JPS6148914B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for self-digestion of bacterial cells, and more specifically, prior to autolysis, the bacterial cells are treated with alkali to cause changes in the bacterial cells to the extent that enzymes necessary for autolysis are not deactivated, and at the same time. This invention relates to a method for suppressing putrefaction during autolysis by stopping or reducing the viability of other contaminating bacteria. In particular, the present invention relates to a method in which at least part of the alkaline treatment is carried out in the presence of common salt, various extracts, and the like. It is known to produce yeast extract by autolyzing bacterial cells, especially yeast. Such autolysis involves adding a high concentration of table salt or an organic solvent such as ethyl acetate to yeast to cause plasma separation and turning the yeast into a slurry.
There is a method that allows autolysis to occur at a temperature of about 30 to 70 degrees Celsius. However, adding a high concentration of salt is not preferable because it increases the salt concentration in the yeast extract obtained, and the use of organic solvents that are not food additives should also be avoided. As a method to solve these difficulties, a method has been proposed in which a relatively small amount of salt and ethanol are used in combination (see Japanese Patent Application Laid-Open No. 49-62675.) The present invention differs from such a method in that live bacteria are heated to pH 10.
An alkaline treatment step in which the bacterial cells are brought into contact with the above alkaline aqueous solution to a state where they can be clearly stained with methylene blue, and the bacterial cells that have undergone the alkali treatment step are
It consists of an autolysis step of self-digestion at pH 5 to 9, and at least a part of the alkali treatment step is selected from sodium chloride, monosodium glutamate, proteolytic amino acids, fish extract, meat extract, vegetable extract, and bacterial cell extract. This is a method for self-digestion of bacterial cells, characterized in that it is carried out in the presence of at least one kind of additive. The present invention will be described in detail below. The targets of the autolysis method of the present invention are live microbial cells such as various yeasts and bacteria. For example, Saccharomyces cerevisiae (Saccharomyces
cerevisiae), Saccharomyces carlsbergensis (Saccharomyces carlsbergensis), Candida genus yeast such as Candida utilis (Candida tropicalis), Candida tropicalis (Candida tropicalis), Pichia miso (Pichia miso), etc. and Brevi-bacterium spp.
Amino acid-fermenting bacteria such as those typified by Corynebacterium sp. and Corynebacterium sp. are used as raw materials. These yeasts and bacteria may be obtained as by-products of various fermentation industries, or may be cultured for the purpose of producing bacterial cells.
For example, brewer's yeast, a byproduct of the beer industry,
This is a typical raw material bacterial cell for the method of the present invention. These microbial cells need to be viable microbial cells. Live bacteria can be easily identified because they are not stained by the methylene blue staining method described below. Of course, viable cells containing some cells that can be stained with methylene blue staining can also be used as the raw material for the method of the present invention. Therefore, the raw material for the method of the present invention may be substantially viable bacterial cells. In the present invention, these bacterial cells are first subjected to an alkali treatment step in which they are brought into contact with an alkaline aqueous solution having a pH of 10 or higher. This step usually requires only adding an alkali to a water slurry of bacterial cells to adjust the pH to a predetermined value and stirring the slurry slightly. As the alkali, any of caustic soda, calcium hydroxide, ammonia, etc. can be used, but usually caustic soda, calcium hydroxide, or both are used in combination. If the pH of the alkaline treatment is low, the alkaline treatment takes a long time and the inactivation of contaminants is insufficient, which may cause spoilage during the subsequent autolysis. On the other hand, if the pH is too high, nucleic acids and proteins are likely to be eluted from the bacterial cells during alkaline treatment, and enzyme activity is likely to be impaired. Therefore, alkaline treatment has a pH of 10.5 to 12.5, especially
