JP2009159853A - Method for creating low pressure-resistant yeast and method for producing fermented food and fermented food and method for sterilizing fermented food - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for creating a low pressure-resistant yeast which can be sterilized by a pressure treatment at a pressure value not deteriorating the advantages of non-heating sterilization, to provide a method for producing a fermented food with the low pressure-resistant yeast, to provide a fermented food produced with the low pressure-resistant yeast, and to provide a method for sterilizing the fermented food produced with the low pressure-resistant yeast. <P>SOLUTION: The method for creating the low pressure-resistant yeast comprises screening of the low pressure-resistant yeast from a wild strain or a strain subjected to a mutation treatment. In the method for producing the fermented food, a food material with the low pressure-resistant yeast is fermented, and then the fermented food material is subjected to a pressure treatment of 1 to 300 MPa. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing low-pressure-resistant yeast that can be sterilized by pressure treatment at a pressure value that does not impair the advantage of non-heat sterilization, a method for producing fermented foods using low-pressure-resistant yeast, and low pressure resistance The present invention relates to a fermented food produced using yeast and a method for sterilizing fermented food produced using low pressure resistant yeast.

  Fermented and brewed foods such as miso, soy sauce, and liquor are all manufactured using the fermenting action of yeast.

  In addition, these fermented and brewed foods are usually subjected to yeast separation and sterilization by filtration or heat treatment to prevent overfermentation after a certain period of fermentation and aging.

  For example, miso is heated at 50 to 70 ° C. for 60 minutes, and soy sauce is heated at 70 to 85 ° C. for 10 to 30 minutes. In addition, the sake that has been pulled and filtered with sake is heat-treated at 60 to 70 ° C. for 10 minutes to sterilize and deactivate the enzyme.

  However, such conventional separation / sterilization methods may change the flavor and color of food due to heat treatment, and require time-consuming cooling, such as quick cooling after heat treatment. Met.

  More uniform heat treatment is necessary, and if this heat treatment is insufficient, the product value is lost due to over-fermentation by remaining yeast and swelling of the packed product due to the generation of carbon dioxide gas. There is also a problem.

  Therefore, in order to solve such a problem, conventionally, as in JP-A-7-213277 (Patent Document 1), a method for suppressing fermentation in a low-temperature range of 0 to 15 ° C. after fermentation and ripening, As described in JP-A-73063 (Patent Document 2), a method for producing fermented foods using a combination of low-temperature sensitive yeast and lactic acid bacteria has been proposed.

  However, since yeast is not sterilized by these methods, temperature control after fermentation is newly required.

JP-A-7-213277 JP 2004-73063 A

  In fermented (brewed) foods, there is a demand for a method that can sterilize yeast without altering useful components of foods after fermentation and ripening, and without losing color.

  The applicant has so far conducted various studies and experiments on foods under the theme of pressure treatment (also referred to as high pressure treatment), and thought that this problem could be solved by this pressure treatment.

  This pressure treatment is a technology that processes and sterilizes food by applying a pressure of about 100 MPa or more, and has traditionally been used to produce fermented foods such as miso and soy sauce in addition to jam, cooked rice, noodle soup, and processed brown rice. There is a track record of being used.

  In addition, this pressure treatment, which is a non-heat sterilization technique, can selectively sterilize only yeast without altering useful components of food. Furthermore, generally, 300 MPa, It has also been found that pressure treatment for 10 minutes or more may be performed.

  Therefore, the applicant has paid attention to this pressure treatment that yeast can be sterilized without deteriorating useful components of food after fermentation and ripening, and without losing color.

  However, when the experiment was repeated, even if pressure treatment was performed at 300 MPa for 10 minutes, there was a possibility of survival depending on the yeast, which was not a reliable result.

  If a pressure of 300 MPa or more is applied, a higher sterilization effect can be obtained, but the processing equipment becomes larger and more expensive, resulting in a higher processing cost. Therefore, a change in hue occurs, and the advantage as non-heat sterilization cannot be utilized. In addition, if the pressure value is too high, in the parallel complex fermented food of yeast and lactic acid bacteria represented by kefir yogurt, not only yeast but also useful lactic acid bacteria may be killed after fermentation.

  In light of the current situation, the applicant has been researching whether there is a method that can sterilize yeast without causing alteration or color change of food by using pressure treatment, which is a non-heat sterilization technique. The idea was changed, and if fermented foods were produced in advance using yeast having low resistance to pressure, it was noted that yeast could be reliably sterilized by subsequent pressure treatment.

