JPS5811200B2 - Manufacturing method for the high-quality button fungus of the tallow - Google Patents

Description

DETAILED DESCRIPTION OF THEINVENTION The present invention relates to a method for obtaining low molecular weight proteins fractionated from yeast in good yield.

Recently, there have been many attempts to use yeast proteins as food, feed and industrial materials.

Accordingly, various methods for extracting proteins from yeast cells have been investigated, including, for example, alkaline solution extraction, urea extraction, enzyme treatment, and mechanical treatment.

Proteins obtained by such methods and then hydrolyzing them with alkali have a wide molecular weight distribution, and have problems in that they have high viscosity and low solubility in solvents when used in industrial applications.

The present inventors conducted research to obtain yeast proteins having a narrow molecular weight distribution suitable for industrial use in good yield, and as a result, arrived at the present invention.

The present invention relates to extracting proteins from yeast, hydrolyzing them, treating them with a cation exchange resin, and then fractionating the proteins and separating substances other than proteins by reverse osmosis to obtain low molecular weight proteins in good yields.

Methods for extracting yeast protein include alkaline solution extraction, urea extraction, enzyme treatment extraction, and mechanical treatment methods. Considering economic efficiency and industrial scale such as mass production, the method of mechanically disrupting yeast and then extracting it with an alkaline solution is generally superior.

The yeasts used in the present invention include baker's yeast (Saccharomyces cerevisiae), nucleic acid yeast (Cancida uteiris), valve yeast (Candida uteiris, Mycotorula japonica, etc.), and yeasts that assimilate petrochemical products (methanol, acetic acid, n-paraffin) (Cancida uteiris, Candida novellas (Microbial Culture Society No. 705, 1929)).
In addition, any yeast cell containing a yeast cell wall can be used as a raw material for the present invention.

In the present invention, dried baker's yeast is used as the raw material, the yeast cell membrane is disrupted using an impact cell membrane disrupter (Patent Application Publication No. 43834, 1972), and then the yeast protein is extracted with a dilute alkaline solution so as not to cause denaturation of the yeast protein.

In this case, sodium hydroxide, potassium hydroxide or the like is used as the alkali, and extraction is performed at an alkali concentration of 0.1% to 1.2% and a pH of 8 to 13, preferably 8 to 10.

The mixture was then centrifuged to separate the yeast protein fraction that was soluble in alkali from the other fractions.

The conditions were 8000 rpm to 110000 rpm.
The reaction was carried out for 30 minutes to 1 hour.

The solution thus obtained is diluted with a relatively dilute acid having a concentration of 5N to 10N (e.g., hydrochloric acid, sulfuric acid, citric acid,
lactic acid, formic acid, etc.) to pH 3.0 to pH 6.0,
Isoelectric precipitation of proteins was carried out preferably within the range of pH 3.0 to pH 5.5 to obtain native yeast proteins.

In addition, a method of obtaining the product by salting out with salt or the like can also be used.

The undenatured yeast protein thus obtained was subjected to protein hydrolysis in an alkaline solution.

The alkali used in this hydrolysis is, for example, sodium hydroxide, potassium hydroxide, aqueous ammonia, sodium carbonate (or potassium carbonate), borax, or potassium cyanide, but any other reagent capable of alkaline hydrolysis of yeast protein may be used, and preferably sodium hydroxide, potassium hydroxide, or aqueous ammonia is used.

The alkali concentration is in the range of 1% or less so as not to cause denaturation of yeast protein, and gentle hydrolysis is carried out.

The range of 0.5% to 1.0% is preferable.

The reaction temperature and reaction time are, for example, 50° C. to 80° C. and 30 minutes to 2 hours, so that the protein does not become discolored by the alkali.

The yeast proteins are then decolorized using oxidizing or reducing agents to remove the inherent color of the yeast proteins.

The oxidizing agent may be hydrogen peroxide, ozone, sodium chlorite, sodium hydrogen sulfite, benzoyl peroxide, bleaching powder, or the like. Any other oxidizing agent that has a decolorizing effect on yeast protein may be used.

