JP2000093166A - Bacillus bifidus growth promoting substance derived from albumen, and food containing the substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject substance capable of promoting the growth of Bacillus bifidus and lactic acid bacteria, and useful for reducing a fermentation time of yogurt, and making the Bacillus bifidus preponderant in intestinal bacterial flora by using albumen treated with a proteolytic enzyme as an active ingredient. SOLUTION: The objective growth promoting substance of Bacillus bifidus and lactic acid bacteria containing albumen treated with a proteolytic enzyme as an active ingredient is obtained by diluting the albumen of a chicken egg two to three times with deionized water, regulating the pH so as to be the optimum pH (8.0) of a proteolytic enzyme, adding the proteolytic enzyme (e.g. actinase) in the proportion of 0.01-1% of the amount of the albumen thereto, reacting the albumen with the enzyme at 37 deg.C while slowly stirring the mixture, stopping the reaction by the heat treatment of 30 min at 80-90 deg.C under stirring, removing a thermocoagulated material from the reaction-stopped product to provide a clear solution and freeze-drying or spray-drying the clear solution. The material enables the fermentation time of yogurt to be shorten, and the Bacillus bifidus to grow in the yogurt, and is useful as a bifidus factor or the like for making the Bacillus bifidus preponderant in intestinal bacterial flora.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a substance for promoting the growth of bifidobacteria and lactic acid bacteria, a food containing the substance, a method for growing bifidobacteria and lactic acid bacteria using the substance, and a method for producing the substance by using bifidobacteria and / or lactic acid bacteria or bifidobacteria. And / or a fermented food obtained by culturing and fermenting with a food containing lactic acid bacteria.

[0002]

2. Description of the Related Art As is well known, eggs are one of the complete nutritional foods along with milk, and many studies have been made on their higher functions. Many of these were primarily aimed at the yolk component, and studies on egg white were lagging behind. In recent years, studies on higher-order functions of egg white have begun to be carried out, and in addition to antibacterial effects, antitumor effects, immunostimulatory effects, antiviral effects, etc. have been revealed, some of which have already been sold as special feed. Also exists.

[0003] The present inventors have been engaged in research on eggs and have clarified the functions of special components of egg yolk and egg white (Japanese Patent Application Nos. 5-529797 and 7-28).
5885, Japanese Patent Application No. 9-173018, Hiroshi Ohna et al., Medicine and Biology, 126 (1), 19-23, 1993).

[0004]

In the above studies, the present inventors had the experience that the oral administration of certain components of egg white to animals clearly improved the fecal properties.
Since the improvement of fecal properties is considered to be due to the improvement of the intestinal environment, egg white is fractionated into various components and their effects on bifidobacteria and lactic acid bacteria are examined. And lactic acid bacteria, especially bifidobacteria, have an effect of promoting the growth very strongly, and have completed the present invention.

Bifidobacterium is generally known to be a useful bacterium in the intestine because of its preventive and therapeutic effects on diarrhea, constipation, infectious diseases and the like, and its inhibitory effect on the growth of harmful bacteria in the intestine.
Since the improvement of the intestinal environment is essential for maintaining health, many foods and pharmaceuticals containing bifidobacteria and their growth factors have been developed. The most common foods containing bifidobacteria are fermented foods represented by yogurt, but the number of bifidobacteria rapidly decreases from immediately after fermentation due to the acid produced by coexisting lactic acid bacteria, It was later pointed out that it could hardly be detected (Hiroya Wakiguchi, Dairy Chemistry and Food Research, vol 33, A2
03-A212, 1984). Against this background, research and development of growth factors that have high growth-promoting activity and can maintain the activity of bifidobacteria have been vigorously continued at many research institutions. The main research and development of oligosaccharides is focused on the source.
However, a large number of substances such as casein hydrolysates and certain types of microbial metabolites have been reported as bifidobacteria, and there has been a demand for the development of bifidobacteria growth factors using cheaper raw materials and a simple production method.