11 to 12 is preferable. Note that the pH will gradually decrease during the alkali treatment, but normally it can be left as is. During alkaline treatment, it is important to ensure that the subsequent autolysis is not inhibited, that is, that the enzymatic activity of the bacterial cells is preserved as much as possible. Therefore, the alkali treatment is preferably carried out at a low temperature, usually from 0 to 40°C, preferably from 5 to 30°C. The alkaline treatment is carried out until the bacterial cells become clearly stained by methylene blue staining. The methylene blue staining method involves diluting an alkali-treated bacterial cell suspension with water, placing a drop on a slide glass, and adding methylene blue solution (a solution of 0.02 g of methylene blue dissolved in 50 ml of distilled water) and Na ₂ HPO. ₄・12H ₂ O: 0.018g
In this method, a drop of a solution prepared by dissolving 2.70 g of KH ₂ PO ₄ in 50 ml of distilled water is added, and the microbial cells are observed under a microscope to see if they are stained. If the methylene blue staining method clearly stains the cells, not only the stained cells but also the unstained cells have already changed, so even if the alkali treatment is stopped, the unstained cells will remain for a short period of time. It will eventually become dyed. Usually, alkaline treatment is carried out until 50% or more of the bacterial cells are stained. The ratio of stained bacterial cells can be easily calculated using a hemocyte disc. The time for the alkaline treatment is usually 1 to 60 minutes, and the stronger the alkalinity or the higher the temperature, the shorter the treatment may be. For example, under strong alkaline conditions with a pH of 12 or higher, staining becomes clearly visible after contact with the alkali for an extremely short period of less than 1 minute. If the treatment time is too long, the enzyme may be deactivated, so treatment should be avoided for an unnecessarily long time. The preferred alkali treatment time is within 20 minutes. In particular, alkali treatment at pH 11 to 12 within 5 minutes is preferred. In the present invention, at least a part of the alkali treatment step is carried out in the presence of at least one selected from sodium chloride, monosodium glutamate, proteolytic amino acids, fish extract, meat extract, vegetable extract, and bacterial cell extract. That is, it is necessary to bring the bacterial cells into contact with an alkaline aqueous solution having a pH of 10 or more in the presence of these additives. For example, the above additives may be added to water-containing microbial cells such as compressed yeast or an aqueous slurry of microbial cells, and then an alkali may be added to adjust the pH to a predetermined level, or the above additives and an alkali may be added at the same time. By setting the pH to a predetermined value, the entire alkali treatment step can be performed in the presence of additives. Alternatively, the second half of the alkali treatment step can be carried out in the presence of the additive by adding an alkali to the water slurry of the bacterial cells to adjust the pH to a predetermined value, and then adding the above-mentioned additives. When using bacterial cells containing a large amount of impurities as a raw material, suspend the bacterial cells in an alkaline aqueous solution in the absence of the above additives,
It is then separated into a supernatant liquid and a slurry containing bacterial cells using a centrifugal separator, and the above-mentioned additives are added to this slurry to maintain it at a predetermined pH of 10 or higher. When the alkali treatment step is carried out in the presence of the above-mentioned additives, the effect of the alkali treatment becomes more pronounced.
Therefore, if the pH and temperature are the same, the bacterial cells can be stained with a short treatment time. Furthermore, if the temperature and treatment time are the same, the treatment can be performed at a lower pH. In addition, these additives have the effect of increasing the preservative ability of the autolytic fluid during the autolysis process. The concentration of the additive is preferably a concentration sufficient to cause autolysis of the bacterial cells. Usually, in the case of sodium chloride, it is added to the bacterial cell slurry at a concentration of 1 to 5% (by weight), and in the case of extracts, the concentration is slightly higher, ranging from several percent to 10% (by weight). The most common of these additives is sodium chloride, which is added to a predetermined concentration of hydrated bacterial cells or an aqueous slurry of bacterial cells to cause plasmolysis of the bacteria, followed by alkali treatment. A method is often used to adjust the pH to a predetermined value by adding . Autolysis involves adding acid to the bacterial cells that have been treated with alkaline to adjust the pH.