  However, conventionally, there is no method for producing low-pressure-resistant yeast, and the applicant first studied a method for producing low-pressure-resistant yeast. And after intensive research and trial and error, it was found that low-pressure yeast has several characteristics, and from this characteristic, a method for screening low-pressure yeast was completed.

  That is, the present invention provides a method for producing a low-pressure-resistant yeast that can be easily sterilized by pressure treatment at a pressure value that does not impair the advantages of non-heat sterilization.

  Furthermore, the present invention uses, for example, a method for producing fermented food using yeast obtained by the method for producing low-pressure-resistant yeast, fermented food produced using low-pressure-resistant yeast, and low-pressure-resistant yeast. It aims at providing the sterilization method of the fermented food manufactured by this.

  The gist of the present invention will be described with reference to the accompanying drawings.

  The present invention relates to a method for producing a low-pressure-resistant yeast, characterized by screening a yeast capable of growing in a low temperature range of 20 ° C. or less for a wild strain or a strain subjected to a mutation treatment.

  In addition, wild strains or strains that have been subjected to mutation treatment are screened by changing the temperature stepwise in a low temperature range from 0 ° C. to 20 ° C. to screen for yeast that can grow in this low temperature range. It concerns on the production method of the low pressure-resistant yeast of Claim 1 characterized by the above-mentioned.

  In addition, for wild strains or mutant strains that have been subjected to mutation treatment, a yeast having a vegetative cell size larger than the average size of the vegetative cells possessed by each yeast in the strain is screened. It relates to the production method of yeast.

  In addition, after culturing and collecting wild strains or strains that have been subjected to mutation treatment, they are suspended in physiological saline, and this suspension is subjected to natural subsidence, centrifugation, density gradient centrifugation, or Eltriator rotor separation. 4. The method according to claim 3, wherein yeast having a large morphological size is screened.

  In addition, screening for wild strains or mutant strains that are difficult to grow under low humidity conditions with a humidity of 20% or less, or that can grow under high humidity conditions with a humidity of 80% or more. The present invention relates to a method for producing a low-pressure-resistant yeast.

  Further, the present invention relates to a method for producing a low-pressure-resistant yeast, characterized by screening a yeast in which colony formation is not recognized by performing a pressure treatment of 1 MPa or more and 300 MPa or less with respect to a wild strain or a strain subjected to a mutation treatment. Is.

  In addition, a wild strain or a mutant-treated strain is enclosed in a container and subjected to a pressure treatment of 1 MPa to 300 MPa at hydrostatic pressure, and after this pressure treatment, it is cultured for about 3 to 7 days to form a colony. 7. A method for producing a low-pressure-resistant yeast according to claim 6, wherein yeasts that are not recognized are screened.

  In addition, the present invention relates to a method for producing a low-pressure-resistant yeast, wherein the yeast is screened together with the method for producing a low-pressure-resistant yeast according to any one of claims 1 to 7.

  In addition, the present invention relates to a method for producing a fermented food, characterized in that a low pressure resistant yeast is screened from a wild strain or a strain subjected to a mutation treatment, and a food material is fermented using the low pressure resistant yeast.

  The fermented food according to claim 9, wherein the food is fermented using the low-pressure-resistant yeast produced by the low-pressure-resistant yeast production method according to any one of claims 1 to 8. It relates to a manufacturing method.

  The fermented food production method according to any one of claims 9 and 10, wherein after the food is fermented using the low-pressure-resistant yeast, a pressure treatment of 1 MPa or more and 300 MPa or less is performed. It is concerned.

  In addition, the present invention relates to a fermented food product characterized by being fermented using a low pressure-resistant yeast screened from a wild strain or a mutant strain.

  The fermented food according to claim 12, wherein the food is fermented using the yeast produced by the method for producing low-pressure-resistant yeast according to any one of claims 1 to 8. It is concerned.

  The fermented food according to any one of claims 12 and 13, wherein the food is fermented using the low pressure-resistant yeast and then subjected to a pressure treatment of 1 MPa to 300 MPa. It is related to.

  Moreover, low-pressure-resistant yeast is screened from wild strains or strains subjected to mutation treatment, and the yeast is obtained by subjecting fermented food obtained by fermenting ingredients using this low-pressure-resistant yeast to a pressure treatment of 1 MPa to 300 MPa. The present invention relates to a method for sterilizing fermented foods characterized by sterilization.

