JPS6148913B2 - - 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 the extent that the enzymes necessary for self-digestion are not deactivated. The present invention relates to a method for suppressing putrefaction during autolysis by causing changes in bacteria and at the same time stopping or weakening the viability of other contaminating bacteria.

It is known to produce yeast extract by autolyzing bacterial cells, especially yeast. Such autolysis methods include adding high concentrations of salt to yeast,
After adding an organic solvent such as ethyl acetate to forcibly cause protoplasm separation and turning the yeast into a slurry,
There is a method of autolysis at around 70°C. However, adding a high concentration of salt is not desirable as it increases the salt concentration in the yeast extract obtained.
Furthermore, the use of organic solvents other than salt 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 methods by converting live bacterial cells into PH
After treating with alkali at a pH of 10.5 or higher to ensure that the bacterial cells are clearly stained with methylene blue,
The present invention relates to a method for self-digestion of bacterial cells, which is characterized by carrying out self-digestion at steps 9 to 9.

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 carlsbergensis
yeasts of the genus Satucharomyces, such as Carlsbergensis),
Candida utilis, Candida tropicalis
yeasts of the Candeida genus, such as Pichia
Yeast such as Pichia miso and amino acid-fermenting bacteria represented by Brevibacterium 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, which is a byproduct of the beer industry, 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 washed 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 treated with alkali at a pH of 10.5 or higher. Normally, it is sufficient to suspend the bacterial cells in an aqueous alkaline solution of a specified pH and stir it 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 11 to 13,
In particular, it is preferable to carry out at 11.5 to 12.5. 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, it is preferable to carry out the alkali treatment 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 O0.018g
In this method, one drop of a solution (combined with a solution of 2.70 g of KH ₂ PO ₄ dissolved 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 stop the alkali treatment and remove the unstained cells for a short period of time. It ended up being 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, in strong alkalinity with a pH of 12 or higher, staining is usually clearly observed in an extremely short time of less than 1 minute. If the treatment time is too long, the enzyme may be deactivated, so it is not preferable to carry out the treatment for a longer time than necessary. The preferred alkali treatment time is within 20 minutes. In particular, treatment within 5 minutes at pH 11.5 to 12.5 is preferred.

After the alkaline treatment, the bacterial cells are centrifuged if desired to remove most of the alkaline aqueous solution, and if necessary, washed with water, and then acid is immediately added to lower the pH and carry out the next autolysis. For example, if the raw material contains a large amount of impurities, or if a large amount of alkali was used in the alkali treatment process and the resulting autolysis solution would contain more than the allowable amount of salts, centrifugation is necessary. It is preferable to do so.

Autolysis is carried out by adding acid to the bacterial cells that have been treated with alkali and adjusting the pH to 5-9. 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. For autolysis, salt, sodium glutamate, proteolytic amino acid solution, fish extract, vegetable extract, meat extract, yeast extract, etc. may be added to the alkaline-treated bacterial cells. When these additives are added, even unstained bacterial cells that have undergone alkali treatment are rapidly stained, and autolysis progresses uniformly. There is also the advantage that the osmotic pressure of the autolyzing fluid is increased and the preservative ability is improved. The amount added is usually
A concentration of about 1 to several (weight) % in the autolysis fluid is sufficient. Once autolysis is complete, 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. After diluting 10kg of commercially available baker's yeast 6 times with tap water to make a slurry, the process of concentrating the yeast using a centrifugal cleaner was repeated 5 times.
Yeast with a solid content of 18% (by weight) was obtained. Sugar 8 (weight)
% (molasses separated from sludge is used as sugar), ammonium sulfate 1% (by weight), KH ₂ PO ₄ 0.2 (by weight)
%, _{NH4H2PO4 0.6} ( _weight )%, _MgSO4・_7H2O
A medium containing 0.03% (by weight), 0.2% (by weight) yeast extract, and 2 μg/bithion was heat sterilized and placed in 100 culture vessels equipped with a stirrer. Add the above-mentioned yeast with a solid content of 18% to this, and pH it with ammonia water.
After culturing at 20°C for 8 hours while maintaining the temperature at 5, it was confirmed that the yeast had split. After static culture was then 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 reduce the solid content to 26% (by weight) and the crude protein content on the dry matter.
It was made into 52.4 (weight)% baker's yeast.

Example 1 100g of baker's yeast, 30ml of water at 30℃ and 1N
NaOH was added to make a slurry of pH 10.7, and the mixture was stirred for 20 minutes. The methylene blue staining rate was over 80%. Next, add 1N HCl to this slurry to adjust the pH.
9.2, and autolysis was performed at 38-40°C for 16 hours. The pH at the end of autolysis was 6.5. 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 centrifugal sedimentation tube, and the precipitate was made up to 500 g by adding water, and then separated into a supernatant liquid and a precipitate using a centrifugal sedimentation tube again. The dry weight of the precipitate is
It was 15.1g. Therefore, 42% of the dry weight of yeast was extracted outside the cells by autolysis (hereinafter, this is referred to as extract extraction rate).

Example 2 100g of baker's yeast, 30ml of water at 20℃ and 1N-NaOH
was added to make a slurry with a pH of 12.4, and the mixture was stirred for 10 minutes. The methylene blue staining rate was over 80%.
Next, 1N-NCl was added to this slurry to adjust the pH to 9.0, and autolysis was performed at 38 to 40°C for 16 hours. The pH at the end of autolysis was 6.5. The autolytic solution was treated in the same manner as in Example 1 to obtain an extract extraction rate of 38%.