[0006]

SUMMARY OF THE INVENTION The present invention provides a growth promoting substance for bifidobacteria and lactic acid bacteria, which comprises, as an active ingredient, egg white treated with a protease.

[0007] The present invention also provides a food containing the growth promoting substance.

Further, the present invention provides a method for growing bifidobacteria and lactic acid bacteria, which comprises using the above-mentioned growth substance.

[0009] In addition, the present invention provides a fermented food obtained by culturing and fermenting the growth promoting substance with a bifidobacterium and / or lactic acid bacterium or a food containing the bifidobacterium and / or lactic acid bacterium.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The egg white hydrolyzate of the present invention remarkably promotes the growth of bifidobacteria, and particularly has the effect of promoting the growth of bifidobacteria even in co-culture with lactic acid bacteria. It was not known at all that such a bifidobacterium growth promoting action was present. INDUSTRIAL APPLICABILITY The present invention enables shortening of fermentation time of yogurt, growth of bifidobacteria in yogurt, and the like, and is a very effective substance for all foods containing bifidobacteria and lactic acid bacteria. In addition, it is considered that the intestinal flora can be expected to be a bifidobacterium alternative to oligosaccharides in order to make bifidobacteria dominant.

When the foodstuffs of the present invention contain a substance for promoting the growth of bifidobacteria and lactic acid bacteria, the substance is contained in an amount of 0.01 to 5.0%, preferably 0.15 to 1%, based on the total amount (weight basis). 0.0%. Target foods are not particularly limited, but include yogurt,
Examples include yogurt drinks, lactic acid bacteria drinks, and other foods containing bifidobacteria and / or lactic acid bacteria.

In the case where the above-mentioned growth promoting substance is cultured and fermented together with bifidobacteria and / or lactic acid bacteria or food containing bifidobacteria and / or lactic acid bacteria, a fermented food is produced. Is 0.01 to 5.0%, preferably 0.15%
To 1.0%, more preferably 0.3 to 0.6%.

[0013]

Next, the present invention will be described in detail with reference to examples.
It should be noted that the examples described below are only for explaining the present invention, and do not limit the enzymes and their reaction conditions to those described in the examples.

[0014]

Example 1 Production of Egg White Bifidus Factor (Egg White BF) Preparation of Egg White BF Chicken egg egg white was diluted 2-3 times with deionized water, and the pH was adjusted to the optimal pH (8.0) for the protease. Thereafter, a proteolytic enzyme (actinase AS; Kaken Pharmaceutical Co., Ltd.) of 0.01 to 1% (preferably 0.1%) of the amount of egg white is added, and 37
The reaction was carried out at 18 ° C. for 18 hours with slow stirring. Next, the enzymatic reaction was stopped by a heat treatment at 80 to 90 ° C. for 30 minutes while stirring. The clarified liquid obtained by removing the heat-coagulated substance was freeze-dried or spray-dried. This actinase-treated egg white was defined as egg white Bifidus Factor (hereinafter abbreviated as egg white BF).

Further, in order to examine whether there is a difference in the activity depending on the type of the protease, trypsin (manufactured by Sigma), papain (manufactured by Sigma, type IV) and pronase K (manufactured by Kaken Kagaku) were used instead of actinase AS. ) Was prepared by treating egg white according to the above method. Trypsin-treated fraction, papain-treated fraction, pronase K
This was the treated fraction.

Preparation of Undegraded Egg White Chicken egg white was diluted with the same amount of deionized water, and heated at 85 ° C. for 30 minutes. Immediately after cooling in ice-cold water, the heat-coagulated material was removed. The obtained clear liquid was spray-dried to obtain undegraded egg white powder.

Preparation of defatted egg white BF After dehydration with acetone, chloroform / methanol (2:
Egg white BF completely defatted and dried in 1) was used.