In particular, hydrogen peroxide has a remarkable effect of decolorizing yeast protein.

Generally, the higher the concentration of hydrogen peroxide, the better the decolorizing effect. However, at the same time, the oxidation process can cause protein molecules to be broken down into smaller molecules, resulting in harmful effects, so it is preferable to use hydrogen peroxide at as dilute a concentration as possible.

It has also been shown that baker's yeast in which the catalase has been inactivated by heat treatment can be sufficiently bleached with 0.2 to 0.3% hydrogen peroxide, whereas uninactivated yeast requires a concentration of 1.5% or more for the bleaching effect to be small.

(Fermentation Engineering Journal 51, 677 Nomura et al.).

On the other hand, the reducing agent is lithium aluminum hydride,
Examples of the reducing agent include sodium borohydride, lithium borohydride, aluminum trichloride, sodium monothiophosphate, dithiothreitol, sodium hypochlorite, and any other reducing agent that has a decolorizing effect on yeast proteins may be used.

In particular, sodium borohydride, sodium aluminum hydride, lithium aluminum hydride, etc. are effective for decolorization.

The above decolorization treatment significantly improves the whiteness of the enzyme protein, but if the subsequent cation exchange resin treatment is carried out directly without this treatment, the whiteness will decrease slightly but other properties will not change.

Next, the solution of the hydrolysate of yeast protein with reduced molecular weight is treated with a cation exchange resin to carry out a desalting reaction.

In this case, the substances to be desalted include table salt, organic acid salts contained in the yeast, and ash.

At the same time, it also adsorbs and removes the pigment components in the yeast protein, thus providing a decolorizing effect.

Therefore, the method is characterized by simultaneously achieving three effects: extraction of high-purity protein, desalting and decolorization.

Other features include, first, that the higher the alkaline concentration of the alkaline hydrolysis solution of yeast protein, the more rapidly the pH of the extract eluted from the cation exchange resin becomes neutral, and thus the faster the extraction is carried out; and, second, because the extract is neutral, the extracted protein is solubilized in the alkaline solution and is not lost.

Thirdly, in the present invention, yeast proteins having low molecular weights due to alkaline hydrolysis are used, so that they are less likely to be adsorbed onto ion exchange resins and the proteins can be effectively extracted.

Fourth, the solid content of the extract is relatively low, ranging from 2% to 10%, and the flow rate is rapid.

Fifth, the pH of the extract is neutral, which has many advantages, such as much less damage to the membrane in the subsequent permeable membrane treatment step than with acid or alkaline solutions.

The cation exchange resins used in the present invention include Dowex -50, -50W, -XI,
, -X2, -X4, -X8, -XI 2, -XI 6,
Nalcite -MX, -HCR
.

-HDR, -HGR, -MCR, Amberlite (Am
berlite) -IR200, -IR112
, -X-Eloo, -MCR, Amberlite
-erlite) -I R200, -IR1], 2, -
XEloo, -IR122, -IR124, -XZ66
-13, -XE69, -XEIII, etc.

Other cation exchange resins suitable for purposes of this invention include carboxylic acid, sulfonic acid and phosphoric acid types.

In particular, strongly acidic cation exchange resins are preferred, but cation exchange resins having ion exchange reactivity of strong acidity, weak acidity, and medium acidity can also be used.

Next, the cation exchange resin was used to elute the 2% to 10%
The aqueous solution of low molecular weight yeast proteins is passed through a membrane by reverse osmosis to fractionate the proteins and separate substances other than proteins.

By using reverse osmosis to pass through a membrane, the size of the solute is 3 to 100%.
It is extremely effective in concentrating low molecular weight yeast proteins since it blocks and separates particles as small as 10 Å.

As described above, the membrane permeation by the reverse osmosis method also blocks particles of about 3 Å such as salt. However, in the present invention, since desalting is performed in advance using a cation exchange resin, there is no risk of salt being mixed into the concentrated yeast protein.

