WO2010061575A1

WO2010061575A1 - Promoter for low temperature fermentation of yeast, and culture medium for growing yeast and method for producing fermented food or beverage each of which utilizes the promoter

Info

Publication number: WO2010061575A1
Application number: PCT/JP2009/006317
Authority: WO
Inventors: 杉山将宏; 北川さゆり
Original assignee: 不二製油株式会社
Priority date: 2008-11-25
Filing date: 2009-11-24
Publication date: 2010-06-03
Also published as: JPWO2010061575A1

Abstract

It is found that a peptide produced by fractionating a fraction rich in β-conglycinin (which is one of globulins) from a soybean protein and enzymatically digesting the fraction has an activity to remarkably enhance the proliferating ability of yeast at lower temperatures. Thus, disclosed is a promoter for low temperature fermentation of yeast, which is characterized by comprising a digestion product of soybean-derived β-conglycinin. The promoter can solve the problem of decrease in fermentation efficiency caused by the slowdown of the proliferation activity of yeast when the yeast is fermented at a low temperature for the production of a sparkling alcoholic beverage or the like.

Description

Yeast low-temperature fermentation promoter, yeast growth medium using the same, and method for producing fermented food and drink

The present invention relates to a yeast fermentation promoter at low temperatures in the production of foods obtained by fermentation at low temperatures with yeast.

Yeast is used in the process of producing many fermented foods and drinks such as soy sauce, miso, bread, and sake. Some yeasts are also used as model organisms for molecular biology. Thus, yeast is a microorganism that is widely used in industry and academia, and active research is being conducted on culture conditions such as temperature and medium composition. One of the important environmental factors for the efficient growth of yeast is temperature. The optimum temperature of yeast varies depending on the type of yeast, but many of them grow well at temperatures around 30 ° C., and at low temperatures of 15 ° C. or less, the ability of yeast to grow is greatly reduced. This is thought to be because the growth of yeast is inhibited by low-temperature stress, which is one of environmental stresses.

In addition, there is a nutrient source outside the fungus that affects the growth of yeast. In order for yeast to grow, in addition to carbon sources such as sugar and nitrogen sources such as amino acids and proteins, trace nutrient sources such as vitamins are required. As the nitrogen source, inorganic substances such as ammonium sulfate and organic substances such as amino acids and proteins are usually used, and soybean peptides obtained by hydrolyzing soybean are also being used as nitrogen sources. For example, soy peptide is used in yeast fermentation in the production of effervescent alcoholic beverages such as beer-like beverages that have low beer and malt usage or do not use malt. However, in effervescent alcoholic beverages, fermentation is often carried out at a low temperature of 15 ° C. or lower, but as mentioned above, the yeast growth activity becomes dull due to low-temperature stress, and as a result, fermentation efficiency decreases, the fermentation time becomes longer. There is a need to. In this case, there is an advantage in terms of flavor, but if the low-temperature fermentation can be completed in a shorter time, the advantage in production efficiency is greater.

As a knowledge related to such a problem, it has been known that when yeast is fermented by adding gum arabic and tragacanth, alcohol production becomes active at 5 ° C. (Patent Document 1). Moreover, it is described that the yeast culture | cultivated by the culture medium containing a soybean peptide raises tolerance with respect to freezing (patent document 2). However, Patent Document 1 looks at the amount of alcohol produced rather than the growth of bacteria, and does not mention the growth activity of the bacteria. Also, the effect at 15 ° C is not shown. Further, the knowledge of Patent Document 2 does not teach yeast growth in a non-freezing region and a low temperature region of 15 ° C. or lower.

JP 2008-99659 A International Publication WO 2008/047596

The object of the present invention is to solve the problem that fermentation efficiency decreases due to slowing of the growth activity of yeast when yeast fermentation is performed at a low temperature during production, such as effervescent alcoholic beverages.

In view of the above problems, as a result of intensive research, the present inventors have fractionated a fraction rich in β-conglycinin, a kind of globulin, from soybean protein, and the peptide obtained by enzymatic degradation of this fraction is capable of low-temperature growth ability of yeast. It has been found that it has an effect of remarkably strengthening. It was found that the low-temperature growth ability of the fraction was based on the action of protecting against so-called low-temperature stress, in which the activity of intracellular enzymes in yeast was reduced by low temperature and yeast activity was suppressed. The present invention has been completed based on such findings.