Do this while adjusting the number from 5 to 9. As the acid, inorganic acids such as hydrochloric acid and phosphoric acid, and organic acids such as acetic acid, citric acid, malic acid, and lactic acid are used. When the alkali treatment is carried out in an aqueous solution containing calcium hydroxide, it is preferable to use phosphoric acid or a combination of phosphoric acid and another acid as the acid to precipitate the calcium as calcium phosphate, which provides an even better result. An autolyzed solution can be obtained. In particular, when brewer's yeast, a byproduct of the beer industry, is used as the living microbial cell, there is a bitterness in the autolysis liquid that is thought to originate from hops, but calcium hydroxide is used as the alkali and phosphoric acid is used as the acid. When calcium is precipitated as calcium phosphate, the bitterness of the autolytic fluid can be removed at the same time. Autolysis reduces bacterial cells to 5-25 (weight)
% suspension, and stir it slightly at 20-50℃, especially
This can be easily done by maintaining the temperature at 30 to 50°C. Note that the pH of the liquid gradually decreases during autolysis, so in the present invention, the pH of the alkali-treated bacterial cells is reduced.
It is also possible to initiate autolysis by neutralizing to a pH slightly above 9, with a substantial portion of the autolysis occurring at a pH of 5-9. However, autolysis is extremely slow outside the pH range of 5 to 9, so from the beginning
It is preferable to adjust the pH to 5 to 9 for autolysis. The time required for autolysis varies depending on the temperature and alkali treatment conditions, but is usually about 10 to 30 hours. Once the fruit itself has been digested, the enzymes are deactivated by heating. After separating the residue, the autolyzed liquid obtained in this manner may be concentrated to produce a yeast extract or the like, or it may be directly concentrated to be used as a seasoning. If desired, bone meal, other extracts, oils, etc. may be added prior to concentration. According to the method of the present invention, autolysis can be carried out without causing spoilage by using only food additives that are allowed, and the resulting autolysis liquid has excellent flavor. EXAMPLES Next, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to the following examples. The baker's yeast used in the examples was obtained by the following method. Commercially available baker's yeast 10
Kg was diluted 6 times with tap water to form a slurry, and the operation of concentrating the yeast using a centrifugal cleaner was repeated five times to obtain yeast with a solid content of 18% (by weight). Sugar 8 (weight) % (molasses separated from sludge is used as sugar), KH ₂ PO ₄ 0.2 (weight %,
NH ₄ H ₂ PO ₄ 0.6 (weight)%, MgSO ₄ 7H ₂ O0.03
(weight)%, yeast extract 0.2% (weight), and biotin 2 μg/L medium was heat sterilized and placed in 100 culture vessels equipped with a stirrer. The above-mentioned yeast with a solid content of 18% was added to this, and while the pH was maintained at 5 with aqueous ammonia, it was cultured at 20°C for 8 hours, and it was confirmed that the yeast had split. Next, after static culture was performed at 10°C for 24 hours, the yeast was collected by centrifugation. The yeast was washed with water five times and then dehydrated using a centrifuge to obtain baker's yeast with a solid content of 26% (by weight) and a dry crude protein content of 52.4% (by weight). Example 1 1 g of sodium chloride was added to 100 g of baker's yeast to cause it to self-thaw. Then add 1N-NaOH
The pH was adjusted to 10.4 and maintained at 30°C for 10 minutes. The methylene blue staining rate was over 70%. to this slurry
1N-HCl was added to adjust the pH to 9, and autolysis was carried out at 38-40°C for 16 hours. The pH at the end of autolysis was 6.7. The resulting autolysis solution was adjusted to pH 6.0 with 1N HCl, water was added to make 500 g, and the solution was kept at 95-99°C for 5 minutes. Next, it was separated into a supernatant liquid and a precipitate using a centrifuge, and the precipitate was made up to 500 g by adding water, and then separated into a supernatant liquid and a precipitate using a centrifuge again. The dry weight of the precipitate was 17.2 g. Therefore, 34% of the dry weight of the yeast was extracted outside the cells by autolysis (hereinafter referred to as extract extraction rate). Examples 2 to 7 The following additives were added to 100 g of baker's yeast. Self-lysed. Next, 0.4N-NaOH was added to adjust the pH to 11, and the mixture was kept at 20°C for 10 minutes. The methylene blue staining rate was over 90%. 1N-HC in this slurry
was added to adjust the pH to 8, and autolysis was carried out at 38°C for 16 hours. The pH at the end of autolysis was 6.7-7. The results are shown in Table 1.