  Moreover, the pressure treatment of 1 MPa or more and 300 MPa or less is applied to the fermented food obtained by fermenting food using the low pressure resistant yeast produced by the method of producing the low pressure resistant yeast according to any one of claims 1 to 8. The method according to claim 15, wherein the yeast is sterilized.

  Since the present invention has been described above, it is extremely practical to be able to produce yeast that can be easily sterilized by pressure treatment of 300 MPa or less, that is, by pressure treatment at a pressure value that does not impair the advantages of non-heat sterilization. This is a revolutionary low-pressure yeast production method.

  In the inventions according to claims 2, 4 and 7, low-pressure-resistant yeast can be produced more easily and reliably.

  In the invention according to claim 8, low-pressure-resistant yeast can be produced with higher accuracy and efficiency.

  Moreover, in invention of Claim 9,10,12,13, various fermented foods, such as sake, beer, wine, bread, soy sauce, and miso, are easily made by the conventional method using low pressure resistant yeast. In addition, the low pressure resistant yeast can be sterilized by pressure treatment using existing pressure treatment equipment, so that a large and expensive pressure treatment equipment is not required and heat treatment is not required. In addition to being sterilized easily, the sterilization process does not alter the useful components or impair the color of the product. It becomes.

  Moreover, in invention of Claim 11, 14, it was excellent in the further practical use which can sterilize yeast reliably without impairing a useful component and hue, without requiring a large-sized and expensive pressure processing equipment. It becomes the manufacturing method of fermented food, and fermented food.

  Further, in the inventions according to claims 15 and 16, the yeast for fermented foods does not require a large and expensive pressure treatment facility, and can be sterilized without impairing useful components and colors, and is extremely practical. This is a revolutionary fermented food sterilization method.

  Embodiments of the present invention that are considered suitable (how to carry out the invention) will be briefly described with reference to the drawings, illustrating the operation of the present invention.

-Invention of Claim 1 The yeast which can be propagated in a low temperature range below 20 degreeC is screened from a wild strain or the strain which gave the mutation process.

  Specifically, for example, the strain is cultured by changing the temperature stepwise in a low temperature range from 0 ° C. to 20 ° C. to screen for yeast that can grow in this low temperature range.

  More specifically, for example, a wild strain (unmutated strain) or a mutant strain is inoculated on a plate such as a YPD agar medium so that the number of colonies formed is about 200. To do.

  The plate is cultured at 0 ° C. for about 7 days to confirm colony formation (proliferative quality). If there is a colony that is favorably formed at 0 ° C., the colony is caught with a platinum loop or the like and cultured again as a candidate for a low pressure resistant yeast.

  If colony formation is not observed at 0 ° C, or if there is no significant difference in the viability of each colony, increase the culture temperature by about 2 ° C and repeat the culture for about 7 days to confirm the formation of colonies. To do.

  In the case of culturing at 2 ° C., if a colony is formed well, the colony is picked and cultured as a candidate for a low pressure resistant yeast.

  In the same manner, the presence or absence of colonies was confirmed after culturing for about 3 to 7 days in an appropriately selected low temperature range from about 0 ° C. to about 20 ° C. If the colony formation is not observed, or if there is no significant difference in the growth ability of each colony, repeat the procedure of increasing the culture temperature step by step, and grow well at low temperature Select the colonies that are

  It has been confirmed by the applicant's experiment that the yeast thus selected is sterilized by applying a pressure treatment of 300 MPa or less. That is, it is found that the optimum temperature for culturing yeast is around 30 ° C., whereas the low pressure resistant yeast has a characteristic that it grows well in a low temperature range of about 0 ° C. to 20 ° C. Using this property, we succeeded in screening low-pressure-resistant strains.

-Invention of Claim 3 The yeast whose size of a vegetative cell is larger than the average size of the vegetative cell which each yeast in the strain has is screened from a wild strain or a strain subjected to a mutation treatment.

  Specifically, for example, a wild strain or a strain subjected to mutation treatment is cultured and collected, and then suspended in physiological saline. From this suspension, a natural settlement method, a centrifugation method, a density gradient centrifugation method, The yeast having a large morphological size is screened by an L-triator rotor (yeast having a large vegetative cell size has a large morphological size).

  More specifically, for example, a wild strain or a strain subjected to a mutation treatment is cultured, and after collection and washing, it is suspended in a physiological saline so as to have an appropriate bacterial concentration.

The bacterial concentration of the suspension is preferably about 10 ³ to 10 ⁴ / ml. In order to dissociate the aggregates of the bacterial cells, the suspension is subjected to an ultrasonic device for about 15 to 30 seconds under low output conditions. Good.