That is, the present invention
(1) A yeast low-temperature fermentation promoter characterized by containing a degradation product of soybean β-conglycinin,
(2) The yeast low-temperature fermentation promoter according to (1) above, wherein the fraction of a molecular weight of 120 to 500 in the degradation product of soybean β-conglycinin is 60% or more,
(3) A yeast growth medium to which the low-temperature fermentation promoter according to (1) is added,
(4) A method for producing a fermented food or beverage, inoculating yeast into a medium containing the low-temperature fermentation promoter according to (1), and fermenting at 15 ° C. or lower,
(5) A yeast low-temperature stress protective agent comprising a degradation product of soybean β-conglycinin.

According to the present invention, by adding soybean β-conglycinin degradation product to the medium, the yeast can be protected from low-temperature stress, and as a result, the growth of the yeast at low temperatures can be promoted. In order to advance the fermentation quickly, it is essential to grow the yeast at a low temperature, but in the present invention, the growth time can be shortened, so that the fermented food can be produced in a shorter time than before.

It is a graph which shows the result of having investigated the growth effect of the yeast in the low temperature by the addition to the culture medium of various soybean protein degradation products (a vertical axis | shaft: the number of bacteria per 1 mL of yeast, a horizontal axis: culture | cultivation time).

The yeast low-temperature fermentation accelerator of the present invention is characterized by containing a degradation product of soybean β-conglycinin. Hereinafter, embodiments of the present invention will be described in detail.

(Soybean β-conglycinin)
Proteins constituting soy protein are classified into 2S, 7S, 11S, and 15S globulins based on sedimentation coefficients obtained by ultracentrifugation analysis. Of these, 7S globulin and 11S globulin are the main constituent protein components of the globulin fraction. Of these, β-conglycinin in the immunological nomenclature substantially corresponds to 7S globulin, and glycinin substantially corresponds to 11S globulin. In addition, the soybean globulin fraction contains a group of minor acid-precipitable soy proteins other than these, and these are accompanied by a large amount of polar lipids such as lecithin and glycolipids. (Hereinafter sometimes abbreviated as “LP”) (refer to International Publication WO2006 / 129647).

Thus, soybean protein can be fractionated into three parts: β-conglycinin (7S globulin), glycinin (11S globulin), and LP (see the above publication). Of these, β-conglycinin is selected as a raw material in the present invention. It is a thing. β-conglycinin is a trimer composed of three subunits α, α ′, and β, and this is known to have an effect of reducing neutral fat in blood.

As means for fractionating a fraction rich in β-conglycinin from soybean protein, known means can be used. In particular, β-conglycinin is preferably contained in the crude protein in an amount of 40% by weight or more, more preferably 50% by weight or more. Preferable production methods include those described in International Publications WO2006 / 129647 and WO2002 / 028198. The purity of β-conglycinin in the fraction rich in β-conglycinin is preferably 60% or more, more preferably 80% or more in the protein.

(Soybean β-conglycinin degradation product)
The β-conglycinin degradation product of the present invention is a peptide mixture obtained by degrading β-conglycinin with a protease. The β-conglycinin degradation product preferably has a high degree of degradation, and the proportion of the fraction having a molecular weight of 120 to 500 in the degradation product is preferably 60% or more, and more preferably 64% or more. Further, according to Non-Patent Document 2, since it is considered that the amino acid is preferably in a peptide form rather than existing in a free form, the proportion of the fraction having a molecular weight of less than 120 is preferably 10% or less, and preferably 5% or less. It is more preferable that Furthermore, since it is desirable that the peptide state be a lower molecule, the proportion of the fraction having a molecular weight of 500 or more is preferably 40% or less, more preferably 35% or less.

