JPS5811200B2 - How can I get the best results? - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for obtaining high-yield low molecular weight proteins fractionated from yeast.

Recently, many attempts have been made to use yeast proteins as foods, feeds, and industrial materials.

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

Proteins obtained by hydrolyzing proteins obtained by such methods with alkali have a wide molecular weight distribution, and when used for industrial purposes, there are problems such as high viscosity and low solvent solubility.

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

The present invention extracts proteins from yeast, hydrolyzes them, treats them with a cation exchange resin, and then uses reverse osmosis to fractionate proteins and separate substances other than proteins to obtain low molecular weight proteins. The goal is to get it quickly.

Extraction methods for yeast proteins include alkaline solution extraction, urea extraction, enzymatic treatment extraction, and mechanical processing methods, but considering economic efficiency and industrial scale such as mass production, mechanically crushing yeast After that, extraction with an alkaline solution is generally better.

Yeasts used in the present invention include baker's yeast (Satucharomyces cerevisiae), nucleic acid yeast (Cancita uterilis), valve yeast (Candida uteilis, Mycotorula japonica, etc.), and petrochemical products (methanol, acetic acid, n-paraffin), etc. Assimilating yeasts (Cancita uteilis, Candida novellas (Feikoken Bacterium No. 705, Showa 4)
(See Patent Application Publication No. 43,877, 2007) (Mycotorula japonica, etc.), but any other materials containing yeast cell walls can be used as the raw material of the present invention.

In the present invention, dried baker's yeast is used as a raw material, and the cell membrane of the yeast is crushed using an impact-type cell membrane crushing device (Patent Application Publication No. 43834 of 1972). Yeast proteins were extracted using the solution.

In this case, the alkali used is sodium hydroxide, potassium hydroxide, etc., and the alkali concentration is 0.1% to 1.2%, and the extraction is carried out at a pH of 8 to 13, preferably PH 8 to 10.

Next, centrifugation was performed to separate the yeast proteins into alkali-soluble yeast proteins and other yeast proteins.

Conditions are 8,000 revolutions (rpm) to 110,000 rpm,
The test was carried out for 30 minutes to 1 hour.

The solution portion thus obtained is mixed with a relatively dilute acid of 5N to 10N (e.g. hydrochloric acid, sulfuric acid, citric acid,
PH3.0 to PH6.0 using lactic acid, formic acid, etc.)
Isoelectric precipitation of the protein was preferably carried out within the range of PH 3.0 to PH 5.5 to obtain native yeast protein.

In addition, a method obtained by salting out salt or the like can also be used.

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

The alkalis used for this hydrolysis include hydric soda, hydric potassium, aqueous ammonia, soda carbonate (or potassium carbonate), borax, potassium cyanide, and other reagents for alkaline hydrolysis of yeast proteins. Any liquid may be used, and preferably sodium chloride, potassium chloride, or aqueous ammonia are used.

The concentration of alkali used is 1% or less, which does not cause denaturation of the yeast protein, and mild hydrolysis is performed.

Preferably, the amount is in the range of 0.5% to 1.0%.

(within the range of PH7 to PH13) The reaction temperature and reaction time are, for example, 50°C to 80°C and 30 minutes to 2 hours to prevent the protein from being colored by alkali.

Next, the yeast protein is decolorized using an oxidizing agent or a reducing agent for the purpose of removing the inherent color of the yeast protein.

Oxidizing agents include hydrogen peroxide, ozone, sodium chlorite, sodium hydrogen sulfite, benzoyl peroxide, bleaching powder, and any other oxidizing agent that has a decolorizing effect on yeast protein can be used.

In particular, hydrogen peroxide has a particularly good decolorizing effect on yeast proteins.

In general, the higher the concentration of hydrogen peroxide, the better the decolorizing effect, but at the same time, oxidation can cause protein molecules to be cut and become too low in molecular weight, and other harmful effects can occur due to oxidation, so use hydrogen peroxide at a dilute concentration as much as possible. It is preferable to use

Furthermore, baker's yeast whose catalase has been deactivated by heat treatment can be sufficiently bleached with hydrogen peroxide at a concentration of 0.2 to 0.3%, whereas uninactivated yeast can be bleached at a concentration of 1.5% or more. It has been shown that the bleaching effect will be less if the bleach is not used.

(Fermentation Engineering Journal 51.677 Nomura et al.).