  From the suspension thus prepared, for example, yeast having a large morphological size is selected by a method such as natural sedimentation, centrifugation, density gradient centrifugation, or L-triator rotor.

  For example, as a natural sedimentation method, a bacterial solution is suspended in a sucrose solution or the like adjusted to an appropriate concentration and allowed to stand for a certain period of time to precipitate a cell having a high cell density. In microorganisms such as yeast, cells having larger vegetative cells generally have a higher cell density, and cells having a lower cell density have a smaller size.

  Therefore, it is possible to isolate yeast having a large size of vegetative cells by collecting the precipitate after standing for a certain period of time.

  In the density gradient centrifugation method, for example, a high-density liquid such as a 60% sucrose solution is placed at the bottom of the centrifuge tube, and a low-density liquid such as 15% sucrose is placed at the top, and the density is changed sequentially between them. Adjust to.

  As another method for creating a density gradient, there is a method in which a density gradient is naturally generated by centrifugal force when a strong centrifugation is performed like a cesium chloride solution.

  Gently overlay the appropriately diluted bacterial solution on the upper part of the density gradient solution formed by such a method, leave it for a certain period of time, and centrifuge it, and the yeast will have a density according to the density of each solution layer. Can be separated.

  In addition, the L triator rotor can be separated as each fraction according to various sizes according to the centrifugal force and the flow rate of flowing the suspension.

  Saccharomyces cerevisiae haploid cells, which are the budding yeast, have an oval shape with a major axis of about 5 μm, and the diploid cells are slightly larger than that and have a lemon shape with both ends slightly cut off. ing.

  When separating yeast according to the size of the vegetative cell by the method as described above, it is necessary to observe the size and form of the yeast mixed with an optical microscope, a micrometer, etc., preferably flow cytometry. It is desirable to measure the particle size distribution of the yeast in the suspension by a method or the like.

  The separation and fractionation of yeast is preferably in the range of about 1 μm. However, since it depends on the method and the device used, an appropriate method and device are used based on the confirmed yeast size and form in the suspension. Will be selected.

  Although there is no particular standard for the size of yeast to be selected, selection is generally made for a range of 10% or less from the larger size of the particle size distribution of the overall cell size.

  It has been confirmed by the applicant's experiment that the yeast thus selected is sterilized by a pressure treatment of 300 MPa or less. That is, the low-pressure-resistant yeast has the characteristic that the size of the vegetative cells is larger than the average size of the vegetative cells of each yeast in the strain. Succeeded in screening.

-Invention of Claim 5 About a wild strain or a strain subjected to a mutation treatment, it is difficult to grow under low humidity conditions with a humidity of 20% or less, or under high humidity conditions with a humidity of 80% or more. Screen for possible yeast.

  Specifically, for example, a wild strain or a strain subjected to a mutation treatment is cultured under a low humidity condition with a humidity of 20% or less and a yeast that is difficult to grow under this low humidity condition is screened, or The strain or the strain subjected to the mutation treatment is cultured under a high humidity condition with a humidity of 80% or more, and a yeast capable of growing under the high humidity condition is screened.

  More specifically, for example, a wild strain or a strain subjected to mutation treatment is inoculated so that the number of colonies formed on a plate such as a YPD medium is about 200.

  The plate is cultured for about 3 to 7 days under a suitable temperature such as 30 ° C. and a low humidity condition of 10% or less, and the strain has poor growth and poor growth, that is, a small colony is formed. To select.

  Alternatively, a strain that grows and grows well under a high humidity condition of 30 ° C. and a humidity of 90% or more, that is, a large colony formed is selected.

  By this method, it is possible to select yeasts that are difficult to grow under low humidity conditions, yeasts that grow well under high humidity conditions, that is, yeasts that have low resistance to drying.

  It has been confirmed by the applicant's experiment that the yeast thus selected is sterilized by a pressure treatment of 300 MPa or less. That is, low pressure-resistant yeast is particularly difficult to grow under low humidity conditions with a humidity of 20% or less, and has good growth under high humidity conditions with a humidity of 80% or more. We found that it has the characteristic that resistance is weak, and succeeded in screening a low-pressure-resistant strain using this characteristic.

-Invention of Claim 6 About the wild strain or the strain which performed the mutation process, the yeast in which colony formation is not recognized is screened by performing a pressure process of 1 MPa or more and 300 MPa or less.

