JPH0768096B2 - Kira-toxin containing powder - Google Patents

Description

TECHNICAL FIELD The present invention relates to a killer toxin-containing powder having good storage stability. The powders of the present invention are stable powders containing various types of temperature-labile killer toxins. Killer toxin-containing powder of the present invention, in the brewing industry, before fermentation, sterilization of wild or contaminating microorganisms in fruit juice and moromi during the fermentation process, termination of fermentation by killing the yeast added as a liquor at the end of fermentation, It is effective in preventing microbial contamination in the storage and aging steps after the completion of fermentation and in the product filled in the container. It is also useful for preventing microbial contamination of various foods and products, and for preventing microbial contamination during aseptic culture such as animal and plant cell tissue culture. Furthermore, it can be used as a therapeutic agent for yeast infectious diseases in the pharmaceutical field.

Prior Art As a method for stabilizing killer toxin, a method using gelatin or albumin [J. Applied Bacteriol., 43 , 425-436]
(1977)] is known.

In addition, it was found by the present inventor that a killer toxin can be stabilized in alcohol by adding sugars and sugar alcohols at the time of wine production, and has been filed (Japanese Patent Application No. 60- 207314). However, there is no report that the purified or unpurified killer toxin is made into a stable powder and is in an easily usable form.

Further, regarding the use of ammonium sulfate in wine production, although it is sometimes used for promoting fermentation of wine, there is no report that it was used for stabilizing killer toxin.

Problems to be Solved by the Invention Killer toxins produced by killer yeasts are classified into 11 types of K _{1 to} K _{11 according} to the killing pattern of killer yeasts (Antonie Van Leuvenhoek). Leeuwenhoek), Vol. 44, p. 59 (197
8)], but recently, some killer toxins that do not belong to any of these types have been found (Japanese Patent Application No. 60-1585).
36, Proceedings of the Annual Meeting of the Society of Agricultural Chemistry 116 pages, 1985).

In the fermentation process of alcoholic beverages, it is possible to secrete killer toxin and prevent microbial contamination by using killer yeast as a mother of liquor [Journal of Fermentation Technology (J.Ferm.Technol) 63
Vol. 5, p. 421, (1985), Wein Wissenschaft, August 1985, No. 4, p. 258].
However, for sterilization of moromi, fruit juice, etc. before fermentation, storage, aging process, and prevention of microbial contamination in the final product,
It is desirable to store the killer toxin itself in a stable form without using killer yeast and add it appropriately. Considering such industrial use of the killer toxin, if the killer toxin is purified and used, the productivity of the killer toxin is very poor. Furthermore, killer toxins have poor temperature stability and are difficult to store. Further, for such a purpose of use, there is no need to use the purified killer toxin. Therefore, killer toxins, purified products,
There is a demand for a technique for making powders which have good storability and are stable with respect to temperature regardless of the unpurified product.

Means for Solving the Problems The present inventor has studied the acquisition of powder of killer toxin having good storage stability, and as a result, polysaccharides such as cyclodextrin, sugars such as glucose, sugar alcohols such as sorbitol. It has also been found that by using ammonium sulfate, the killer toxin can be made into a powder having good storage stability. Furthermore, the obtained killer toxin-containing powder is fruit juice, sterilization of wild yeast in moromi, moromi during fermentation, prevention of microbial contamination of alcoholic beverages during storage, prevention of microbial contamination of alcoholic beverages, foods, etc. The present invention has been completed, finding that it is useful for stopping fermentation by killing the yeast used.

The present invention will be described in detail below.

The present invention is a solution containing a killer toxin, a polysaccharide, a saccharide,
1 selected from sugar alcohols and ammonium sulfate
A killer toxin-containing powder obtained by concentrating and pulverizing in the presence of one kind or two or more kinds.

Examples of the solution containing killer toxin include a culture solution of killer yeast or an aqueous solution of purified killer toxin.

Examples of the killer yeast that produces the killer toxin used in the present invention include those belonging to the genera Saccharomyces, Candida, Hansenula, Cliveromices, Pichia, and Cryptocox.

