JP3672258B2 - Method for producing fermented product containing significant amount of nisin using barley shochu distillation residue - Google Patents

Description

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【表３】

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【発明の効果】
以上詳述したように、本発明のナイシンを含有する発酵生産物の製造方法は、大麦焼酎蒸留残液を固液分離することにより得られる液体分（A）からなり、Brix濃度を0.5乃至6.0の範囲に調整した液体に糖を添加したものを培地として使用し、該培地にナイシン生産能を有する乳酸菌を加えて培養を行い、該培養は、培養液の糖濃度を少なくとも0.1g/L以上に制御し且つそのpH値を4.0乃至7.0の範囲に制御しながら行うことを特徴とし、ナイシンを著量含有する発酵生産物の効率的製造を可能にする。そして、得られるナイシンを著量含有する発酵生産物は、優れた呈味性を有し、食品として好適に使用することができる。[0001]
[Technical field to which the invention belongs]
The present invention is a method for producing a fermented product containing nisin for culturing lactic acid bacteria having nisin-producing ability using a liquid component obtained by solid-liquid separation of shochu distillation residue as a medium, A liquid component (A) obtained by solid-liquid separation of the barley shochu distillation residue, and a solution prepared by adding sugar to a Brix concentration adjusted to a range of 0.5 to 6.0. Culturing is performed by adding lactic acid bacteria having a production ability, and the culturing is performed while controlling the sugar concentration of the culture solution to at least 0.1 g / L or more and controlling the pH value in the range of 4.0 to 7.0. The present invention relates to a method for producing a fermented product containing a significant amount of nisin. The method for producing a fermented product containing a significant amount of nisin according to the present invention includes an embodiment in which the culture is performed by a batch culture method and / or a continuous culture method. In addition, the method for producing a fermented product containing a significant amount of nisin of the present invention comprises a part of the liquid (A) obtained by solid-liquid separation of the barley shochu distillation residue, and the rice shochu distillation residue. An embodiment in which the liquid component (B) obtained by solid-liquid separation is replaced is included. The fermented product containing a significant amount of nisin obtained by the present invention has excellent taste and can be used as a food.
[0002]
[Prior art]
What is generally known as nisin is a polypeptide consisting of 34 amino acids, the amino acid sequence whose 27th position is histidine is called nisin A, and the amino acid sequence whose 27th position is asparagine. Say Nisin Z. Such nisin is a bacteriocin produced by Lactococcus lactis subsp. Lactis, and has antibacterial activity against Gram-positive bacteria such as Streptococcus, Bacillus, Clostridium, Staphylococcus and is currently used in certain foods in about 50 countries. Limited use as a preservative is permitted. Examples of such foods include cheese, canned food, milk, cream and mayonnaise.
[0003]
For the manufacture of nisin, the following method has been proposed. That is, in Japanese Laid-Open Patent Publication No. 4-75596 [hereinafter referred to as “Reference 1”], in the fermentation production of nisin using lactic acid bacteria, the growth rate of the produced bacteria is caused by metabolites other than nisin produced by nisin-producing bacteria. Before the growth or the growth stops, the culture solution containing the cells is continuously filtered using a membrane, and separated into a solution containing the cells and a solution containing nisin without the cells, The liquid containing the bacterial cells is returned to the culture tank, the liquid containing no nisin and containing nisin is extracted, and a fresh liquid medium having the same volume as the amount of the extracted liquid is added to the nisin-producing bacterial culture liquid in the culture tank. Dilution rate 0.1 ~ 10hr ^-1 The nisin is characterized in that nisin is separated from the liquid that does not contain the cells and that contains nisin, the culture solution in the culture tank, or the cells themselves that are contained in the culture solution. A manufacturing method is described. More specifically, Document 1 describes that MRS medium is used as a medium, Streptococcus lactis IFO12007 is used as a lactic acid bacterium strain capable of producing nisin, and fermentation is performed using the above filtration culture method. The nisin concentration in the culture solution obtained by the fermentation is 1.72 × 10 ^Five It is stated that it is U / L.
[0004]
Japanese Patent Laid-Open No. 4-126093 [hereinafter referred to as “Document 2”] describes that when producing nisin by culturing Streptococcus lactis strain SBT1212 having nisin-producing ability, serine or phenylalanine is added to the culture solution. A method for producing nisin is described which comprises culturing by adding 0.1 to 10 mg / ml to enhance nisin production. Reference 2 describes that a nisin production amount of a fermented product obtained by the production method using a medium composed of whole milk to which serine is added is 1000 (IU / ml).