  Specifically, for example, a wild strain or a mutant-treated strain is enclosed in a container and subjected to a pressure treatment of 1 MPa or more and 300 MPa or less under hydrostatic pressure, and cultured for about 3 to 7 days after this pressure treatment. And screening for yeast in which colony formation is not observed.

  More specifically, for example, a wild strain or a strain subjected to mutation treatment is appropriately diluted and inoculated so that about 200 colonies are formed on the plate, and this is used as a master plate.

  A replica is produced from the master plate by the replica plate method.

  The plate inoculated with the bacterial solution is placed in a soft plastic pouch, heat sealed so as not to mix air as much as possible, and then subjected to pressure treatment at 200 MPa, for example.

  After the pressure treatment, the pouch is opened, transferred aseptically to a sterile petri dish, etc., and cultured at 30 ° C. for about 3 to 7 days.

  The other of the produced replica plates is cultured at 30 ° C. for 3 to 7 days as an untreated section where no pressure treatment is applied.

  From this, colonies are formed in the untreated section, and those that do not form colonies in the pressure-treated section are selected as low-pressure resistant yeast.

  In addition, each colony is collected from the master plate with a platinum loop or the like and suspended in a liquid medium or physiological saline in a plastic resin tube.

  Prepare two tubes for each colony and fill with liquid medium or physiological saline so that air does not remain in the tube as much as possible.

  The tube used here may be any tube that can withstand pressure treatment, such as a microtube.

  The tube in which the bacterial solution is suspended is subjected to pressure treatment, for example, at 200 MPa, and the treated bacterial solution is inoculated on the plate.

  The cells are cultured at 30 ° C. for about 3 to 7 days, and those having no colony formed are selected as low-pressure-resistant yeast.

  In any of the above methods, when the pressure treatment is performed at 200 MPa and no growth is observed, the pressure condition is lowered to 100 MPa, etc., and how much pressure treatment the separated low-pressure yeast is killed. Limit as necessary.

  The yeast thus selected does not form colonies when subjected to a pressure treatment of 200 MPa or less. That is, this yeast is sterilized by pressure treatment of 200 MPa or less.

  This method is an effective method for directly obtaining a low-pressure-resistant yeast, but it is necessary to handle a large number of strains and cannot be said to be efficient compared to the other methods described above. Therefore, for example, when two or more production methods described later are used in combination, it is desirable that the other screening method is a primary screening and the method is used as a secondary screening.

-Invention of Claim 8 If yeast is screened together using any two or more of the above four methods for producing low-pressure yeast, low-pressure yeast can be produced more accurately and effectively.

  In addition, the number of combined use and order of these production methods are not particularly limited. Further, there is no need to perform the selected two or more creation methods in order, and for example, the two methods can be performed simultaneously.

-Invention of Claim 9,12 Low-pressure-resistant yeast is screened from the wild strain or the strain which gave the mutation process, and foodstuffs are fermented using this low-pressure-resistant yeast, and it is set as fermented food.

  Specifically, for example, low-pressure-resistant yeast produced by the above-mentioned five production methods can be used, and low-pressure-resistant yeast produced by other production methods can also be used. And using this low pressure-resistant yeast, fermented foodstuffs can be produced according to a conventional method to produce various fermented foods such as sake, beer, wine, bread, soy sauce, and miso.

  In addition, the "fermented food" as used in the field of this invention is used in the meaning including the brewed food manufactured using the fermenting action by yeast.

-Invention of Claim 15 Fermented food manufactured using low pressure-resistant yeast (for example, low pressure-resistant yeast produced by the above-mentioned five production methods) (for example, fermentation produced by the invention of Claim 9) The food or the fermented food of the invention according to claim 12 can sufficiently sterilize the yeast by performing a pressure treatment of 1 MPa or more and 300 MPa or less, for example.

  The pressure treatment at such a pressure value can be carried out without any problem with existing pressure treatment equipment, so that no additional equipment investment is required, and there is no change in food quality or color.

  That is, according to the present invention, yeast of fermented foods can be sterilized without damaging useful components and colors without newly requiring large and expensive equipment.

  A specific embodiment 1 of the present invention will be described with reference to FIG.

  As a target strain in this example, for example, a yeast strain belonging to Saccharomyces cerevisiae, which has not been subjected to mutation treatment, or a mutant treatment strain can be used. Mutation treatment is carried out by some method such as X-ray, UV irradiation, ethylmethanesulfonic acid (EMS), nitrosoguanidine (NTG) treatment. Usually, the treatment is performed so that the survival rate after the mutation treatment is 10% or less. If the survival rate is further reduced, an extra mutation occurs, which is not preferable. Although the diploid yeast may be used for the mutation treatment, it is more efficient to use a haploid spore strain.