The killer toxin used in the present invention may be a K _{1 to} K ₁₁ type killer toxin or Candida vinea SW55 (FE
There are various types of killer toxins such as new type killer toxin produced by RMP-8350).

The powdering is roughly classified into a step of adding one or more kinds selected from polysaccharides, sugars, sugar alcohols, and ammonium sulfate to a culture solution of a killer yeast or an aqueous solution of a killer toxin and mixing them, and then concentrating and powdering them. It consists of two steps:

Examples of the polysaccharide used in the present invention include various polysaccharides such as cyclodextrin, dextrin, starch, dextran, cellulose, mannan, and arabinogalactan. Further, commercially available cyclodextrin powder candy (CD powder candy, manufactured by Toyo Brewery Co., Ltd.) and the like can also be used. As sugars, glucose, galactose, sucrose, fructose, xylose, arabinose,
Various sugars such as lactose, melezitose, maltose, raffinose, rhamnose, maltotriose and fructo-oligomers can be mentioned. Also, an isomerized sugar which is a mixture of glucose, fructose and sucrose can be used. As sugar alcohols,
Various sugar alcohols such as sorbitol, xylitol, arabitol and mannitol can be mentioned.

The amount of the above substances added to the solution containing the killer toxin is 2 to
It is 70% (W / V), preferably 15 to 35% (W / V). When a culture solution is used as the solution containing the killer toxin, it may be added directly to the culture solution, or these substances may be added in advance to the medium before culturing the killer yeast.

Examples of the method for concentration and pulverization include various methods such as vacuum concentration with a rotary evaporator, freeze-drying, and pulverization with a spray dryer.

In the case of vacuum concentration with a rotary evaporator, reduce the pressure with an aspirator, etc., and evaporate to dryness while maintaining the water bath temperature at 20 to 48 ° C. In the case of freeze-drying, the decompression degree is about 100 mmHg and the shelf temperature is -40 ° C to 50 ° C. Preferably 5 at -20 ° C
For 6 hours, stop adjusting the shelf temperature and let the temperature rise naturally overnight at room temperature (about 20
(C) for 5 to 6 hours. In the case of powdering with a spray dryer, the inlet temperature is 130-250 ℃, preferably 18
0 ℃, outlet temperature 60 ~ 120 ℃, preferably 80 ℃, spray compressor pressure 2kg / cm ² , processing speed 500ml / h
Powdering can be performed by preferably treating at 400 ml / h.

The killer toxin-containing powder produced by the method of the present invention,
It is stable against temperature and retains killer activity after being stored at 30 ° C for 6 months. Further, when added to moromi, fruit juice or sake, it exhibits a bactericidal effect or a microbial contamination preventing effect, and when added at the end of fermentation of moromi, it exhibits a fermentation stopping effect.

Examples of the present invention will be shown below to specifically describe the present invention.

Example 1 YEPD-CP medium (glucose 2%, peptone 2%, yeast extract 1% was dissolved in 0.1M citrate-phosphate buffer, and pH was adjusted to
Saccharomyces cerevisiae NCYC232 (K ₁ type killer), which had been cultured for 2 days at 25 ° C.
Saccharomyces cerevisiae AST2043 (FERM P-840
7, K ₂ type killer), Saccharomyces cerevisiae N
CYC761 (K ₃ type killer), Hansenula Mulaki NCYC
500 (K ₉ type killer), Candida Vinaire SW55
(FERM P-8350, new type killer), 500 ml of YEPD-C
10 ⁵ each in Erlenmeyer flask containing P medium (pH 4.8)
The cells were inoculated at a bacterial concentration of cells / ml and statically cultured at 20 ° C. for 3 days. After culturing, each culture solution was filtered through a 0.45μ cellulose acetate membrane filter, and the resulting culture solution was 20% (W / V) cyclodextrin powder candy (Toyo Brewing Co.) and 5% ( W / V) glucose was added, and 150 ml each was divided into 3 sections (Category I to II).
It was divided into I) and pulverized. Category I was transferred to a petri dish with a diameter of 25 cm and freeze-dried using a freeze dryer (pressure reduction of 100 m
mHg, shelf temperature −20 ° C., 6 hours, spontaneous heating 9 hours).
For Category II, the pressure was reduced by an aspirator using a rotary evaporator and evaporated to dryness (water tank temperature 41 ° C., 2 hours).
Category III is a spray dryer (inlet temperature 180
° C., outlet temperature of 80 ° C., spray compressor pressure 2 kg / cm ^2, dried at a processing speed 400 ml / h, 25 min treatment), and powdered. Put each type killer toxin-containing powder obtained by the three methods into a 5 ml glass vial (clear glass),
It was stored at 3 ° C, 22 ° C and 30 ° C, and changes in killer activity over time were traced by the following method. The results are shown in Table 1.

