JPH0793871B2 - Yeast extract and its manufacturing method - Google Patents

Description

[Detailed Description of the Invention] Technical Field The present invention relates to a method for extracting natural 5'-IMP from an extract component containing RNA extracted from live yeast cells, which has a high RNA content.
(inosine monophosphoric acid), 5'-GM
The present invention relates to a method for producing a yeast extract containing a high percentage of P (guanine monophosphoric acid), etc., the yeast extract obtained, and a yeast mutant strain with a high RNA content used therein.

BACKGROUND ART Currently, there are a wide variety of yeast extracts available on the market, and the yeasts commonly used as raw materials are baker's yeast and brewer's yeast. The extracts are extracted by hot water extraction, enzymatic decomposition of the yeast cells, mechanical cell destruction, or autolysis of the yeast cells themselves, and are then commercialized as powder or paste. Other natural seasonings include:
There are natural extracts from meat, fish, vegetables, seaweed, etc. On the other hand, RNA extracted and purified from Torula yeast, which has a high RNA content,
is chemically and enzymatically decomposed, and the resulting decomposition product, 5'-
5'-IMP and 5'-GMP are fractionated and purified from the nucleotide mixture and are widely used as nucleic acid seasonings.

However, the nucleotides contained in the commercially available yeast extracts are mainly 3'-AMP (adenine monophosphoric acid) and 3'-GMP, and contain almost no 5'-IMP or 5'-GMP, which have excellent flavors, and therefore are not fully satisfactory seasonings.

On the other hand, when extracting and decomposing the RNA in the yeast cells to produce 5'-IMP and 5'-GMP, a method is used in which a large amount of alkali, salt, surfactant, etc. is used to efficiently extract RNA from the cells.
After extraction of NA, these additives are washed and removed, so the RNA
The degradation products, i.e., the nucleotide components, do not contain yeast extract components.

As described above, conventional commercially available yeast extracts have little umami flavor as a seasoning, so in order to produce a seasoning with a high level of general umami flavor, it has been necessary to add separately produced artificial seasonings such as 5'-IMP and 5'-GMP to regular yeast extract to create an artificial seasoning.

3. Detailed Description of the Invention (Means for Solving the Problems) The present inventors have conducted extensive research to solve the above problems and have arrived at the present invention.
A raw yeast cell solution with a high content of A is heated at 80-120°C, and then 5'-phosphodiesterase is applied to it.
The present invention relates to a method for producing a natural yeast extract having a high content of 5'-IMP, 5'-GMP, or a high content of 5'-GMP, with or without the action of deaminase, as well as the resulting yeast extract and a yeast mutant strain having a high content of RNA.

The yeast extract obtained by the present invention has a unique taste with a long-lasting umami flavor, a rich taste, and little yeast odor.

The present invention will be described in detail below.

The origin of the 5'-phosphodiesterase, deaminase, protease, etc. used in the present invention does not need to be particularly limited, and commercially available products are sufficient.

First, the yeast strain is inoculated into a known medium and cultured. The resulting cells are washed thoroughly and then suspended in water in an amount approximately 5 to 30 times the dry cell volume. The suspension is then heated at 80 to 120°C, preferably 90 to 100°C, to inactivate the RNase enzymes present in the cells.

As the yeast cells used in the present invention, live yeast is used, and dry yeast has been subjected to severe heat treatment in advance, so the flavor of the product is undesirable.

Furthermore, the yeast cells used had an RNA content of 10
% or more of the strain is preferred, and 15% or more by weight is even more preferred.

Examples of yeast strains with high RNA content include Candida
Examples include Candida utilis strain CS7529 (FERM BP-1656) and Candida utilis strain CSB6316 (FERM BP-1657).

The mutant strain CSB6316 was obtained by applying the mutagen nitrosoguanidine to the cold-sensitive parent strain Candida utilis CS7529, and was obtained by selecting a strain that can grow in a synthetic medium containing a boric acid concentration at which the parent strain CS7529 cannot grow, and that has a high RNA content in the bacterial cells.