Enzymatic degradation is carried out using the soybean protein slurry or aqueous solution as a substrate for protease treatment. Proteases used here are of animal origin, plant origin or microbial origin, and are classified as “metal protease”, “acid protease”, “thiol protease”, “serine protease” in the classification of protease, preferably “metal” The protease can be appropriately selected from proteases classified as “protease”, “thiol protease”, and “serine protease”. In particular, a degradation method in which enzymes belonging to two or more, or three or more different categories are sequentially or simultaneously acted is preferable because the ratio of peptides having a relatively low molecular weight such as dipeptides and tripeptides can be increased.

This classification of protease is a classification method based on the type of amino acid at the active center, which is usually performed in the field of enzyme chemistry. As representatives of each, "Metal protease" includes Bacillus neutral proteinase, Streptomyces neutral proteinase, Aspergillus neutral proteinase, Samoaase, etc. Examples of “serine protease” such as bromelain, papain, and the like include trypsin, chymotrypsin, subtilisin, Streptomycins alkaline protease, alkalase, bioprase and the like. The classification of other enzymes can also be confirmed by the working pH and reactivity with inhibitors. Since the site of action on the substrate is greatly different between enzymes having different active centers, the “uncut residue” can be reduced, and an enzyme degradation product can be obtained efficiently. Alternatively, enzymatic degradation products can be produced more efficiently by using enzymes of different origins (origin organisms) in combination. Even in the same classification, if the origin is different, the site of action of the substrate protein is also different, and as a result, the proportion of dipeptides and tripeptides can be increased. These proteases are preferably those with low exo activity.

The reaction pH and reaction temperature may be set in accordance with the characteristics of the protease to be used. Usually, the reaction pH is carried out near the optimum pH, and the reaction temperature may be carried out around the optimum temperature. In general, the reaction can be carried out at a reaction temperature of 20 to 80 ° C., preferably 40 to 60 ° C. After the reaction, the enzyme is heated at a temperature sufficient to inactivate the enzyme (about 60 to 100 ° C.) to inactivate the remaining enzyme activity.
The obtained reaction solution can be used as it is or after being concentrated, but it can usually be sterilized and used in the form of a dry powder by mist drying, freeze drying or the like. Further, the reaction solution can be adjusted to an arbitrary pH, and precipitates and suspensions generated during pH adjustment can be removed by centrifugation, filtration, or the like. Further, purification can be performed with activated carbon or an adsorption resin.

(Molecular weight distribution measurement method)
When measuring the molecular weight distribution of the obtained β-conglycinin degradation product, it is measured by the HPLC method using the following gel filtration column. A gel filtration system for peptides is assembled using one column, a known peptide serving as a molecular weight marker is charged, and a calibration curve is obtained in the relationship between molecular weight and retention time (Table 1). The supernatant obtained by centrifuging the enzymatically decomposed product (1%) at 10,000 × g for 10 minutes was diluted 2-fold with a solvent for gel filtration, and 5 μl thereof was applied. About the content ratio% of each molecular weight fraction, it calculated | required by the ratio of the area of the specific molecular weight range (time range) with respect to the chart area of the whole light absorbency. (Column: Superdex Peptide 7.5 / 300GL, solvent: 1% SDS / 10 mM phosphate buffer, pH 8.0, 25 ° C., flow rate: 0.25 ml / min, detection: OD220 nm).

(Table 1) Molecular weight reference materials

(Yeast low temperature fermentation promoter)
The present invention is characterized in that the β-conglycinin degradation product obtained as described above is used as a yeast low-temperature fermentation promoter. Usually, the optimum temperature of yeast used for food production is about 20 to 30 ° C., and the growth rate of yeast is remarkably reduced in a low temperature range of 15 ° C. or lower. In the process of producing fermented foods, the growth state of yeast is an important key, and the days spent for fermentation are relatively long. However, the low-temperature fermentation promoter of the present invention can increase the growth rate of yeast at low temperatures and shorten the number of production days.
Here, in the present invention, “low temperature” refers to a temperature lower than the room temperature (around 20 ° C.), specifically a temperature of 15 ° C. or lower, and more specifically 12 ° C. or lower. However, since the yeast cannot proliferate in the first place at the temperature of the freezing region where the culture solution is frozen, it also leads to a decrease in the activity of the yeast. Specifically, it is 0 ° C. or higher, more specifically 3 ° C. or higher.