<Killer Activity Assay Method> 10 mg of each killer toxin-containing powder stored at each temperature was dissolved in 0.5 ml of sterilized 0.1 M citric acid-phosphate buffer of pH 4.8 to obtain a killer toxin-containing solution. YEPD-MB medium (glucose 2) inoculated with a killer-sensitive Saccharomyces cerevisiae Epane strain (preserved wine yeast of Geisenheim Wine Institute, West Germany) at a concentration of 10 ⁵ cells / ml
%, Peptone 2%, yeast extract 1%, methylene blue 0.
04% and agar 2% were dissolved in 0.1M citric acid-phosphate buffer solution, pH was adjusted to 4.8 with 1N HCl) Killer toxin was placed in a 10 mm diameter hole on an agar plate containing 20 ml. 200 μl of each containing solution was injected, and the mixture was cultured at 25 ° C. for 2 days. The killer activity was represented by the diameter (mm) of a circle formed by a susceptible bacterium that incorporated methylene blue generated around a hole (a bacterium that was killed by a killer toxin and incorporated methylene blue).

In the table, -indicates no activity.

Both types of killer toxin were powdered by the method of the present invention. Freeze-drying and spray-drying were almost the same as the concentrating methods, and the concentration of the compounds under reduced pressure resulted in a relatively low recovery rate of the activity from the culture solution.

In all the killer types, the powders obtained by the three methods were remarkably stabilized with respect to temperature and had killer activity even after storage at 30 ° C. for 6 months. In addition, as shown in Table 2, after the culture medium was previously concentrated with an ultrapermeabilization membrane (exclusion limit of 10,000), cyclodextrin powder candy 20% (W / V) and glucose 5% (W / V) were used.
By carrying out the pulverization by adding the above, the specific activity (killer activity per unit concentration of powder) of the obtained powder can be increased.

Example 2 Saccharomyces cerevisiae strain AST2043 (FERM P-8407, K) cultured in YEPD-CP medium (pH 4.8) at 25 ° C. for 2 days
₂ type killer) was inoculated into 3 L of YEPD-CP medium at 10 ⁵ cells / ml, and statically cultured at 20 ° C. for 3 days. After culturing, the culture solution was passed through a 0.45μ cellulose acetate membrane filter, and the obtained culture solution of 2.9 l was concentrated to 200 ml with an ultrapermeabilization membrane (exclusion limit 10,000), and then freeze-dried to give a powder, A small amount of 0.1M citrate-phosphate buffer (pH
4.8, containing 0.2M NaCl) and dissolved in Sephadex G-
Using 50 columns (Pharmacia), 0.1M citric acid-
Elution with phosphate buffer. The active fractions were collected, freeze-dried, dissolved in a small amount of deionized distilled water, and eluted with deionized distilled water using a Sephadex G-50 column. The active fractions were collected, dialyzed in running water, dialyzed in deionized distilled water, and lyophilized to give a white powder, 2.4 mg.
(Hereinafter abbreviated as K ₂ powder) was obtained. Since K ₂ powder has a strong killer activity and showed a protein-like single band by SDS-polyacrylamide gel electrophoresis, it is considered to be a K ₂ type killer toxin produced by the AST2043 strain.