Example 3 Beer pre-fermentation yeast (solid content 13 (weight)%) 1Kg
Add 6N-NaOH to make a slurry with pH 12.7,
Stir for 10 minutes at °C. Methylene blue staining rate is 90
% or more. 6N-HCl was added to this slurry to adjust the pH to 8.0, and autolysis was performed at 38°C for 20 hours. The pH at the end of autolysis was 6.2. The autolysis solution was heated to 70°C or higher for 10 minutes and then separated into a supernatant and a precipitate using a centrifugal sedimentation machine. Precipitate with boiling water 600
After stirring for 1 hour, the mixture was again separated into a supernatant liquid and a precipitate using a centrifugal sedimentation machine. Extract extraction rate is 34%
It was hot.

Example 4 Beer pre-fermentation yeast (solid content 13 (weight)%) 1Kg
6N-NaOH was added to adjust the pH to 8.0, calcium hydroxide was further added to adjust the pH to 12.3, and the mixture was stirred at 8°C for 10 minutes. The methylene blue staining rate was over 80%. 2 kg of cold water was added to this slurry, and the slurry was separated into a supernatant liquid and a precipitate using a centrifugal sedimentation machine, and the precipitate was separated into a supernatant liquid and a precipitate using a centrifugal sedimentation machine again after adding 2 kg of cold water. Add 1N-H ₃ PO ₄ to this precipitate to adjust the pH to 7.5,
Autolysis was performed at 40°C for 16 hours. The pH at the end of autolysis was 6.8, and the extract extraction rate was 37%.

Example 5 Beer post-fermenting yeast (solid content 15% (weight)) 1Kg
1N-NaOH was added to adjust the pH to 7.5, calcium hydroxide was added to adjust the pH to 11.5, and the mixture was stirred at 10°C for 10 minutes. The methylene blue staining rate was over 90%. This slurry was separated into a supernatant liquid and a precipitate using a centrifugal sedimentation machine, and the precipitate was separated into a supernatant liquid and a precipitate again by adding 500 g of cold water. 1N―H ₃ PO ₄ to this precipitate
was added to adjust the pH to 7.5, and autolysis was performed at 40°C for 16 hours. The extract extraction rate was 33%.

Example 6 Candida utilis was cultured in a molasses medium, and yeast was collected from the culture solution. This was washed with water to obtain yeast (solid content 20% (weight)) containing almost no impurities. 1N for this yeast
NaOH was added to adjust the pH to 12.5 and the mixture was stirred at 10°C for 10 minutes. The methylene blue staining rate was over 90%.
1N-HCl was added to this to adjust the pH to 7.5, and autolysis was carried out at 40°C for 16 hours while maintaining this pH. Then, it was treated in the same manner as in Example 3 to obtain an extract extraction rate of 31%.
Obtained.

Example 7 Beer pre-fermentation yeast (solid content 16 (weight)%) 234
The same amount of water was added to form a slurry, and 1N-NaOH was further added to adjust the pH to 10.9, followed by treatment at 11°C for 5 minutes. The methylene blue staining rate was 20-30%. Centrifuge this slurry and discard the supernatant, leaving 114 g of precipitate.
0.5% hydrochloric acid was added to adjust the pH to 8.4, 1.2 g of common salt was added, and autolysis was carried out at 40°C for 18 hours. At the end of autolysis, the pH was 5.7 and the extract extraction rate was 43%. In addition, the methylene blue staining rate 5 minutes after adding the salt was 80% or more.

Example 8 Beer pre-fermentation yeast (solid content 16 (weight)%) 234
Add the same amount of water at 5-7℃ to g, and pH it with 1N-NaOH.
12.3 and held for 20 seconds. The methylene blue staining rate was approximately 50%. Immediately centrifuge, discard the supernatant, add 1% hydrochloric acid to the precipitate to adjust the pH to 8.0, and add 1% hydrochloric acid to the precipitate.
Autolysis was performed for 18 hours at ℃. At the end of autolysis, the pH was 5.6 and the extract extraction rate was 46%.

Claims (1)

[Scope of Claims] 1. A bacterium characterized by treating live bacterial cells with an alkali at a pH of 10.5 or higher so that the bacterial cells are clearly stained with methylene blue, and then autolyzing the cells at a pH of 5 to 9. The body's self-digestion method. 2. The method for autolysis of bacterial cells according to claim 1, characterized in that the autolysis is carried out after 50% or more of the bacterial cells are stained. 3. The method for self-digestion of bacterial cells according to claim 1 or 2, characterized in that viable bacterial cells are treated with alkali at a pH of 11.5 to 12.5. 4. The method for self-digestion of bacterial cells according to any one of claims 1 to 3, characterized in that the viable bacterial cells are treated with alkali at 5 to 30°C. 5. The 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. In the autolysis method for bacterial cells according to any one of claims 1 to 5, most of the alkaline aqueous solution is separated from the bacterial cells after alkali treatment, and then acid is added to adjust the pH to 5 to 9. A method characterized by causing autolysis. 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 pH is adjusted using an acid containing phosphoric acid. 5～
A method characterized by making the number 9.