JP3389683B2 - Fermentation method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for promoting fermentation using lactic acid bacteria, bifidobacteria, yeasts and the like.

[0002]

2. Description of the Related Art The applicant of the present application has applied for an oligopeptide (described in JP-A-63-164841) that promotes lactic acid fermentation.

Japanese Patent Publication No. 51-36340 discloses a method for producing a soymilk-fermented beverage by simultaneously fermenting a soybean enzyme decomposition product with lactic acid bacteria and yeast. The soybean protein enzyme decomposition product is the present invention. It does not show a specific molecular weight range fractionated as above, and the fermentation promoting effect is inferior.

Further, JP-A-58-152498 discloses a low molecular weight peptide mixture containing 15% by weight or less of free amino acids and 20% by weight or less of a high molecular weight fraction having a molecular weight of 700 or more. It is described that the concentration operation is repeated using a reverse osmosis membrane having a salt inhibition rate of 40% or less, preferably 5 to 20%. However, not only is it different from the present invention in that the internal liquid of the reverse osmosis membrane is collected because it is a work of concentrating using the reverse osmosis membrane, and there is no description of using the collected low molecular weight peptide mixture for fermentation, There is no suggestion of a fermentation promoting effect.

[0005]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention
An object of the present invention is to provide a fermentation method which is more excellent in the fermentation promoting effect than the invention described in Japanese Patent No.

[0006]

Means for Solving the Problems In the present inventors, in the earnest research for further enhancing the fermentation effect of Japanese Patent Application Laid-Open No. 63-164841, the oligopeptide mixture was separated by a reverse permeable membrane. It was found that the drawn external solution (low molecular weight fraction) showed a high fermentation effect. That is, the present invention has been completed by obtaining knowledge that lactic acid bacteria, bifidobacteria, yeasts and the like have an excellent effect of promoting fermentation and the amount of substances (such as lipase) produced by these microorganisms increases.

That is, according to the present invention, a peptide mixture obtained by enzymatically decomposing soybean protein is fermented with an external solution obtained by fractionating with a reverse permeable pressure permeable membrane having a salt inhibition rate of 25 to 80% or a dried product of the external solution. Is the way. The fraction of the external liquid or the dried product of the external liquid having a molecular weight of 1,000 or less can be 90% or more.
For fermentation, fermentation using lactic acid bacteria, bifidobacteria, fermentation using yeast and the like are suitable.

The protein used for the enzymatic degradation of the present invention is soybean protein. Animal proteins such as casein and other peptones used in the medium are inferior in fermentation promoting effect to peptides obtained from soybean protein.

The enzyme is not particularly limited, but an enzyme having a strong decomposing power, for example, an enzyme derived from mold is suitable. The enzymatic hydrolysis method is also not particularly limited.

It is important to collect the external solution by fractionating the enzymatic decomposition product of soybean protein with a reverse permeable pressure permeable membrane having a salt inhibition rate of 25 to 80%. The salt inhibition rate is preferably 30 to 75%,
Or 35-70%, or 35-75%, or 40
An external solution (low molecular weight fraction) fractionated by a reverse osmosis permeable membrane of ˜65% or 45-65% is suitable.

The external solution or the dried product of the external solution of the present invention preferably has a molecular weight of 1,000 or less and 90% or more. 100
The greater the ratio of 0 or less, the more excellent the effect of promoting fermentation. On the contrary, since the fermentation of amino acid is not promoted by the mixture of amino acids, it is presumed that the peptide having a molecular weight of 1000 or less coexists appropriately with the amino acid to exert an excellent fermentation promoting effect.

According to the present invention, the external solution or the dried product of the external solution is 0.003% by weight (hereinafter "%") or more, preferably 0.004% or more, preferably 0.005% or more, with respect to the medium.
Preferably 0.01% or more, preferably 0.05% or more,
It is preferably 0.1% or more, preferably 0.5% or more, usually less than 20%, preferably less than 15%, preferably less than 10%, preferably less than 5%, preferably 3%.
%, Preferably less than 2%.

[0013] The addition of a small amount of the external liquid or the dried product of the external liquid exhibits an excellent effect of promoting fermentation. The fermentation of the present invention is lactic acid bacteria,
Fermentation of bacteria such as Bifidobacterium and yeast can be performed.

In lactic acid bacterium fermentation, not only the growth of lactic acid bacterium cells can be promoted but also the amount of lactic acid produced by lactic acid bacterium can be increased.