1.6 mg of K ₂ powder was added to 300 ml of 0.1 M citrate-phosphate buffer (pH
After dissolving in 4.8), 150 ml was divided into two equal portions. On the other hand, cyclodextrin powder candy 20% (W / V), glucose 5% (W
/ V) was added, and pulverized by a spray dryer (the powder referred to as CD-K ₂ powder). K ₂ powder, CD-K ₂
Powder and K ₂ powder 0.1M citric acid-phosphate buffer solution (0.8mg / 150ml) was divided into 2 equal parts and stored at 3 ℃ and 22 ℃ for 1 month. The results are shown in Table 3.

It was found that the purified K ₂ type killer toxin was also stabilized by pulverizing with the method of the present invention.

Example 3 Saccharomyces cerevisiae AST2043 (FERM P-8407, K ₂ ) cultivated in YEPD-CP medium (pH 4.8) at 25 ° C. for 2 days
Type Killer), Candida Vinaire SW55 (FERM P
-8350, new type killer) was inoculated into an Erlenmeyer flask containing 1 of the same medium at a bacterial concentration of 10 ⁵ cells / ml, and statically cultured at 20 ° C. for 3 days. After culturing,
Pass through a 0.45μ cellulose acetate membrane filter, and add 200 ml of each culture solution to 4 of I to IV.
It was divided into two categories. Cyclodextrin powder candy 25% (W / V), glucose 5% (W / V) for Category I, Cyclodextrin powder candy 30% (W / V) for Category II, Glucose 30% (W / V), sorbitol 30% for Category IV
After adding (W / V), each was pulverized with a spray drier, each specific activity, solid content recovery rate [weight of powder obtained / solid content added to 200 ml of culture solution (60 g in this case)]
× 100 (%)], the properties of the produced powder were examined. The results are shown in Table 4. In the category that did not use cyclodextrin, the recovery rate of solid content was extremely low, and the obtained powder showed a considerable hygroscopicity. Also, the specific activity of the powder was considerably lower than that of the cyclodextrin group. However, stabilization of killer activity was observed even when powdered without cyclodextrin.

Example 4 Koshu Seed Juice and Koshu Seed Wine [Alcohol 9%, which had been sterilized in advance by passing through a 0.45μ membrane filter
(V / V)] in, Saccharomyces cerevisiae FY-3
Strains [Agr.Biol.Chem.] 44 (6) 1215-1222 (19
80)] was added at 10 ⁴ cells / ml, and then each was divided into three equal parts. To this, the spray-dried powder of the SW55 strain obtained in Example 1 (which was powdered after being concentrated 1000 times with an ultrafiltration membrane) was added to 0 (no addition), 5 or 20 μg / ml. After culturing at 30 ° C. for 7 days, the number of viable bacteria in each treatment section was counted. As is clear from Table 5, the growth of FY-3 strain in fruit juice and wine was almost completely suppressed by the addition of 5 μg / ml or more of spray-dried powder.

Example 5 The same spray-dried powder as used in Example 4 was
Dissolved in 3% sodium alginate solution (pH 6.5 0.1M phosphate buffer) to give μg / ml, then 5% CaC
The killer toxin-immobilized gel was prepared by dripping into the ₁₂ solution. All these operations were performed at 5 ° C.

After packing this gel in a 100 ml column, Koshu seed juice containing 10 ⁶ cells / ml of Saccharomyces cerevisiae FY-3 strain at 5 ° C. was poured from above. The flow rate was 25 ml / hr for 3 consecutive days, and the juice flowing out from the lower part of the column was sampled with time to measure the viable cell count.

As shown in Table 6, by passing through the column immobilized with killer toxin, the viable cell count of FY-3 strain in the fruit juice was extremely reduced, and this bactericidal effect was observed during the 3-day experimental period. It was kept quite stable.