On the other hand, as a reducing agent, lithium aluminum hydride,
Sodium borohydride, lithium borohydride, aluminum trichloride, sodium monothiophosphate, dithiothreitol, sodium hypochlorite, or any other reducing agent that has a decolorizing effect on yeast protein. good.

In particular, sodium borohydride, sodium aluminum hydride, lithium aluminum hydride, and the like are effective for decolorizing.

The above decolorization treatment significantly improves the whiteness of the enzyme protein, but even if you directly perform the next cation exchange resin treatment without this treatment, the whiteness will decrease slightly but other properties will not change. .

Next, a solution of a hydrolyzate of yeast protein whose molecular weight has been reduced is treated with a cation exchange resin to perform a desalting reaction.

In this case, things that are desalted include common salt, organic acid salts contained in yeast, and ash.

In addition, it also adsorbs and removes pigment components in yeast proteins, so it also has a decolorizing effect.

Therefore, it is characterized in that it simultaneously performs the three effects of extracting high-purity proteins, desalting, and decolorizing.

In addition, other characteristics include, firstly, that the higher the alkaline concentration of the yeast protein alkaline hydrolysis solution, the more rapidly the pH of the extract eluted from the cation exchange resin becomes neutral, and the faster the extraction. Second, since the extract is neutral, the extracted proteins will not be solubilized in an alkaline solution and will not be lost.

Thirdly, since the present invention uses yeast protein whose molecular weight has been reduced by alkaline hydrolysis, it is less likely to be adsorbed to the ion exchange resin and the protein can be extracted effectively.

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

Fifth, since the pH of the extract is neutral, it has many advantages, such as much less damage to the membrane in the subsequent permeable membrane treatment process compared to 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,
Na1cite -MX, -HCR
.

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

Other cation exchange resins suitable for the purpose of the present invention are carboxylic acid type, sulfonic acid type and phosphoric acid type.

In particular, strongly acidic cation exchange resins are preferred, but in addition to strongly acidic ion exchange reactivity, weakly acidic or moderately acidic cation exchange resins can also be used.

Next, 2% to 10% concentration was eluted with a cation exchange resin.
An aqueous solution of low molecular weight yeast protein is permeated through a membrane using reverse osmosis to fractionate proteins and separate substances other than proteins.

If membrane permeation is performed using reverse osmosis, the size of the solute will be 3~
Since it blocks and separates particles as small as 10 Å, it is extremely effective in concentrating low molecular weight yeast proteins.

As mentioned above, membrane permeation by the reverse osmosis method also blocks particles of about 3 Å such as salt, but in the present invention, desalting is performed in advance using a cation exchange resin, so salt is mixed in the concentrated yeast protein. There is no risk of it happening.

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

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

From the above, the features of the present invention include reducing the molecular weight 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). It consists of a combination of a series of separation steps.

The permeable membrane used in the present invention is 08 manufactured by Osmoex.
M0-3319.08M0-334-〇, 08M0-3
34-97, M-600, M-800, M-870 membranes from Walhart Brothers Japan Limited,
Nippon Shinku Co., Ltd.'s M'C-4, G-05H, etc. are used.
Preferably, acetyl cellulose membranes manufactured by Osmoex, 08M0-334-0 (molecular weight cut 1000 or more) and 08M0-334-97 (molecular weight cut 20
0 or more), and when permeated through a 08M0-334-0 membrane, it can be separated into a concentrated protein solution and a solution containing nucleic acids.

Furthermore, when O5MO-334-97 is used, nucleic acid molecules and low-molecular-weight polypeptides pass through the membrane, and low-molecular-weight yeast proteins are concentrated.

The thus concentrated low molecular weight yeast protein solution 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 white powdery low molecular weight protein.

In this case, drying is carried out at a temperature of 70°C, which does not cause deterioration of the protein.
Hereinafter, the temperature was preferably 30°C to 50°C.

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

This yield is 40% (by weight) when other treatment methods are used in the same manner as the present invention without treatment with a cation exchange resin or treatment with membrane permeation.
It is.

) will be higher than that of

While the molecular weight of yeast protein without alkali hydrolysis is several hundred thousand, 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 a low viscosity, is easily soluble in general organic solvents, water, etc., and has a high solubility.

Furthermore, the coloration inherent to yeast protein is improved, and a white powder is obtained.

That is, in the present invention, since proteins are fractionated by membrane permeation, low-molecular-weight yeast proteins with relatively uniform molecular weights, that is, with narrow molecular weight distributions, can be obtained.