  Moreover, as a culture medium used for a present Example, the normal culture medium containing a carbon source, a nitrogen source, an inorganic ion, and the organic trace nutrient as needed can be used. For example, YPD agar medium (yeast extract, glucose, polypeptone, agar, distilled water), YM agar medium (yeast extract, malt extract, peptone, glucose, agar, distilled water, pH 5-6), etc. are used (in liquid medium) Except agar). As a culture method, for example, it is cultured at 30 ° C. for 3 days, collected by centrifugation (3000 rpm, 10 minutes), washed twice with physiological saline, and suspended in physiological saline to obtain an appropriate bacterial concentration. It becomes cloudy.

  The culture may be performed under general conditions, and the culture temperature is generally 20 to 30 ° C., preferably around 30 ° C., and the culture days are 1 to 7 days, preferably about 3 days.

When mutation treatment is performed, this suspension is treated with a mutation agent such as UV, NTG, EMS or the like so that the survival rate is about 10% or less. After centrifugation, the number of viable bacteria is about 4 ×. Suspend in a liquid medium to about 10 ⁷ and culture with shaking at 30 ° C. for 12 hours.

  In addition, the above point is the same also in Examples 2 to 5 described later.

  Saccharomyces cerevisiae NBRC1136 (Biotechnology Research Institute) was initially cultured in YM medium at 30 ° C. for 3 days.

  The cells were collected by centrifugation at 3000 rpm for 3 minutes, and then washed by adding 5 ml of sterilized water.

  This washing operation was repeated twice and then suspended in physiological saline so as to obtain an appropriate concentration.

  After the suspension was diluted, it was inoculated so that the number of colonies formed on the plate was about 200.

  Cultivation was performed at 0 ° C. for about 3 to 7 days, and the presence or absence of colony formation was confirmed every day.

  When colony formation was not observed, the cells were cultured in a low temperature range up to 20 ° C. while gradually increasing the temperature by 2 ° C. to form colonies capable of growing in the low temperature range.

  As a result, colonies of a degree that can be visually confirmed at 10 ° C. for 5 to 7 days are formed. Among them, a plurality (about 10 to 20) of particularly good growth are collected with platinum ears. Then, the cells were again cultured in YM medium at 30 ° C. for 3 days.

  Thereafter, pressure resistance was evaluated by pressure treatment at 200 MPa, and a low pressure resistant strain was obtained.

  FIG. 1 is a graph in which the pressure resistance of each of the yeast screened in this example and the untreated yeast under pressure conditions was verified.

  From this result, it was confirmed that untreated yeast requires a pressure treatment of about 300 MPa for sterilization, but the yeast screened in this example can be sterilized by a pressure treatment of about 200 MPa.

  In addition, the pressure process here means the hydrostatic pressure process which used liquids, such as water, as a medium, and all the pressure processes of Examples 2-5 described below also mean this.

  A second embodiment of the present invention will be described with reference to FIGS.

The suspension prepared in the same manner as in Example 1 is an L triator separation system consisting of a CR22GII type high speed cooling centrifuge (manufactured by Hitachi Koki Co., Ltd.) and an R5E type L triator rotor (manufactured by Hitachi Koki Co., Ltd.). Thus, fractionation was performed for each fraction depending on the size of vegetative cells, and yeast (low pressure resistant strain) was obtained in which the size of vegetative cells was larger than the average size of vegetative cells present in the strain ( (See FIG. 3). The processing conditions are a temperature of 4 ° C., a rotational speed of 3000 rpm, and a flow rate of 9 ml / min to 23 ml / min (see Table 1).

  FIG. 2 is a graph in which the pressure resistance of each of the yeast screened in this example and the untreated yeast under the pressure conditions was verified.

  From this result, it was confirmed that untreated yeast requires a pressure treatment of about 300 MPa for sterilization, but the yeast screened in this example can be sterilized by a pressure treatment of about 200 MPa.

  A specific third embodiment of the present invention will be described with reference to FIG.

  The plate is inoculated so that the number of colonies forming the bacterial solution prepared in the same manner as in Example 1 is about 200. Cultivation was started at 30 ° C. and a humidity of 90%, and colony formation was confirmed over time.