The mycological properties of the Candida utilis strain SC7529 and the Candida utilis strain CSB6316 are shown below. Both strains have exactly the same mycological properties except for boric acid resistance, and also have exactly the same general mycological properties as Candida utilis except for boric acid resistance and low temperature sensitivity.

Mycological properties of Candida utilis CS7529 and CSB6316: 1. On YM agar, they form soft, cream-colored colonies with entire margins, semi-lenticular protrusions, a smooth surface, and a dull luster.

Liquid medium containing glucose/yeast extract/peptone was turbid and precipitated, but no pellicle or ring was observed.

2. Pseudohyphae are formed on the Dalmor plate.

3. No ascospore formation was observed in the cultures on sodium acetate agar slants, malt extract agar slants, V8 juice agar slants, and corn milk agar slants.

4. It ferments glucose, sucrose, and raffinose, but does not ferment galactose, maltose, trehalose, or lactose.

5. Assimilates glucose, sucrose, maltose, cellobiose, trehalose, raffinose, melezitose, xylose, ethanol, mannitol, lactic acid, citric acid, and KNO3.

6. Grows well in vitamin-free medium.

7. After 3 days of incubation at 30°C, ovoid or cylindrical cells (3.5-4.5)
×(7 to 13) μm.

8. At 20°C, there is little or no growth.

9. Grows well at temperatures between 30 and 40°C.

The boric acid resistance of the CS7529 and CSB6316 strains was as follows:

Each strain was incubated in a solution of 3% by weight of glucose, 0.3% by weight of monopotassium phosphate, 0.7% by weight of ammonium sulfate, and 0.0% by weight of magnesium sulfate.
3% by weight, ferric sulfate 5 ppm, zinc sulfate 9 ppm, manganese chloride 4 ppm
Batch culture was carried out on plates containing 2% agar at 30°C ^for 7 days. The resistance of each strain was as shown in Table 1.

Yeast extracts with a low RNA content have low amounts of flavor components such as 5'-IMP and 5'-GMP, and when used as a seasoning, the balance with other extract components results in a strong yeast odor and a short flavor development.

If the heating temperature is less than 80°C, the RNase is not completely inactivated, and in the subsequent process, the RNA is converted into 2'- or 3'-nucleotides or nucleosides, inhibiting the production of 5'-nucleotides, which are preferred as taste components.
Also, temperatures above 120°C are not desirable as they produce a burnt smell.

In this way, the aqueous suspension of bacterial cells whose intracellular RNA-degrading enzymes have been inactivated is treated with 5'-phosphodiesterase, either directly or after being treated with protease, or after adding alkali to extract the RNA extract components, and then, if desired, with deaminase.

The amount of protease added is sufficient at 1 g/L or less.
The reaction is allowed to proceed at 0 to 50°C for 1 to 20 hours. Addition of more than 1 g/L does not significantly increase the effect and is therefore not practical. Addition of a smaller amount results in a correspondingly lower effect, and the amount of 5'-nucleotides, which are RNA degradation products, obtained in the subsequent step, is reduced.

When alkaline treatment is performed, caustic soda, caustic potash, etc. can be used as the alkali, and the action conditions are pH =
The preferred temperatures are 8 to 12°C, preferably 9 to 10°C, 40 to 50°C, and 1 to 3 hours. If the pH is less than 8, RNA extraction will be insufficient and there will be little point in using alkali. If the pH is 12 or higher, alkaline decomposition of RNA will occur, which is not preferred.
It is preferable to add as little alkali as possible to minimize the influence of salt remaining in the final product on the taste.

Next, when 5'-phosphodiesterase is used to decompose the RNA in the extract into 5'-nucleotides,
Typically, 0.1 to 0.5 g/L of 5'-phosphodiesterase is added and allowed to act at 50 to 80°C for 30 minutes to 3 hours. It is preferable to add the enzyme after first heating it to 50 to 80°C, preferably 60 to 70°C. Adding the enzyme at a temperature below 50°C is undesirable because the phosphatase often present in trace amounts in 5'-phosphodiesterase preparations will act on the enzyme, which is likely to produce impurities as by-products. If desired, the 5'-AMP in the 5'-nucleotide-containing solution obtained as described above can be converted to 5'-IMP by adding 0.1 to 0.5 g/L of deaminase and allowing it to act at 35 to 45°C for 30 minutes to 2 hours.