In JP-A-6-9690 [hereinafter referred to as “Reference 3”], Lactococcus lactis subspecies lactisNRRL-B-18809, MRS medium supplemented with 1% yeast extract, or 0.5% yeast extract are added. A method for producing bacteriocin (nisin) cultured using a medium consisting of 7% whey is described, and the nisin titer of the fermented product obtained by the production method is 1600 AU / ml. ing.
[0005]
However, since the nisin concentration in the fermented product achieved by the nisin production method described in References 1 to 3 is extremely low as described above, these nisin production methods are useful for industrially producing nisin in large quantities. It is not suitable. Incidentally, at present, these nisin production methods are not industrially implemented.
[0006]
By the way, it has been proposed to use a shochu distillation residue as a culture medium for lactic acid bacteria. That is, JP 2000-236891 A [hereinafter referred to as “Document 4”. ] Describes a method for producing lactic acid, characterized in that lactic acid bacteria are added to a medium obtained from solids by solid-liquid separation of rice shochu distiller and subjected to fermentation. JP 2000-245491 A (hereinafter referred to as “Reference 5”) describes a lactic acid bacterium that produces L lactic acid in a medium obtained from a solid content obtained by solid-liquid separation of a shochu-distilled distilled spirit in an open system that is not steam sterilized. A method for producing high-purity L-lactic acid, characterized by being added and subjected to fermentation, is described.
JP-A-2000-342247 (hereinafter referred to as “Reference 6”) discloses a liquid component obtained by solid-liquid separation of a shochu distillation residue obtained as a by-product in the production of shochu using barley as a raw material. Filtration to obtain a clarified liquid, concentrating the clarified liquid to obtain a concentrated liquid, and for microorganisms containing, as an active ingredient, a non-adsorbable fraction obtained by subjecting the concentrated liquid to an adsorption treatment using a synthetic adsorbent It is described that the medium is excellent as a culture medium for yeast, lactic acid bacteria, and bifidobacteria, and when the microorganism medium is used for culturing yeast, lactic acid bacteria, and bifidobacteria, the amount of cultured cells is remarkably increased. .
[0007]
Japanese Patent Laid-Open No. 2002-369672 [hereinafter referred to as Document 7]. ] Describes a food sterilization method using a lactic acid fermentation broth containing bacteriocin such as nisin. And it is described that the antibacterial action which this lactic acid fermentation liquid has can be maintained long by adding this lactic acid fermentation liquid and calcium ion to food.
[0008]
[Patent Document 1]
JP-A-4-75596
[Patent Document 2]
Japanese Patent Laid-Open No. 4-126093
[Patent Document 3]
Japanese Unexamined Patent Publication No. 6-9690
[Patent Document 4]
JP 2000-236891 A
[Patent Document 5]
JP 2000-245491 A
[Patent Document 6]
JP 2000-342247 A
[Patent Document 7]
JP 2002-369672 A
[0009]
However, the solid content of the rice shochu distillation residue and the solid content of the koji shochu distillation residue used as the medium raw material in Reference 4 and Reference 5 are the secondary ingredients in the production of shochu using barley as the raw material used in the present invention. What is objective is the liquid content obtained by solid-liquid separation of the barley shochu distillation residue produced and the liquid fraction obtained by solid-liquid separation of the rice shochu distillation residue produced as a by-product in the production of shochu using rice as a raw material The distinction is clearly different. In addition, the inventions described in Document 4 and Document 5 are intended to produce lactic acid by culturing lactic acid bacteria using a medium consisting of a solid content obtained from rice shochu distillation residue or rice cake shochu distillation residue. Therefore, there is no suggestion about a method for producing lactic acid bacteria fermentation broth containing nisin and having excellent taste.
[0010]
Literature 6 describes that the microorganism medium is effective for the growth of lactic acid bacteria belonging to the genus Lactobacillus, i.e., Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, etc., but Lactococcus lactis subsp. Having nisin-producing ability. There is no description about the cell growth effect of lactic acid bacteria belonging to Lactis. Further, in Document 6, since the microorganism medium is described as being effective for lactic acid bacteria belonging to the genus Lactobacillus, that is, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus fermentum, the microorganism medium is used. It may be easy to infer that when the lactic acid bacteria belonging to Lactococcus lactis subsp. However, the amount of nisin produced in the culture solution by culturing the lactic acid bacterium having the ability to produce nisin cannot be inevitably increased only by increasing the amount of cells in the culture solution. This is the same even when the microorganism medium having the above-mentioned cell growth effect is used. Incidentally, the present inventors used the microorganism medium described in Document 6 to culture each of several strains of lactic acid bacteria belonging to Lactococcus lactis subsp. Lactis having the ability to produce nisin by changing the culture conditions. However, the amount of microbial cells and nisin concentration in the obtained fermentation product were examined. In any case, no universal correlation was found between the amount of microbial cells and the nisin concentration. I couldn't.