Preparation of fat-soluble fraction The above organic solvent-soluble matter was treated under reduced pressure (evaporator) to completely remove the organic solvent. This was dissolved in a small amount of dimethyl sulfoxide (DMSO) and used as a fat-soluble fraction.

Preparation of Sugar Chain Fraction Egg white BF was thoroughly decomposed with an enzyme to prepare a sample in which only the sugar chain was left. That is, 10% of the defatted egg white BF described
Dissolved in deionized water to a concentration and adjusted the pH to 8.0.
Papain (Sigma, Type IV) and Pronase K
(Manufactured by Kaken Chemical Co., Ltd.) was added and the protein was digested with enzymes. Trichloroacetic acid (TCA) was added to the decomposition product to a final concentration of 10%, followed by cooling in ice-cold water for 10 minutes. The precipitate was removed by centrifugation and freeze-dried. 99.5% on dry matter
Ethanol was added to remove TCA.

Measurement of Bifidobacterium growth promoting activity Culture was carried out using a yogurt starter ABT-1 (CHR. Hansen
Bifidobacterium lact
is) using 10% skim milk medium (3 ml)
Filtered sterilized egg white BF solution (final concentration 0.6
%), The precultured bacterial solution was diluted 10-fold, 0.1 ml was added, and sterilized liquid paraffin was overlaid.
The cells were cultured (37 ° C., 24 hours). The skim milk medium used was deaerated by quenching in running water after autoclaving.

The number of cells in the culture was measured by appropriately diluting 100 μl of the culture after the completion of the culture with a selective plate medium for bifidobacteria (MRS agar medium containing 5% NNLP solution; Merck KGaA).
) And detected by culturing at 37 ° C for 48 hours under anaerobic conditions. The composition of the NNLP solution is shown in Table 1 below.

[0022]

[Table 1] result

[Table 2] The above-mentioned Table 2 shows the bifidobacterium growth-promoting effects of egg white BF, egg white treated with each enzyme, undegraded egg white, defatted egg white BF, fat-soluble fraction and sugar chain fraction. No activity was observed in the sugar chain fraction, the fat-soluble fraction and undegraded egg white, but the actinase-treated egg white BF, the protease-treated fraction and the defatted egg white BF showed an activity of promoting the growth of bifidobacteria. Egg whites treated with various proteases have almost the same activity as egg white BF treated with actinase. Therefore, egg whites show bifidobacterial growth promoting activity when treated with proteases. However, it was confirmed that it was not a protein degradation product (peptide). Up to now, some peptide-derived bifidobacterial growth promoting substances have been reported to be enzymatically decomposed products of milk casein, but those derived from egg white have not been reported yet.

As is evident from the above test, there is almost no difference in the effect of promoting the growth of bifidobacteria depending on the type of protease. Therefore, in the following examples, the test was carried out using actinase-treated egg white (egg white BF). Was.
The examples described below are only for explaining the present invention, and do not limit the samples, the conditions of each test, and the like to the descriptions of the examples.

[0024]

Example 2 Effect of Egg White BF on Proliferation of Various Bifidobacteria The effect on various Bifidobacteria was examined by single culture.

Bacteria used Five strains of bifidobacteria derived from human intestine were used. All strains were purchased from RIKEN. After pure culture, they were pre-cultured (repeated at least 3 times) in each synthetic medium and used for main culture. Table 3 below shows the strains and the synthetic medium for preculture.

[0026]

[Table 3] The test was performed in substantially the same manner as the test in the culture destination. That is, filter-sterilized egg white B was added to 10% skim milk medium (3 ml).
0.2 ml of F solution (final concentrations 0.6 and 0.3%)
added. Further, the pre-cultured bacterial solution was diluted 10-fold, and after adding 0.1 ml, sterilized liquid paraffin was overlaid,
The cells were cultured (37 ° C., 24 hours). The skim milk medium used was deaerated by quenching in running water after autoclaving. Measurement of the number of bacteria The culture solution was appropriately diluted 10-fold, smeared on each selective medium, cultured, and the number of bacteria per 1 ml of skim milk medium was calculated from the number of appearing colonies. Table 3 shows the agar medium for detection of each strain.