Example 6 Koshu seed juice (reducing sugar 21.2% (W / V)) of Example 1 was inoculated with Saccharomyces cerevisiae Epane strain at 5 × 10 ⁵ cells / ml and fermented at 15 ° C. Extract is 4.
When the concentration became 5% (W / V), the same spray-dried powder as that used in Example 4 was added so as to have a concentration of 10 μg / ml, stirred lightly, and allowed to stand for 2 hours for observation. Gas production stopped and fermentation stopped.

Example 7 The same spray-dried powder as used in Example 4 was added at 30 μg /
The solution was dissolved in a citric acid-phosphate buffer solution having a pH of 5.0 so as to be ml.

The strains shown in Table 7 were used as test strains in place of the Saccharomyces cerevisiae Epane strains in the "killer activity assay" shown in Example 1, and the effect of the above spray-dried powder solution on these strains was evaluated as "killer". It was examined in the same manner as in the “activity assay method”.

As shown in Table 7, the solution of the above powders has a bactericidal action against Saccharomyces cerevisiae, Candida glabrata, Crivellomyces lactis, Pichia membranefaciens, Rodotorula rubra and Hansenula anomala. showed that.

Example 8 Saccharomyces cerevisiae AST2043 (FERM P-8407, K) cultured in YEPD-CP medium (pH 4.8) at 25 ° C. for 2 days each
₂ type killer), Candida Vinaire SW55 (FERM
P-8350, a new type killer) in a Erlenmeyer flask containing 500 ml of YEPD-CP medium (pH 4.8) at 10 ⁵ cells / m 2 respectively.
The cells were inoculated to a bacterial concentration of 1 and cultured at 25 ° C for 3 days. After culturing, each culture solution was filtered through a 0.45μ cellulose acetate membrane filter. The obtained culture broth was previously concentrated 10 times with an ultrafiltration membrane, and then 30% (W /
V) Ammonium sulfate was added, the pressure was reduced with an aspirator using a rotary evaporator, and the mixture was evaporated to dryness (water bath temperature 40 ° C., 2 hours). The obtained powder was placed in a desiccator and dried by a vacuum pump for 24 hours to obtain two types of killer toxin-containing powder. Put each of these powders into glass vials (clear glass) of 5 ml at 3 ° C and 3
It was stored at 0 ° C., and changes in killer activity over time were followed by the same method as in Example 1. The results are shown in Table 8.

The two killer toxin-containing powders obtained by this method are both significantly stabilized against temperature,
It had a strong killer activity even after storage for 2 months. Also,
None of the powders had hygroscopicity.

EFFECTS OF THE INVENTION By the method of the present invention, a killer toxin-containing powder having good storage stability can be obtained.

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Claims (7)

[Claims] 1. A killer toxin-containing powder obtained by concentrating and pulverizing a solution containing a killer toxin in the presence of one or more selected from polysaccharides, saccharides, sugar alcohols and ammonium sulfate. 2. The killer toxin-containing powder according to claim 1, wherein the solution containing the killer toxin is a culture solution of killer yeast or an aqueous solution of purified killer toxin. 3. The killer yeast according to claim 2, wherein the killer yeast is a yeast belonging to the genera Saccharomyces, Candida, Hansenula, Cliveromyces, Pichia or Cryptocox. Killer toxin containing powder. 4. The killer toxin is K ₁ , K ₂ , K ₃ , K ₄ , K ₅ , K ₆ ,
_{K 7, K 8, K 9} , K 10, K 11 Type killer toxin or killer toxin containing powder Claims second term, wherein it is a new type killer toxin produced by Candida & Vinea SW55 . 5. The killer toxin-containing powder according to claim 1, wherein the polysaccharide is cyclodextrin, dextrin, starch, dextran, cellulose, mannan or arabinogalactan. 6. The saccharide is glucose, galactose, sucrose, fructose, xylose, arabinose, lactose, melezitose, maltose, raffinose, rhamnose, maltotriose or fructo-oligomer. The killer toxin-containing powder according to item 1. 7. The killer toxin-containing powder according to claim 1, wherein the sugar alcohol is sorbitol, xylitol, arabitol or mannitol.