[0011]
Document 7 describes a method for sterilizing food with a lactic acid fermentation broth containing bacteriocin such as nisin. Moreover, when obtaining the said lactic acid fermentation broth, it describes that shochu can be used as a nitrogen source used for a culture medium. However, Document 7 does not describe at all that the productivity of nisin is remarkably increased when the shochu is used as a nitrogen source, and that the obtained lactic acid fermentation broth has excellent taste.
[0012]
As is clear from the above description, even if the above-mentioned literature is scrutinized, the liquid obtained by solid-liquid separation of the barley shochu distillation residue or rice shochu distillation residue is used as the medium. When lactic acid bacteria having nisin-producing ability are cultured, nisin production is remarkably increased, and the obtained fermentation product contains a significant amount of nisin and has an excellent taste. .
[0013]
[Problems to be solved by the invention]
As described above, in the conventional nisin production technology, the above-mentioned various culture media are used, but the nisin concentration in the obtained fermentation product is extremely low even when any of these conventional culture media is used. . For these reasons, in order to industrially produce nisin in large quantities, early provision of means for further increasing the amount of nisin produced in the fermented product is strongly demanded.
Apart from this, as described above, in Reference 7, lactic acid fermentation broth containing nisin obtained by culturing lactic acid bacteria capable of producing nisin using a culture medium using shochu as a nitrogen source has antibacterial activity. A method for sterilizing food by adding this lactic acid fermentation broth to food is described. However, a lactic acid fermentation broth containing nisin obtained by culturing lactic acid bacteria capable of producing nisin using such a conventional medium has a low nisin concentration as described above, and its taste is satisfactory. It will never be the case. When such a lactic acid fermentation broth is used by adding it to food, it is highly desired that the lactic acid fermentation broth contains a significant amount of nisin and has an excellent taste.
[0014]
The present invention has been completed as a result of further research in view of the above-described problems in the prior art. An object of the present invention is to solve the above-mentioned problems in the conventional nisin production technology and to provide an industrial production method of nisin that enables mass production of nisin. Another object of the present invention is to provide a method that enables efficient production of a fermented product that contains a significant amount of nisin, has excellent taste, and is extremely suitable for use in foods. is there.
[0015]
[Means for Solving the Problems]
The present inventors use the shochu distillation residue more effectively to solve the above-mentioned problems in the conventional nisin manufacturing technology, that is, the use of the shochu distillation residue as a medium enables an increase in nisin production. In order to develop a method to make it.
As a result, it comprises a liquid component (A) obtained by solid-liquid separation of the barley shochu distillation residue, and uses a liquid prepared by adding sugar to a liquid in which the Brix concentration is adjusted to a range of 0.5 to 6.0. Culturing by adding lactic acid bacteria capable of producing nisin to the medium, and culturing while controlling the sugar concentration of the culture solution to at least 0.1 g / L and controlling the pH value in the range of 4.0 to 7.0 It has been found that the production amount of nisin is remarkably increased and a fermented product containing a significant amount of nisin can be obtained. And when the fermented product was subjected to a sensory test, it has excellent taste compared to the fermented product obtained by using a conventional medium, and is extremely suitable for use as a food product. understood. The present invention has been completed based on the above findings.
[0016]
The present invention makes it possible to produce a large amount of nisin by using a barley shochu distillation residue that has never been used as a medium in the conventional nisin production technique. That is, the present invention comprises a liquid component (A) obtained by solid-liquid separation of barley shochu distillation residue, and uses as a culture medium a sugar added to a liquid having a Brix concentration adjusted to a range of 0.5 to 6.0. Then, lactic acid bacteria capable of producing nisin are added to the culture medium, and the culture is controlled by controlling the sugar concentration of the culture solution to at least 0.1 g / L and controlling the pH value within the range of 4.0 to 7.0. The present invention provides a method for producing a fermented product containing a significant amount of nisin. The culture in the production method of the present invention may be performed by a batch culture method and / or a continuous culture method. The fermented product containing a significant amount of nisin obtained according to the present invention has excellent taste and can be suitably used as a food.