(Yeast culture medium)
The low-temperature fermentation promoter of the present invention can be blended in a medium as one of the main nitrogen sources of yeast to obtain a yeast growth medium. For example, it can be obtained by adding 0.1 to 10% in a yeast growth medium. The type of the yeast growth medium may be a natural medium, a synthetic medium, or a semi-synthetic medium, or may be a liquid medium or a solid medium. Peptone, yeast extract, yeast nitrogen base, ammonium sulfide, amino acids, nucleotides and the like can be added as other nitrogen sources, and glucose, galactose, raffinose, dextrose and the like can be added as carbon sources.
When yeast is cultured in this yeast growth medium at a low temperature of 15 ° C. or lower, the growth becomes active even at low temperatures. This is due to the action of a β-conglycinin degradation product as a low-temperature stress protective agent that protects yeast from low-temperature stress experienced by yeast.

(Manufacture of fermented food and drink)
The low-temperature fermentation promoter of the present invention can be added as a medium component of a commercially available yeast growth medium as described above. In addition, in the production of fermented foods and beverages produced by yeast fermentation at low temperatures, it is mixed as a raw material before fermentation. It is also effective to inoculate and ferment yeast.
Examples of fermented foods and beverages that can be produced by yeast fermentation at low temperatures include effervescent alcoholic beverages such as beer-like beverages with low or 0% use of beer and malt as liquors, wine, whiskey, sake, shochu, etc. Examples of seasonings include soy sauce, miso, and vinegar. Examples of general foods include bread, fermented milk, fermented butter, kefir, and pickles, but are not limited to these. Applicable to all fermented foods and beverages to be performed.
The amount of the low-temperature fermentation accelerator added to the fermented food / beverage product can usually be 0.1 to 10%.

Hereinafter, more specific embodiments of the present invention will be described by way of examples.

(Production Example 1) Preparation of various soybean protein fractions According to the method of Samoto et al. (M Samoto etc, Food Chemistry, 102, 317-322, 2007.) A soy protein fraction was prepared.
1) A hot water extraction slurry of defatted soybean (NSI 70%) that has been subjected to heat treatment on low-denatured defatted soybean to reduce NSI (water-soluble nitrogen index, according to AOCS official analysis method BA-11-65 NSI) in a centrifuge The okara fraction was removed to obtain defatted soy milk.
2) Next, the pH of the defatted soymilk was adjusted to 5.8, and the precipitated curd fraction was collected with a centrifuge. This fraction is the “glycinin fraction”.
3) Next, the pH of the remaining supernatant was adjusted to 5.0, allowed to stand at 55 ° C. for 10 minutes, then adjusted to pH 5.5, and then the precipitated card fraction was collected with a centrifuge. This fraction is an “LP fraction” containing a large amount of lipophilic protein.
4) Next, the pH of the remaining supernatant was adjusted to 4.5, and the precipitated card fraction was collected with a centrifuge. This fraction is the “β-conglycinin fraction”.
5) Each obtained fraction was neutralized, heat-treated at 140 ° C. for 10 seconds, and then spray-dried.
The purity of each fraction was 96% for the glycinin fraction and 91% for the β-conglycinin fraction.

(Manufacture example 2) Preparation of isolation | separation soybean protein Separately, isolation | separation soybean protein was prepared from the low modified | denatured defatted soybean as follows.
1) Warm water extraction slurry of low-denatured defatted soybeans was defatted soymilk by removing the okara fraction with a centrifuge.
2) The obtained defatted soymilk was adjusted to pH 4.5 and subjected to isoelectric precipitation, and an acid precipitation card was obtained and neutralized with a centrifuge.
3) Next, this neutralized solution was spray-dried to obtain separated soybean protein (SPI).