After the above reaction, the enzyme is inactivated by 90 to 100
After heat treatment at °C for about 30 to 60 minutes, the solid content is removed by centrifugation or other methods, and the supernatant is then concentrated to form a paste or further dried to form a powder. The concentration and drying methods are not particularly limited, but drying methods that do not result in excessively high temperatures, such as vacuum concentration and spray drying, are particularly preferred.

FIG. 1 is a chart showing the results of quantifying the nucleotides in the yeast extract obtained in Example 3 by high performance liquid chromatography.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 Candida utilis CSB6316 strain was cultured in advance in a flask as a seed culture medium, and this was then inoculated into a 30 L fermenter at a concentration of 2%.

The medium consisted of 3% glucose, 0.3% monopotassium phosphate, 0.7% ammonium sulfate, and magnesium sulfate.
Batch culture was carried out for 16 hours using a medium containing 0.03 wt.%, ferric sulfate 5 ppm, zinc sulfate 9 ppm, and manganese chloride 4 ppm.

The culture conditions were: volume of liquid in the tank: 15 L, culture temperature: 30°C, aeration rate: 15 L/min,
The stirring rate was 500 rpm and pH was 4.0 (automatic control with ammonia). The parent strain of the above mutant strain was also cultured under the same conditions. The results of measuring the obtained bacterial cells are shown in Table 2.

Example 2 The boric acid-resistant mutant Candida utilis CSB6316 and its parent strain were batch cultured in a 30 L fermenter in the same manner as in Example 1, and then transferred to continuous culture during the logarithmic growth phase. The medium described in Example 1 was continuously added, and the culture was maintained at pH 4.0, a culture temperature of 30°C, a liquid volume in the tank of 15 L, an agitation rate of 500 rpm, and an aeration rate of 15 L/min.

The parent strain of the mutant strain was also continuously cultured under the same conditions. The culture results are shown in Table 3.

As can be seen from the results of Examples 1 and 2 above, the boric acid resistant mutant strain had a significantly improved RNA content.

Example 3: Using a 30 L jar fermenter, 15 L of the following medium was cultured.
1 L of the pre-culture solution of Candida utilis CS7529 strain was inoculated into the medium, and the mixture was aerated and cultured at 30°C for 24 hours to obtain yeast cells. The medium composition was 5% by weight of glucose, 0.2% by weight of ammonium phosphate, 0.2% by weight of ammonium sulfate, 0.1% by weight of magnesium sulfate, and 0.1% by weight of ammonium sulfate.
The aqueous solution contains 0.17% by weight of potassium chloride, 5 ppm of ferric sulfate, 9 ppm of zinc sulfate, and 4 ppm of manganese chloride, and the pH was adjusted to 4.5.

After the cultivation was completed, the cells were collected using a Sharpless centrifuge to obtain wet yeast. This was resuspended in water and centrifuged twice. This method yielded approximately 300 g of dry cells. Water was added to the yeast and the total volume was reduced to 150 ml.
The mixture was then heated in a water bath until the temperature exceeded 80°C, after which it was heated in the range of 80-100°C for 30 minutes. After this, it was immediately cooled in running water to a temperature of 40°C, and 1.5 g of Protin PC-10 (a protease preparation manufactured by Daiwa Kasei Co., Ltd.) was dissolved in a small amount of water and added, followed by reaction at 30-50°C for 10 hours with stirring.