  The formation of colonies can be confirmed visually by culturing for about 3 to 7 days. Among them, about 10 to 20 samples having particularly good growth are collected with platinum ears, and again in YM medium. Culturing was carried out at 3 ° C. for 3 days.

  Thereafter, the pressure resistance of the bacterial solution was evaluated by pressure treatment up to 200 MPa, and a low pressure-resistant strain was obtained.

  FIG. 4 is a graph in which the pressure resistance under each pressure condition of the yeast screened in this example and the untreated yeast is verified.

  From this result, it was confirmed that untreated yeast requires a pressure treatment of about 300 MPa for sterilization, but the yeast screened in this example can be sterilized by a pressure treatment of about 200 MPa.

  A specific embodiment 4 of the present invention will be described with reference to FIG.

  For the suspension prepared in the same manner as in Example 1, in order to break up aggregates and separate mature budding daughter cells from their mother cells, the diluted solution of the bacteria was gently sonicated (low 10 to 15 seconds at output), then transferred to a sterile petri dish and irradiated with ultraviolet light.

  For ultraviolet irradiation, 5 ml was collected in a sterilized petri dish and subjected to a germicidal lamp with an output of 15 W from a distance of 5 cm. As the germicidal lamp, a GL-15 germicidal lamp manufactured by Matsushita Electric Works was used.

  The treated bacterial solution was inoculated after serial dilution in YM agar medium and cultured at 30 ° C. for 3 days.

  A replica was prepared using the plate on which the colony was formed as a master plate.

  The replicated plate medium was aseptically sealed in a pouch and heat sealed while discharging air as much as possible.

  After the sealed flat plate medium was pressure treated at 200 MPa, it was opened aseptically, transferred again to a sterile petri dish, and cultured at 30 ° C. for about 3 days to form colonies.

  Compared with the untreated section, the one in which no colony was formed was obtained as a low pressure resistant yeast.

In this example, the viable cell count after irradiation with ultraviolet rays is 2.3 × 10 ² (6.3 × 10 ⁶ before treatment), and the sterilization rate by ultraviolet irradiation is 99.9%.

  In addition, 100 colonies formed after ultraviolet irradiation were replicated, and 2 colonies that died by pressure treatment of 200 MPa were obtained.

  In addition, two colonies obtained were proliferated, and 0 were killed by pressure treatment of 100 MPa.

  FIG. 5 is a graph in which the pressure resistance of each of the yeast screened in this example and the untreated yeast under pressure conditions was verified.

  From this result, it was confirmed that untreated yeast requires a pressure treatment of about 300 MPa for sterilization, but the yeast screened in this example can be sterilized by a pressure treatment of about 200 MPa.

  A specific embodiment 5 of the present invention will be described with reference to FIG.

  The present embodiment is a case where the first embodiment and the fourth embodiment are combined.

  The plate is inoculated so that the number of colonies forming the bacterial solution prepared in the same manner as in Example 1 is about 200.

  Cultivation was carried out at 10 ° C. for about 7 days, and about 300 colonies with particularly good growth among the formed colonies were collected and transferred to a plate to obtain a master plate.

  A replica was prepared from the master plate and subjected to pressure treatment in the same manner as in Example 4 to obtain a low pressure resistant yeast.

  In this example, about 300 colonies were isolated by the method of Example 1 as a primary screening for low-pressure resistant yeast.

  Thereafter, four low-pressure-resistant yeasts were obtained by the method of Example 4 as secondary screening.

  Compared with the screening method of Example 1 or Example 4 alone, it was possible to screen low-pressure-resistant yeast with high probability.

  FIG. 6 is a graph that verifies the pressure resistance of each of the yeast screened in this example and the untreated yeast under pressure conditions.

  From this result, it was confirmed that untreated yeast requires a pressure treatment of about 300 MPa for sterilization, but the yeast screened in this example can be sterilized by a pressure treatment of about 200 MPa.

  A specific embodiment 6 of the present invention will be described.

  A present Example is a manufacturing method of fermented foods using the low pressure resistant yeast.

  Specifically, for example, the low pressure-resistant yeast produced by the above-mentioned five production methods is used.

  Using this low pressure resistant yeast, various fermented foods such as sake, beer, wine, bread, soy sauce and miso can be produced by fermenting food ingredients according to a conventional method.

  Subsequently, the yeast is sterilized by subjecting this fermented food to a pressure treatment of 200 MPa or less.

  The pressure treatment at such a pressure value can be carried out without any problem with existing pressure treatment equipment, so that no additional equipment investment is required, and there is no change in food quality or color.