[Table] Amino acids: Powder of amino acid mixture obtained by acid decomposition of gluten, composition is 58% sodium chloride.
(weight)%, amino acids and other 42% (weight) Fish meat extract: Powder of hot water extract of Japanese sardine Beef extract: Crude protein 60% (weight), water 17% (weight), sodium chloride 3% (weight) Yeast Extract: Sodium chloride 47% (weight), other extracts 53% (weight) Vegetable extract: Powdered broth from canned asparagus, composition: ash 7% (weight), total nitrogen 2.1
(weight) %, soluble nitrogen-free matter 81 (weight) % Example 8 45 g of beer pre-fermentation yeast (solid content 23 (weight) %, methylene blue staining rate 5% or less) washed in advance with an alkaline aqueous solution and 30 ml of water. to make a slurry. The pH of this slurry was 6.8. 1.5 g of sodium chloride and 1N-NaOH were added to this to adjust the pH to 11.5, and the mixture was maintained at 20°C for 3 minutes. The methylene blue staining rate was over 90%. Then 1N-
The pH was adjusted to 8 with HCl and autolysis was performed at 38°C for 24 hours.
In addition, 1N-NaOH was occasionally added during the autolysis to maintain the pH in the range of 8 to 6.5. Extract extraction rate
34% was obtained.

Claims (1)

[Scope of Claims] 1. An alkaline treatment step in which viable bacterial cells are brought into contact with an alkaline aqueous solution with a pH of 10 or more to bring the bacterial cells into a state where they can be clearly stained with methylene blue; At least a part of the alkali treatment step includes sodium chloride, monosodium glutamate, proteolytic amino acids, fish meat extract, meat extract,
A method for self-digestion of bacterial cells, characterized in that the method is carried out in the presence of at least one additive selected from vegetable extracts and bacterial cell extracts. 2. The method for self-digestion of bacterial cells according to claim 1, characterized in that the alkali treatment step is carried out until 50% or more of the bacterial cells can be stained. 3. In the method for self-digestion of bacterial cells according to claim 1 or 2, the alkali treatment step involves suspending live bacterial cells in an alkaline aqueous solution and then using a centrifuge to remove the supernatant liquid and the bacterial cells. Separate into slurry and
The slurry is characterized by adding at least one additive selected from sodium chloride, monosodium glutamate, proteolytic amino acids, fish extract, meat extract, vegetable extract, and bacterial cell extract to maintain the pH at 10 or above. How to do it. 4. A method for self-digestion of bacterial cells according to any one of claims 1 to 3, characterized in that viable bacterial cells are brought into contact with an alkaline aqueous solution having a pH of 11 to 12. 5. A method for autolysis of bacterial cells according to any one of claims 1 to 4, characterized in that the autolysis is carried out at 30 to 50°C. 6. The method for self-digestion of bacterial cells according to any one of claims 1 to 5, characterized in that the alkali treatment is carried out at 5 to 30°C. 7. In the method for self-digestion of bacterial cells according to any one of claims 1 to 6, the alkali treatment is performed in an alkaline aqueous solution containing calcium hydroxide, and then the alkaline aqueous solution containing the bacterial cells is treated with phosphoric acid. A method characterized by neutralizing using an acid containing the acid and autolyzing it to a pH of 5 to 9.