethylene laminated paper container with a capacity of 200 ml, and seal.
Stored in a refrigerator at ℃. A sample prepared in the same manner as above without adding lactoperoxidase was used as a control. The microorganisms used were all commercially available yogurt.
It can be easily obtained as a culture or from ATCC.

2) Test method The viable cell count of each microorganism was measured in the same manner as in Test Example 1 and the changes in viable cell count and acidity were compared and tested. However, the acidity was not measured, and the test period was set to 14 days after production.

3) Test Results The results of this test are shown in Table 2, and the addition of lactoperoxidase to sour milk significantly reduced the survival number of Bifidobacterium (B. breve) during storage. Was confirmed to be suppressed by L. acidophilus
It was confirmed that the decrease in the viable cell count of () was significantly suppressed. In addition, when lactoperoxidase was added, no production of hydrogen peroxide was observed during storage of the sample, but when lactoperoxidase was not added, significant production of hydrogen peroxide was observed. It should be noted that almost the same result was obtained even when the test was performed by changing the test conditions (for example, the microorganism used, lactoperoxidase, etc.).

[0030]

[Table 2]

Test Example 3 This test was carried out to examine the effective concentration of lactoperoxidase. 1) Preparation of sample A sample was prepared by the same method as in Test Example 1 except that lactoperoxidase was added at the concentration shown in Table 3.

2) Test Method The same method as in Test Example 1 was used. However, the number of lactic acid bacteria and the concentration of hydrogen peroxide were not measured.

3) Test Results The results of this test are shown in Table 3. As is clear from Table 3, the sample containing lactoperoxidase in an amount of 10 ppm or more maintained a viable cell count of about 10 ⁷ per 1 ml even after storage for 21 days. The number of viable bacteria was 1/10. Therefore, it was proved that the added amount of lactoperoxidase was at least 10 ppm. The upper limit of the amount of lactoperoxidase added is 200 ppm from the viewpoint of manufacturing cost and flavor, and almost the same results can be obtained by changing the test conditions (for example, the microorganism used, lactoperoxidase, etc.). Was given.

[0034]

[Table 3]

Reference Example 1 1000 kg of unheated skim milk was passed through a column (diameter 20 cm, length 32 cm) packed with 10 liters of CM-Sephadex C-50 (trademark, manufactured by Pharmacia) to obtain Sephadex resin. Lactoperoxidase was adsorbed on the column, and then Sephadex resin was washed with 50 kg of water, and 20 liters of 0.02 mol disodium phosphate-0.3 mol salt buffer solution (pH 7.0) was passed through the column to adsorb Lactoperoxidase on the Sephadex resin. The oxidase was eluted. The eluate was concentrated and desalted at the same time with an ultrafiltration device with a molecular weight fraction of 10,000 (made by DDS), and about 2 liters of the obtained concentrated liquid was freeze-dried by a conventional method to contain powdered lactoperoxidase. About 22 g of the product was obtained.

The purity of lactoperoxidase in the obtained powdery lactoperoxidase-containing material was about 45%.

Reference Example 2 A powdery lactoperoxidase-containing material was obtained in the same manner as in Reference Example 1 except that 1000 kg of unheated whey was used.
18 g (purity about 50%) was obtained.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0039]

【Example】

Example 1 1000 kg raw milk (milk fat content 3.5%, non-fat milk solids content 9.2%)
Homogenize, sterilize at 90-92 ℃ for 10 minutes, cool to 42-43 ℃, commercially available Streptococcus thermophilus, Lactobacillus bulgaricus, and Bihidobacteria.
Milk culture of longum (ATCC15707) 15K each
g, 10 kg, and 15 kg were added. Furthermore, a 2% aqueous solution of lactoperoxidase prepared by the same method as in Reference Example 10
Kg was aseptically filtered with a membrane filter (Millipore) and added. Stir for about 10 minutes, fill each 500 ml container (polyethylene / paper laminate container), seal, and
Ferment in a constant temperature room at ~ 39 ° C for 4 hours, cool, and then yogurt
I got 2000 pieces.

The number of bifidobacteria, the number of lactic acid bacteria, and the acidity of the obtained yogurt after 1 day of production were 2.2 × 10 ⁸ respectively.
/ ml, 5.9 × 10 ⁸ / ml, and 0.77%, and the flavor was good.

Example 2 1000 kg of milk with adjusted components (milk fat content 1.5%, non-fat solids content 8.5%) was homogenized and sterilized at 130 ° C. for 2 seconds to about 5
After cooling to ℃, commercially available Lactobacillus acidophilus and Bihitobacterium infantis (ATCC
15697) milk culture was added at 10 kg each. Further, 5 kg of a 2% aqueous solution of lactoperoxidase prepared by the same method as in Reference Example 1 was sterilized at 70 ° C for 15 seconds and added. The mixture was stirred for about 10 minutes, 180 ml each was filled in a glass bottle, and a paper cap was applied to obtain 5600 milk drinks.

The number of bifidobacteria and lactic acid bacteria one day after the production of the obtained milk drink was 2.2 × 10 ⁷ / ml, and
It was 1.7 × 10 ⁷ / ml, and the flavor was good.

[0043]

The effects of the present invention are as follows. 1) Useful microorganisms can survive for a long period of time. 2) Since only a small amount of natural products is added, the manufacturing cost is low and hygiene is safe.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12N 1/38 7236-4B 9/08 7823-4B // (C12N 1/20 C12R 1:46 1: 225 1 : 01) (C12N 1/20 C12R 1:23 1:01)

Claims (2)

[Claims] 1. A liquid composition containing a viable bacterium, which comprises at least a viable bacterium of a useful microorganism and lactoperoxidase. 2. The viable cell-containing liquid composition according to claim 1, which contains at least viable cells of bifidobacteria and / or viable cells of lactic acid bacteria, and lactoperoxidase at a concentration of 10 ppm or more.