The operating pressure is generally 34 to 102 kg/cm'g, and the permeation rate (flow rate) is generally 0.625 to 21.51/m"
/hr/(kg/cm'), but in the present invention, the pressure is 40 to 80 kg/cm'.g and the flow rate is 0.625 to 0.
The test was performed at 7001/m/hr/(kg/cm').

The reason why the flow rate is relatively good despite the high pressure is believed to be due to the fact that the yeast protein of the present invention has a low molecular weight.

From the above, the feature of the present invention is the combination of a series of steps including the degradation of yeast proteins by alkaline hydrolysis, desalting with a cation exchange resin, decolorization treatment, fractionation of yeast proteins, and separation of substances other than proteins (nucleic acids, low molecular weight peptides).

The permeable membrane used in the present invention is Osmoex 08
M0-3319.08M0-334-〇, 08M0-3
34-97, Walhart Brothers Japan Limited's M-600, M-800, M-870 membranes,
We use Japan Vacuum's M'C-4, G-05H, etc.
Preferably, acetylcellulose membranes manufactured by Osmoex Corporation, 08M0-334-0 (molecular weight cut-off 1000 or more) and 08M0-334-97 (molecular weight cut-off 20
0 or more), and when it is passed through a 08M0-334-0 membrane, it can be separated into a protein concentrate solution and a solution containing nucleic acids.

Furthermore, when O5M0-334-97 is used, nucleic acid molecules and low molecular weight polypeptides are allowed to permeate the membrane, and low molecular weight yeast proteins are concentrated.

Such a concentrated solution of low molecular weight yeast protein can be used as it is as a foaming agent, fire extinguishing liquid, etc.

On the other hand, this concentrated protein solution was treated with an organic solvent such as alcohols, ketones, ethers, etc., preferably acetone or methanol, and then dried to obtain a low molecular weight protein in the form of a white powder.

In this case, drying is done at a temperature of 70°C, which is a temperature at which protein does not denature.
The following steps were preferably carried out at 30°C to 50°C.

The yield is 50% to 70% by weight based on dry yeast.
% (nitrogen recovery rate).

This yield is based on the case where the treatment with the cation exchange resin and the treatment by membrane permeation were not performed, and other treatment methods were the same as those of the present invention (the yield in this case was 40% (by weight)).
It is.

) is higher than that.

The molecular weight of yeast protein not hydrolyzed with alkali is several hundred thousand, whereas the molecular weight of the protein obtained by the present invention is extremely low, ranging from 12,000 to 40,000.

Therefore, the obtained protein has low viscosity, is easily dissolved in common organic solvents and water, and has high solubility.

Furthermore, the inherent coloring of the yeast protein is improved, and the product is obtained in the form of a white powder.

That is, in the present invention, protein fractionation is carried out by membrane permeation, so that yeast proteins having a relatively uniform molecular weight, that is, low molecular weight proteins having a narrow molecular weight distribution, can be obtained.

At the same time, nucleic acids, low molecular weight peptides, etc. are separated and removed by permeation through the membrane.

The yield of nucleic acid is 0.05% when membrane permeation is not performed.
4% (by weight), whereas when membrane permeation treatment is performed, the value is 0.41 or more, reaching as high as 0.8%.

The low molecular weight yeast protein obtained in this way has the properties of milk casein that could not be imparted by conventional methods, and is useful for paper coatings, adhesives, synthetic leather, resin-like moldings, and
It is used as an additive in the field of water-soluble polymers, for example in industrial materials such as protective colloids and dispersants for paints and inks.

In addition, nucleic acids that are not easily digested have been removed,
The yeast protein of the present invention can also be suitably used in the fields of food and feed.

In this way, the present invention makes it possible to extract proteins from yeast and at the same time separate components other than proteins, and each of them can be effectively utilized, which is economical.

The present invention will now be described in detail with reference to examples.