At this time, nucleic acids, low molecular weight peptides, etc. are simultaneously separated and removed by membrane permeation.

The yield of nucleic acid is 0 when membrane permeation is not performed.
．． While it is 4% (by weight), in the case of membrane permeation treatment, it is 0.41 or more and reaches a high value of up to 0.8%.

The low molecular weight yeast protein obtained in this way has the properties of milk casein, which cannot be imparted by conventional methods, and can be used in paper coated products, adhesives, synthetic leather, resin molded products, etc.
It is used as an additive in the field of water-soluble polymers, such as protective colloid agents for paints and inks, and industrial materials such as dispersants.

In addition, since nucleic acids with poor digestibility are removed,
The present yeast protein can also be suitably used in the fields of food and feed.

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

The present invention will be specifically explained with reference to Examples.

Example 1 Baker's yeast (Saccharomyces cerevisiae) was subjected to hot water extraction treatment (90°C, 1 hour) to remove components that can be used as raw materials for food and feed, and further deactivated catalase.
According to the method of Patent Application Publication No. 43834 of 1972,
A yeast cell suspension containing 1 kg of yeast cells with a concentration of 7.9% was used to disrupt yeast cell membranes using an impact cell membrane disruption method to obtain a yeast cell suspension with a concentration of 0.9%.
The pH was adjusted to 9.5 with a 1-1.2% dilute caustic soda aqueous solution to elute soluble and insoluble proteins, which were separated in a centrifuge at 10,000 rpm for 30 minutes to separate the solution portion. Isoelectric precipitation was performed with 6N hydrochloric acid at pH 4.5.

Next, centrifugation (6000 rpm, 20 minutes) was performed, and water washing was repeated three or more times for purification.

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

Yeast protein with low molecular weight due to hydrolysis at a concentration of 1%
The alkaline aqueous solution containing the low molecular weight protein was then treated with a cation exchange resin (Dow
Ex-50) is used to decolorize and desalinate (e.g. remove salt, organic acid salts contained in yeast, ash, etc.), and then centrifuge (6000 rpm, 20 minutes), and the extract is reversely Membrane permeation method using osmotic pressure method, that is, acetylcellulose membrane with molecular weight cut of 1000 or more (OS
MC)-334-0 (manufactured by Osmonix) to obtain a concentrated yeast protein aqueous solution.

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

Example 2 Baker's yeast (Saccharomyces cerevisiae) was used in Example 1.
Using a reverse osmosis method, the yeast protein solution 501 was extracted using a cation exchange resin treated in a similar manner to , 12.51 per hour
When the yeast protein is processed and permeated through a membrane in a relatively short period of time, it 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 an acetone solvent 10 times the volume of the dried yeast, and then dried under reduced pressure at a temperature of 40°C to obtain a powdered 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 hydrated carbon are compared to those not treated with the cation exchange resin of the present invention and the reverse osmosis method. Very few.

In addition, the molecular weight is 12,000 to 40,000, and the yield of low molecular weight proteins is 53 (weight)% to 70 (weight)% (nitrogen recovery rate). When yeast proteins are extracted with alkaline solution after physically crushing yeast, the protein yield is
The amount is 40% (by weight) or more, and the method according to the present invention is excellent in that the yield of yeast protein can be significantly improved.

In addition, it is readily soluble in organic solvents and water and can be obtained as a low-viscosity white powder.

This yeast protein, which has many characteristics as described above, is used as a paper coating agent, adhesive, synthetic leather, molded products, dispersant, water retention agent, protective colloid agent, dyeing aid, fiber treatment agent, and more. Proteins with relatively high molecular weights, for example those with a molecular weight of 10,000 or more, can be used as thickeners.

In this way, in addition to being used as a wide variety of industrial materials, since nucleic acids are removed at the same time by membrane permeation, the digestibility of yeast proteins is improved and it can also be used in the food and feed fields.

The yield of nucleic acid is 0.4 (weight) without membrane permeation.
% or more, whereas in the present invention, which permeates through the membrane, it is 0.4%.
(weight)% to 0.8 (weight)%.

Claims (1)

[Claims] 1 Extract protein from yeast, mildly hydrolyze it with alkali at a concentration of 1% or less, and treat the resulting solution containing low-molecular-weight protein with a cation exchange resin to desalt it and obtain high-purity protein. 1. A method for producing a low molecular weight yeast protein, which comprises obtaining a solution and then collecting a yeast protein having a molecular weight of 12,000 to 40,000 at a high yield by reverse osmosis.