(Production Example 3) Production of various soybean protein degradation products From the β-conglycinin, glycinin, LP and the separated soybean protein obtained in Production Examples 1 and 2, with reference to International Publication WO2006 / 134752, the following proteases were used. Enzymatic degradation products were prepared.
2% Samoaase (source: Bacillus thermoproteolyticus, metalloprotease, Daiwa Kasei) was added to the 3% soy protein solution and allowed to act at pH 9.0, 58 ° C. for 60 minutes. Next, 1% of biopulase (origin: Bacillus sp., Serine protease, Nagase Chemtech) was added per protein and allowed to act at pH 7.5, 58 ° C. for 60 minutes. Sumiteam FP (source: Aspergillus sp., Metalloprotease, Shin Nippon Chemical Industry) was added at 1% per protein and allowed to act at pH 7.5, 58 ° C for 60 minutes. After the above treatment, the reaction was stopped at 90 ° C. for 20 minutes, and then used as a sample.
The molecular weights of the obtained four types of enzyme degradation products were measured according to the method described in Non-Patent Document 2. The measurement results are shown in Table 2.

(Table 2) Molecular weight distribution of β-conglycinin degradation product

In the β-conglycinin degradation product, the fraction with a molecular weight of 120 to 500 containing a large amount of dipeptides and tripeptides was 66.9%, accounting for 60% or more. On the other hand, the ratio of the fraction containing a large amount of free amino acids and having a molecular weight of less than 120 was 3.1%, which was less than 10%. The proportion of the relatively high-molecular peptide fraction having a molecular weight of 500 or more was 30%, which was less than 40% (Table 2). The fraction ratios in the other degradation products were almost the same as those in the β-conglycinin degradation product, and there was no significant difference in molecular weight distribution, indicating that the same enzymatic degradation was performed in all cases.

(Example 1)
In order to observe the low-temperature growth promoting effect of the β-conglycinin degradation product obtained in Production Example 3, a low-temperature culture experiment of yeast was performed. As comparative objects, the glycinin degradation product, LP degradation product, and separated soybean protein degradation product obtained in Production Example 3 were used.
As a medium, various soybean protein degradation products were added as a nitrogen source to an SD medium for yeast growth. Specifically, Yeast Nitrogen Base w / o amino acids and anmoniuum sulfate 0.17%, glucose 2%, adenine 14.6 mg / L, uracil 20 mg / L, lysine hydrochloride 30 mg / L, leucine 90 mg / L, histidine 20 mg A medium was prepared by mixing various soy protein digests with the / L mixture at 1% by dry weight. The budding yeast "YPH499 strain" in this medium the cells per 1mL was inoculated so that the ^106, over time the number of cells at 10 ° C. (0 h, 24 h, 72 h) were seen.

As shown in the results of FIG. 1, when the β-conglycinin degradation product was added, a significant difference was observed in the number of yeasts at a fermentation time of 72 hours, compared with the case where other soybean protein degradation products were added. Yeast growth was significantly promoted in the low temperature range. On the other hand, the other glycinin degradation products and LP degradation products are equivalent to the growth rate of yeast in the isolated soybean protein degradation product, and they do not show a significant yeast low-temperature fermentation promoting effect like β-conglycinin degradation products. It was.
As described above, it was revealed that β-conglycinin peptide, which is one of the fractions of soybean protein, has a remarkable effect of promoting low-temperature fermentation of yeast, although it is a peptide derived from the same soybean protein.
In addition, it was shown that by adding β-conglycinin degradation product, yeast can grow even at low temperatures, but this is a phenomenon that the activity of intracellular enzymes decreases and the activity of yeast is suppressed at low temperatures. Is considered to be relaxed by β-conglycinin. In that sense, it can be said that β-conglycinin degradation products have an effect as a low-temperature stress protective agent.

▲ Total: Isolated soy protein degradation product ○ 7S: β-conglycinin degradation product □ 11S: Glycinin degradation product * LP: LP (lipophilic protein) degradation product

Claims

A yeast low-temperature fermentation promoter comprising a degradation product of soybean β-conglycinin.
The yeast low-temperature fermentation promoter according to claim 1, wherein the proportion of the fraction having a molecular weight of 120 to 500 in the degradation product of soybean β-conglycinin is 60% or more.
A yeast growth medium to which the low-temperature fermentation promoter according to claim 1 is added.
The manufacturing method of the fermented food / beverage products which inoculate yeast to the culture medium containing the low-temperature fermentation promoter of Claim 1, and ferment at 15 degrees C or less.
A yeast low-temperature stress protectant comprising a degradation product of soybean β-conglycinin.