Example 1 Baker's yeast (Saccharomyces cerevisiae) was subjected to hot water extraction (90°C, 1 hour) to remove ingredients that can be used as raw materials for food and feed, and then catalase was inactivated.
According to the method of Patent Application Publication No. 43834 of 1972,
A yeast cell suspension containing 1 kg of yeast cells and having a concentration of 7.9% was subjected to an impact cell membrane disruption method to disrupt the yeast cell membranes, resulting in a concentration of 0.
The pH was adjusted to 9.5 with 1-1.2% dilute aqueous caustic soda solution to separate soluble and insoluble proteins, which were then centrifuged at 10,000 rpm for 30 minutes, and the solution was subjected to isoelectric precipitation with 6N hydrochloric acid at pH 4.5.

Then, the mixture was centrifuged (6000 rpm, 20 minutes) and washed with water three or more times for purification.

Next, yeast protein was hydrolyzed using a dilute aqueous solution of caustic soda having a concentration of about 1% under the conditions shown in Table 1.

Hydrolyzed low molecular weight yeast protein at a concentration of 1%
The low molecular weight protein was decolorized with 100% hydrogen peroxide, and then the alkaline aqueous solution containing the low molecular weight protein was immersed in a cation exchange resin (Dow
The extract was then subjected to a membrane permeation method using a reverse osmosis method, i.e., an acetyl cellulose membrane (OSM) with a molecular weight cutoff of 1000 or more.
The yeast protein was permeated through a membrane using a cellulose acetate ester (MC)-334-0 (manufactured by Osmonix Co.) to obtain a concentrated aqueous solution of yeast protein.

This product can be used for industrial purposes such as foaming agents and digestive fluids.

Example 2 Baker's yeast (Saccharomyces cerevisiae) was cultured in the same manner as in Example 1.
The extract from the cation exchange resin treated in the same manner as in Example 1 was subjected to reverse osmosis using an Osmonix membrane (O8MO-334-97) with a molecular weight cutoff of 200 or more, and 50 L of the yeast protein solution was subjected to a reverse osmosis process at a rate of 12.5 L per hour.
By passing the mixture through a membrane after a relatively short period of time, the mixture can be separated into a solution containing concentrated low molecular weight yeast proteins and nucleic acid components.

The concentrated yeast protein was washed three times with acetone solvent having a volume 10 times that of the dried yeast, and then dried under reduced pressure at a temperature of 40° C. to obtain a powder of yeast protein.

The properties of this yeast protein are as shown in Table 2. The protein content is 90% (by weight) or more, and the crude ash and crude carbohydrate contents are extremely low compared with those not treated with the cation exchange resin and reverse osmosis method of the present invention.

In addition, the molecular weight is 12,000 to 40,000, and the yield of low molecular weight protein is 53% (by weight) to 70% (by weight) (nitrogen recovery rate). When yeast is simply physically disrupted by alkaline extraction or impact cell membrane disruption method, and then yeast protein is extracted with an alkaline solution, the protein yield is
The yield of yeast protein is 40% (by weight) or more, and the method according to the present invention is excellent in that it can remarkably improve the yield of yeast protein.

In addition, it is obtained as a white powder with low viscosity that is easily soluble in organic solvents and water.

The yeast protein, which has such many characteristics, can be used as a paper coating agent, adhesive, synthetic leather, moldings, dispersant, water retention agent, protective colloid, dyeing assistant, and fiber treatment agent. Furthermore, proteins with relatively high molecular weight, for example those with a molecular weight of 10,000 or more, can be used as thickeners, etc.

In addition to the wide range of applications as an industrial material, the nucleic acids are simultaneously removed by membrane permeation, improving the digestibility of yeast protein, and the yeast can also be used in the food and feed industries.

The yield of nucleic acid was 0.4 (by weight) without membrane permeation.
% or more, whereas in the present invention where the membrane is permeated, the
(by weight)% to as high as 0.8 (by weight)%.

Claims (1)

[Claims] 1. A method for producing low molecular weight yeast protein, comprising extracting protein from yeast, mildly hydrolyzing the protein with an alkali having a concentration of 1% or less, treating the resulting solution containing low molecular weight protein with a cation exchange resin for desalting to obtain a high purity protein solution, and then recovering yeast protein having a molecular weight of 12,000 to 40,000 in high yield by reverse osmosis.