The mixture was then heated to 80°C or higher for 30 minutes and then cooled to 65°C. 0.2 g of ribonuclease P (a 5'-phosphodiesterase preparation manufactured by Amano Pharmaceutical Co., Ltd.) dissolved in a small amount of water was added, and the mixture was gently stirred at this temperature for 3 hours. The mixture was then cooled to 45°C, and 0.2 g of Deamizyme (a deaminase preparation manufactured by Amano Pharmaceutical Co., Ltd.) was added in the same manner as above, and the mixture was maintained at this temperature for 2 hours with stirring.
The insoluble solids were then removed by centrifugation to obtain the extract. The extract was then heated to 90-95°C for 30 minutes, allowed to cool, and then powdered in a spray dryer to obtain approximately 75g of powdered yeast extract. The extract contained 12% by weight of 5'-IMP, 8.3% by weight of 5'-GMP, and 7.5% by weight of 5'-CMP.
The 5'-nucleotides were 6% by weight and 11.8% by weight, respectively. The quantification of each 5'-nucleotide was carried out by high performance liquid chromatography under the conditions shown below.

Conditions: Packing material: TSK-GEL ODS-80TM Column: 15 cm x φ4.6 mm Elution solvent: 0.05 mol KH ₂ containing 7.0 wt% methanol
_PO4 elution rate: 0.5 ml/min Sample injection volume: 20 μl Detection: UV ₂₅₄ The same applies to the following examples. Furthermore, this 1% aqueous solution had little yeast odor and had a complex and uniquely pleasant taste that combined the so-called umami flavor of dried bonito flakes with the taste of shiitake mushrooms in addition to the taste of amino acids and peptides derived from yeast.

Example 4 Approximately 300 g of bacterial cells were obtained in a dry weight in the same manner as in Example 3. Water was added to this to make the total volume 1500 ml, and after heat treatment in the same manner as in Example 3, the liquid temperature was brought to 50°C. Next, 6N caustic soda was added dropwise while stirring to adjust the pH to 10, and stirring was continued for a further 3 hours. Next, the bacterial cells were removed by centrifugation, and the pH of the resulting clear liquid was immediately adjusted to 6 by adding 6N hydrochloric acid, and the liquid was further heated to a temperature of 65°C.
The yeast extract was treated with 5'-phosphodiesterase and deaminase in the same manner as in Example 3 and powdered using a spray dryer to obtain approximately 70 g of powdered yeast extract. The contents of 5'-IMP in this extract were 10.2% by weight, 5'-GMP 7.1% by weight, 5'-CMP 5.1% by weight, and 5'-UMP 10.0% by weight.

This 1% aqueous solution had a slightly salty taste but had almost the same taste as that of Example 3.

Example 5 Candida utilis CSB6316 strain was cultured in the same culture medium as in Example 3 at 30°C for 24 hours under aeration to obtain yeast cells.

After the cultivation was completed, wet yeast was obtained as in Example 3. This was resuspended in water and centrifuged twice. This method yielded approximately 360 g of cells in dry weight. The yeast thus obtained was reacted with protin PC-10, ribonuclease P, and deamizyme in the same manner as in Example 3. The insoluble solids were then removed by centrifugation to obtain an extract. This extract was then heated to 90-95°C for 30 minutes, allowed to cool, and then powdered using a spray dryer to obtain approximately 98 g of powdered yeast extract. The 5'-IMP content of this extract was 15.0% by weight, and that of 5'-GMP was 1.
The content of each 5'-nucleotide was determined in the same manner as in Example 3.

This 1% aqueous solution contained a mixture of the so-called bonito flake-like umami flavor and the shiitake mushroom flavor in addition to the amino acid and peptide flavors derived from yeast, and had a stronger umami flavor than Examples 3 and 4.

Example 6 A yeast extract was produced in the same manner as in Example 6, except that deamizyme was not added.
The 5'-GMP content in the obtained extract was 12.5% by weight, and although there was no bonito flavor, there was a strong shiitake mushroom flavor.

Comparative Example 1 The CS7529 strain cells were treated in exactly the same manner as in Example 3, except that they were not subjected to heat treatment at 80 to 100°C for 30 minutes before extracting the extract. Approximately 65 g of powdered yeast extract was obtained from approximately 300 g of cells in dry weight.

The 5'-IMP, 5'-GMP, 5'-CMP and 5'-U
The MP content was less than 0.5% by weight in all cases. A 1% aqueous solution of this extract had an amino acid-like taste but almost no umami flavor.