In the fermentation of bifidobacteria, not only the growth of bifidobacteria can be promoted but also the amount of lactic acid produced by bifidobacteria can be increased and the survival rate of bifidobacteria can be increased.

In the fermentation of yeast, not only the growth of yeast cells but also the production of substances produced by yeast (for example, lipase and alcohol) can be promoted.

[0017]

The embodiments of the present invention will be described below with reference to examples. Example 1 Isolated soy protein (“Fuji Pro-R” manufactured by Fuji Oil Co., Ltd.) 1
Proteolytic enzyme (Kaken Pharmaceutical Co., Ltd. "Actinase A")
S ”) 1 part, and enzymatically decomposing at 50 ° C. for 5 hours under pH 7 conditions, readjusting to pH 7 and heating at 70 ° C. for 30 minutes to inactivate the enzyme, and centrifuging at 5000 RPM for 20 minutes to obtain. The supernatant was spray-dried to obtain a soybean protein hydrolyzate.

2, a 5% aqueous solution of this soy protein hydrolyzate,
A reverse osmosis permeable membrane (“DRS-50” manufactured by Daicel Corp.) with a salt rejection rate of 50% was used as 000 ml (membrane area: 0.1).
04 square meters, pressure: 15 kg / square centimeter, flow rate: about 50 ml / min, temperature: 18-22
Outside liquid (low molecular weight fraction) and internal liquid (high molecular weight fraction)
Fractionated into In addition, DRS-50 with ion-exchanged water 1,4
After washing with 00 ml, the obtained external solution and internal solution were combined with the previously obtained external solution and internal solution to make total external solution (low molecular fraction) and total internal solution (polymeric fraction), respectively. These were freeze dried.

The above soybean protein hydrolyzate, low molecular weight fraction,
High-performance liquid chromatography (H
PLC: TSK G3000Pw), 45% acetonitrile, 0.1% TFA (trifluoroacetic acid) was used as a buffer solution, and fractionated at a flow rate of 0.3 ml / min.
Lysozyme with a molecular weight of 14,000 and 3500 as a marker
As a result of measuring the molecular weight using Bacitracin, 1060 Bradykinin, and 245 tripeptide (Leu-Gly-Gly),
It was as shown in FIG.

The following Table 1 shows the results of calculating the areas of the molecular weights of 1,000, 500, and 300 from FIG. 1 and measuring the ratios.

[0021]

[Table 1] (Unit is%) ----------------------------------------- ------------------------ Molecular weight 1000 or less 500 or less 300 or less ------------------- ---------------------------------------------- Soybean protein hydrolysis Material 85.0 68.5 49.7 External liquid 96.6 68.5 49.7 Internal liquid 72.1 45.7 34.2 ------------------ ------------------------------------------------ More than The external liquid, which is a low molecular weight fraction, has a molecular weight of 1000.
The feature was that the following ratios were high. Example 2 Soy protein hydrolyzate, dry powder of total external solution (low molecular weight fraction) obtained in Example 1, and dry powder of total internal solution (high molecular weight fraction) were added to 300 ml of milk respectively. 12g (0.
Heated to 50-60 ° C.,
After homogenizing at 150 kg / square centimeter using a homogenizer, heating at 80 to 90 ° C. for 10 minutes, cooling to 42 ° C., culture starter (Lb.bulgaricus) and Stearo thermophilus (Str. mixed culture of thermophilus)
Cultured in milk medium for 17 hours, acidity 1, bacterial count 9 × 100
Lactic acid fermentation was carried out at 42 ° C by adding 3 ml of 000000 / ml. The control was a milk-only medium. When the change in acidity with time was observed by neutralization titration with 1/10 N NaOH, total external liquid (low molecular weight fraction), soybean protein hydrolyzate, total internal liquid (high molecular weight fraction)
The promotion effect was high in the order of.

The results of acidity measurement over time are shown in FIG. Example 3 Soy protein hydrolyzate, dry powder of total external solution (low molecular weight fraction) or dry powder of total internal liquid (high molecular weight fraction) was added to milk.
5% by weight was added, and the mixture was heated in an autoclave at 110 ° C for 10 minutes to remove Bifidobacterium longum.
× 10,000,000 was added and anaerobic culture was performed at 37 ° C.
The control was the same as in the culture medium containing only milk supplemented with bifidobacteria. As a result of taking out pH and acidity at intervals of time, the accelerating effect was highest in the order of total external solution (low molecular fraction), soybean protein hydrolyzate, and total internal solution (high molecular fraction).