  Therefore, according to the present invention, it is possible to selectively sterilize yeasts of fermented foods without altering useful components and without impairing the hue, without requiring new large and expensive equipment.

  In addition, this invention is not restricted to Examples 1-6, The concrete structure of each component can be designed suitably.

It is the graph which verified the pressure | voltage resistance in each pressure conditions of the yeast screened in Example 1 and untreated yeast. It is the graph which verified the pressure | voltage resistance in each pressure conditions of the yeast screened in Example 2 and an untreated yeast. It is a comparative microscope picture which shows the yeast (yeast with a large average particle shape) screened by Example 2, and the yeast with a small average particle shape. It is the graph which verified the pressure | voltage resistance in each pressure conditions of the yeast screened in Example 3 and untreated yeast. It is the graph which verified the pressure | voltage resistance in each pressure conditions of the yeast screened in Example 4 and an untreated yeast. It is the graph which verified the pressure | voltage resistance in each pressure conditions of the yeast screened in Example 5 and an untreated yeast.

Claims (16)

  A method for producing a low-pressure-resistant yeast, comprising screening a wild strain or a strain subjected to a mutation treatment for yeast capable of growing in a low temperature range of 20 ° C. or lower.   Screening yeast that can grow in this low temperature range by culturing wild strains or strains that have undergone mutation treatment in a low temperature range from 0 ° C to 20 ° C in stages. The method for producing a low-pressure-resistant yeast according to claim 1, wherein   A low-pressure-resistant yeast characterized by screening a yeast having a vegetative cell size larger than the average size of vegetative cells of each yeast in the strain for wild strains or mutant strains. Production method.   After culturing and collecting wild strains or mutant strains, they are suspended in physiological saline. From this suspension, the form size is determined by natural subsidence, centrifugation, density gradient centrifugation, or L-triator rotor separation. The method for producing a low-pressure-resistant yeast according to claim 3, wherein yeast having a large size is screened.   Screening for wild strains or mutant strains that are difficult to grow under low humidity conditions with a humidity of 20% or less, or that can grow under high humidity conditions with a humidity of 80% or more A method for producing a low-pressure-resistant yeast characterized by   A method for producing a low-pressure-resistant yeast, comprising screening a yeast in which colony formation is not recognized by performing a pressure treatment of 1 MPa to 300 MPa for a wild strain or a strain subjected to a mutation treatment.   A wild strain or a strain subjected to mutation treatment is enclosed in a container and subjected to a pressure treatment of 1 MPa or more and 300 MPa or less under hydrostatic pressure. After this pressure treatment, it is cultured for about 3 to 7 days, and colony formation is observed. The method for producing low-pressure-resistant yeast according to claim 6, wherein yeast that cannot be screened is screened.   A method for producing a low-pressure-resistant yeast, wherein the yeast is screened together with the method for producing a low-pressure-resistant yeast according to any one of claims 1 to 7.   A method for producing a fermented food comprising screening a low-pressure resistant yeast from a wild strain or a strain subjected to a mutation treatment, and fermenting food using the low-pressure resistant yeast.   The method for producing a fermented food according to claim 9, wherein the food is fermented using the low-pressure-resistant yeast produced by the method for producing a low-pressure-resistant yeast according to any one of claims 1 to 8. .   The fermented food production method according to any one of claims 9 and 10, wherein after the food is fermented using the low-pressure yeast, a pressure treatment of 1 MPa or more and 300 MPa or less is performed.   A fermented food comprising fermented food using a low-pressure-resistant yeast screened from a wild strain or a mutation-treated strain.   The fermented food according to claim 12, wherein the food is fermented using the yeast produced by the low pressure-resistant yeast production method according to any one of claims 1 to 8.   The fermented food according to any one of claims 12 and 13, wherein the food is fermented using the low pressure-resistant yeast and then subjected to a pressure treatment of 1 MPa to 300 MPa.   A low-pressure-resistant yeast is screened from a wild strain or a strain subjected to a mutation treatment, and the yeast is sterilized by performing a pressure treatment of 1 MPa to 300 MPa on a fermented food obtained by fermenting ingredients using the low-pressure resistant yeast. A method for sterilizing fermented foods.   A fermented food obtained by fermenting food using the low-pressure resistant yeast produced by the method for producing a low-pressure resistant yeast according to any one of claims 1 to 8 is subjected to a pressure treatment of 1 MPa to 300 MPa. 16. The method for sterilizing fermented food according to claim 15, wherein the yeast is sterilized.