Comparative Example 2: The CS7529 strain cells were treated in the same manner as in Example 3, except that the liquid temperature was not adjusted to 50°C beforehand, but was added at room temperature and then heated to 65°C for enzymatic reaction. Approximately 75g of powdered yeast extract was obtained from approximately 300g of cells in dry weight. The 5'-IMP content of this extract was 2.5% by weight, 1.7% by weight of 5'-GMP, 1.3% by weight of 5'-CMP, and 0.01% by weight of 5'-UMP.
The concentration was 2.5% by weight. This 1% aqueous solution had an inferior taste compared to the extract obtained in Example 3.

INDUSTRIAL APPLICABILITY The present invention relates to the decomposition of RNA into nucleotides in bacterial cells with a high RNA content, by pre-heat treatment of the 2'
- or 3'-nucleotides and can be converted to 5'-nucleotides,
Unlike conventional methods, this method does not require the use of large amounts of alkali, salt, or surfactants, and the entire amount of the extracted extract can be converted directly into a seasoning product with excellent flavor.

In particular, yeast extracts obtained using yeast mutants with an RNA content of 10% or more, more preferably 15% or more, are seasonings with particularly excellent umami.

Reference to the deposited microorganisms under Rule 13bis of the Regulations 1. Candida Utilis CS7529
7529) A. Name and address of the depository institution that deposited the microorganism: Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Address: 1-1-3 Higashi, Tsukuba, Ibaraki Prefecture, 305, Japan
Date of deposit at the depository institution in Japan: January 18, 1988 (Showa 63). However, this deposit is the same as the Bacterial Pathogen Deposit No. 3340 deposited in Japan.
The deposit was changed from No.

The deposit was made under the accession number FERM BP-1656 by the depository institution in Japan. 2. Candida utilis CSB6316
B 6316) A. Name and address of the depository institution that deposited the microorganism: Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Address: 1-1-3 Higashi, Tsukuba, Ibaraki Prefecture, 305, Japan
No. Date of deposit at the depository in Loi January 18, 1988 Accession number assigned by the depository in Hi is FERM BP-1657

──────────────────────────────────────────────────── Continued from the front page (56) References: JP 52-90684 (JP, A) JP 43-28831 (JP, B1) JP 57-22313 (JP, B2) Agric. Biol. Chem., Vol. 38, No. 4 (1974) T. Yamash ita et al., "Studies on Substances Which Increase RNA Content of Yeast Cells" P. 724-734

Claims (7)

[Claims] [Claim 1] Candida utilis CS-7529 (FERM BP
-1656) and/or Candida utilis CSB-6316 (F
A method for producing a natural yeast extract with a high content of 5'-GMP, comprising heating an aqueous solution containing yeast extract (ERM BP-1657) at 80-120°C, extracting RNA and extract components, and then treating the extract with 5'-phosphodiesterase. Claim 2: 5'-GM in the obtained yeast extract component
2. The method for producing yeast extract according to claim 1, wherein the P content is 7% by weight or more. Claim 3: Candida utilis CS-7529 (FERM BP
-1656) and/or Candida utilis CSB-6316 (F
A method for producing 5'-IMP and 5'-GMP by heating an aqueous solution containing 5'-IMP and 5'-GMP (ERM BP-1657) at 80 to 120°C, extracting RNA and extract components, and then treating the extract with 5'-phosphodiesterase and then with deaminase.
A method for producing natural yeast extract with a high content of 4. The 5'-IM in the obtained yeast extract component
4. The method for producing yeast extract according to claim 3, wherein the contents of P and 5'-GMP are each 7% by weight or more. 5. A method for producing yeast extract according to claim 1 or 3, characterized in that the extraction conditions are pH 8 to 12 and 40 to 50°C. Claim 6: Candida utilis CS-7529 (FERM BP
-1656) and/or Candida utilis CSB-6316 (F
The enzyme was allowed to act on the cell extract of the fungus (E. coli BP-1657), and 5'-IMP and 5'-GMP were obtained without adding any external components.
or 7% by weight or more of 5'-GMP
Yeast extract containing the above. [Claim 7] A Candida utilis CSB-6316 mutant strain (FERM BP-1657) that is resistant to boric acid and has a high RMA content.