The results of acidity measurement over time are shown in FIG. EXAMPLE 4 Soy protein hydrolyzate, dry powder of total external liquid (low molecular weight fraction) or dry powder of total internal liquid (high molecular weight fraction) was added to milk.
After adding 5% by weight and heating at 110 ° C. for 10 minutes in an autoclave, bifidobacteria are added and anaerobic culture is performed at 37 ° C. After that, the product storage condition of yogurt is 8 to 10
It was stored at ℃ and tested for the viability of Bifidobacterium. As a control, Bifidobacteria was added to a medium containing only milk.

The results are shown in Table 2.

[0025]

[Table 2] ---------------------------------------------- ------------ Directly 20 days later ---------------------------------- ------------------------ Control 3 × 100,000,000 22 × 100,000 Total external solution 22 × 100,000,000 24 × 10,000,000 Soy protein hydrolyzate 21 × 100,000,000 15 × 10,000,000 total internal solution 13 × 100,000,000 10 × 100,0000 -------------------------------------- -------------------- From Table 2, the total internal liquid (polymer fraction) had almost no effect on the survival of Bifidobacterium, but the total external liquid The liquid (low molecular weight fraction) showed the same or higher effect as the soybean protein hydrolyzate. Example 5 Yeast: S. Cerevisiae AH-
22 strains) were cultivated in a commercially available agar medium for yeast, and one platinum loop was inoculated into 5 ml of YNB-Histidine medium in a test tube, and after one day of pre-culture, 50 ml of YPD medium in a flask was added. Inoculate 0.25 ml into
The culture was performed at 30 ° C with shaking. The casein hydrolyzate (“Casitone” manufactured by Difco Co., Ltd.), the soybean protein hydrolyzate obtained in Example 1, and the total external solution (low molecular weight fraction) were used for 2.0% in the YPD medium at this time. I used the one I had.

The composition of the YNB-Histidine medium is shown in Table 3 and the composition of the YPD medium is shown in Table 4.

[0027]

[Table 3] -------------------------------------------------- ------ Difco Yeast-Nitrogen base 0.67% Glucose 2.0% Histidine 50 μg / mL -------------------------------------------------- ------

[0028]

[Table 4] The time-dependent lipase activity at this time is shown in FIG.

The method for measuring the lipase activity at this time is as follows. First, 2 parts of 3 parts for 1 part of olive oil
% Polyvinyl alcohol and homogenized to obtain 5 ml of an emulsion, 4 ml of a pH 7 phosphate buffer and 1 ml of the yeast culture (50
After removing the cells by centrifugation at 00 RPM for 5 minutes) and reacting at 37 ° C for 30 minutes, 10 ml of a mixed solution of acetone and ethanol was added to stop the reaction, and Na
The fatty acid released was measured by the method of raising the pH with OH and then back titrating with hydrochloric acid. Therefore, the unit was 37 ° C, and 1 unit was defined as the activity of releasing 1 µmol of fatty acid in 1 minute.

[0030]

[Effect] According to the present invention, the present inventors have a higher fermentation effect than the oligopeptide mixture described in Japanese Patent Application Laid-Open No. 63-164841, that is, an excellent fermentation promoting effect in lactic acid bacteria, bifidobacteria, yeasts and the like. The effect of increasing the amount of substances (such as lipase) produced by the microorganisms was obtained.

[Brief description of drawings]

1 is a drawing showing the molecular weight distribution by high performance liquid chromatography in the method of Example 1. FIG.

FIG. 2 is a drawing showing changes over time in the acidity of lactic acid fermentation by lactic acid bacteria by the method of Example 2.

FIG. 3 is a drawing showing changes over time in acidity of lactic acid fermentation by Bifidobacterium by the method of Example 3.

FIG. 4 is a drawing showing changes over time in the degradation activity of lipase produced by fermentation by yeast according to the method of Example 5.

[Selection diagram] None

Claims (5)

(57) [Claims] 1. A method of fermenting a peptide mixture obtained by enzymatically degrading soybean protein using an external solution fractionated with a reverse osmosis permeable membrane having a salt inhibition rate of 25 to 80% or a dried product of the external solution. 2. A molecular weight 10 of external liquid or a dried product of the external liquid.
The method according to claim 1, wherein the fraction of 00 or less is 90% or more. 3. The method according to claim 1 or 2, wherein the fermentation is carried out using lactic acid bacteria. 4. The method according to claim 1 or 2, wherein the fermentation is carried out using Bifidobacterium. 5. The method according to claim 1 or 2, wherein the fermentation is performed using yeast.