Result

[Table 4] The results for Bifidobacterium are shown in Table 4. Although a considerable difference was observed depending on the type of Bifidobacterium, a growth promoting activity of 10 times or more was observed for all strains. In particular, strong activity was observed for B. adolescentis and B. infntis.

From these results, since egg white BF grows bifidobacteria at a level of 10 to 100 times, it is expected that intestinal flora predominant in bifidobacteria is predominant.

[0029]

Example 3 Co-culture with Lactic Acid Bacteria The results of Examples 1 and 2 revealed that egg white BF had a high growth promoting effect on Bifidobacteria. Example 1 describes how the egg white BF according to the present invention acts on bifidobacteria co-cultured with lactic acid bacteria.
The growth promoting effect of the egg white BF obtained in Example 2 on the growth of bifidobacteria and lactic acid bacteria was compared with that of undegraded egg white. Test sample Egg white BF and undegraded egg white obtained in Example 1 were used.

Test cells The yoghurt starter ABT-1 (CHR.
Hansen).

Lactobacillus acidophilus (L. acidophilus) Streptococcus thermophilus (S. thermophilus) Bifidobacterium bifidum (B. bifidum) Bifidobacterium lactis (B. lactis) The above ABT-1 is suspended in sterile physiological saline to a concentration of 1% and a starter solution is prepared. And Culture To 3 ml of 10% skim milk medium (110 ° C., 10 minutes, autoclave treatment), 0.2 ml of egg white BF solution prepared at each concentration (final concentration: 0.3 and 0.6%) was added, and the starter solution was added to 0 ml. .1 ml was added. After stirring, 40
Cultured at 6 ° C for 6 hours. In addition, what added the same density | concentration undegraded egg white solution instead of the egg white BF liquid was set as the control.

Measurement of Number of Bacteria The number of Bifidobacteria was measured according to the method described in Example 1.

L. acidophilus was detected by culturing at 37 ° C. for 48 hours using MRS maltose agar medium. S.ther
mophilus was detected by using Lee's agar medium at 43 ° C. for 48 hours. The composition of Lee's agar medium is shown in Table 5 below.

[0034]

[Table 5] result

[Table 6] Egg white BF in skim milk medium, 0.3 and 0.6%
Table 6 shows the results of cultivation after adjusting to a concentration. Enzymatically hydrolyzed egg white BF showed a growth promoting activity. The growth-promoting effect on Bifidobacteria is very remarkable, reaching about 20,000 times the addition of 0.6% and the addition of undegraded egg white, confirming that it is a highly effective Bifidobacterium growth-promoting substance. Was done. In addition, the addition of 0.6% to lactic acid bacteria showed a 1.5- to 2.5-fold growth promoting effect.

[0035]

Example 4 Change in Bifidobacterium Growth-Promoting Activity According to Egg White BF Concentration As shown in Example 3, egg white BF has a remarkable bifidobacterium growth-promoting activity. Therefore, the activity was examined in more detail.

Test sample The egg white BF obtained in Example 1 was used.

Test cells As in Example 3, the yoghurt starter ABT-1 (CHR.
Hansen).

Culture Each concentration (final concentration: 0, 0.015, 0.0375,
0.075, 0.15, 0.3, 0.6, 1.2, 2.4
And 5.0%) of the egg white BF was measured for the activity of promoting the growth of bifidobacteria in accordance with Example 3.

Measurement of the number of bacterial cells The measurement was performed according to Example 3.

Result

[Table 7] Egg white BF in skim milk medium, 0.015 to 5.0%
Table 7 shows the results obtained by culturing the cells after adjusting the concentration. Bifidobacterium growth promoting activity was observed in a concentration-dependent manner from 0.15% to 0.6%, and the growth reached a plateau at a concentration of 0.6% or more. The addition of 0.6% reached about 20,000 times that of the control, confirming that the substance is a very effective bifidobacteria increase promoting substance.

[0041]

Example 5 Production of Yogurt Production of Yogurt Egg white BF was added to commercially available unregulated milk in an amount of 0.6%, dissolved, and heat-sterilized (80 ° C., 15 minutes). After sterilization, the mixture was cooled to 40 ° C. in ice-cold water, and about 300 mg of starter (ABT-1) was added per liter of milk. Immediately after stirring, the mixture was dispensed into a 100 ml cup, sealed with an aluminum seal, and fermented (40 ° C., 7 hours). Also, egg white BF
A control without adding was used as a control.

Measurement of pH The pH was measured with a pH meter (pH METER F-22:
Using Horiba, Ltd.), the time when the starter was added was set to 0 hour, and the measurement was made over time every hour.

Measurement of acidity The acidity was measured by a titration method and converted to lactic acidity. The time at which the starter was added was set to 0 hour, and the measurement was made over time every hour.

Measurement of the Number of Bacteria The number of cells was measured in the same manner as in Example 3 with the starter added at 0 hour and hourly over time.

Results Changes in pH and acidity are shown in FIG. 1, and changes in the number of bacteria are shown in FIG. It was confirmed that when the egg white BF was added, the pH, the acidity and the number of bacteria increased more clearly. In particular, in the case of bifidobacteria, the number of bacteria decreased with the passage of time in the control, whereas the number of bacteria increased when egg white BF was added, and as shown in FIG. Showed a growth promoting activity about 200 times that of the control. In addition, the growth promoting activity of lactic acid bacteria was confirmed to be two to three times.

From these results, the addition of egg white BF not only imparts acid resistance to bifidobacteria and enables the growth of bifidobacteria even during the production of yogurt, but also adds to the starter lactic acid bacteria (L. acidophilus, S. thermophilus). Also show a growth promoting effect, resulting in a rapid decrease in pH and an increase in acidity.

[0047]

As described above, the egg white-derived bifidobacterium growth-promoting substance of the present invention has a unique activity against bifidobacteria. By utilizing this, it is possible to shorten the fermentation time of yogurt and to grow bifidobacteria in yogurt, etc., and it is a very effective substance for all products containing bifidobacteria and lactic acid bacteria. In addition, it can be expected as a bifidogenic factor replacing oligosaccharides in order to make the intestinal flora dominant in bifidobacteria.

[Brief description of the drawings]

FIG. 1 is a graph showing changes in pH and acidity during yoghurt fermentation.

FIG. 2 is a graph showing a change in the number of cells during fermentation of yogurt.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Ichizo Oishi 3-15-24 Nishisunacho, Tachikawa-shi, Tokyo F-term (reference) 4B001 AC05 AC31 AC50 BC14 EC99 4B018 LB07 MD72 MD87 ME11 MF13 4B065 AA21X AA30X AA49X BB19 BB34 CA42

Claims (7)

[Claims] 1. A growth-promoting substance for bifidobacteria and lactic acid bacteria, comprising as an active ingredient egg white treated with a protease. 2. The method according to claim 1, wherein the substance is 0.01 to 5.0.
% Food. 3. The food according to claim 2, containing 0.3 to 1.0% of said substance. 4. The food according to claim 2, wherein the food is yogurt. 5. A method for growing bifidobacteria and lactic acid bacteria, comprising using the substance according to claim 1. 6. A fermented food obtained by culturing and fermenting the substance according to claim 1 together with a bifidobacterium and / or lactic acid bacterium or a food containing the bifidobacterium and / or lactic acid bacterium. 7. The fermented food according to claim 6, wherein the fermented food is yogurt.