[0017]
In light of the fact that the amount of cultured cells obtained when the lactic acid bacteria of the genus Lactobacillus are cultured using the culture medium for microorganisms described in Reference 6, the Document 6 describes the above-mentioned for microorganisms. Lactococcus lactis subsp. That has the ability to produce nisin using a culture medium There is no mention of culturing lactic acid bacteria belonging to the genus Lactis, but this lactic acid bacterium that has the ability to produce nisin is cultured using the microorganism medium Regardless of the degree of increase in the amount of cultured cells, the present inventors have conducted intensive studies through experiments, assuming that significant production of nisin would be brought about. That is, the present inventors solid-liquid-separated shochu distillation residue (hereinafter referred to as “barley shochu distillation residue”) produced as a by-product in the production of shochu using barley as described in Document 6. To obtain a liquid component, filter the liquid component to obtain a clarified liquid, concentrate the clarified liquid to obtain a concentrated liquid, and subject the concentrated liquid to an adsorption treatment using a synthetic adsorbent. After adjusting the fraction and the liquid to a Brix concentration of 8.0, add the same amount of glucose to each, then inoculate the lactic acid bacteria belonging to Lactococcus lactis subsp. It was attached to. As a control, an MRS medium supplemented with the same amount of glucose as described above was used, and everything else was subjected to batch culture in the same manner as described above.
[0018]
The amount of lactic acid bacteria and nisin concentration were measured for the fermentation products obtained in each batch culture. As a result, when using the medium composed of the liquid described in Reference 6 and when using the medium composed of the non-adsorbing fraction, the amount of lactic acid bacteria in the fermentation product obtained in these two cases and nisin Concentrations were found to be substantially the same. Further, it was found that the amount of lactic acid bacteria and the concentration of nisin were substantially the same, as compared with the case where the control MRS medium was used. That is, it has been clarified that nisin productivity notably higher than that of the control MRS medium cannot be achieved simply by using the microorganism medium described in Document 6.
[0019]
Therefore, the present inventors conducted an examination through experiments on nisin production using a medium composed of a liquid obtained by solid-liquid separation of the barley shochu distillation residue. That is, in producing nisin using the medium composed of the liquid, it is assumed that the concentration of Brix in the liquid will affect the production of nisin, and the Brix in the liquid suitable for nisin production is estimated. The study was conducted through experiments with the aim of determining the concentration. That is, the Brix concentration of the liquid is adjusted to 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, and 15.0, and each has the same amount. Glucose was added to obtain liquid media adjusted to the optimum pH value for nisin production of lactic acid bacteria having the following nisin-producing ability, and lactic acid bacteria having nisin-producing ability belonging to Lactococcus lactis subsp. Inoculated and subjected to batch culture separately. As a control, MRS medium added with the same amount of glucose as described above and adjusted to the same pH value as described above was used, and everything else was subjected to batch culture in the same manner as described above. As a result, when a liquid medium obtained by adjusting the Brix concentration of the liquid to a range of 0.5 to 6.0 was used, compared with the case where the control MRS medium was used, nisin in the obtained fermentation product A tendency to increase the concentration was observed.
[0020]
Therefore, in the case of subjecting to culture for the purpose of nisin production using the medium composed of the above liquid, examination was conducted through experiments for the purpose of determining optimum pH conditions for mass production of nisin. That is, a predetermined amount of glucose was added to the liquid component adjusted to the Brix concentration of 4.0 to obtain a plurality of liquid culture media having pH values adjusted to 3.0 to 8.0. Each liquid medium having a different pH value was inoculated with a lactic acid bacterium having an ability to produce nisin belonging to Lactococcus lactis subsp. Lactis, and separately subjected to batch culture while maintaining the adjusted pH value. As a control, an MRS medium adjusted to pH 6.8 by adding the same amount of glucose as described above was used, and everything else was subjected to batch culture in the same manner as described above. As a result, when the culture was performed while maintaining the pH value of the liquid medium in the range of 4.0 to 7.0, compared with the case where the culture was performed using the control MRS medium (maintained at pH 6.8). It was found that the concentration of nisin in the resulting fermentation product was significantly increased.
[0021]
From the above, the Brix concentration of the liquid obtained by solid-liquid separation of the barley shochu distillation residue is adjusted to a range of 0.5 to 6.0, and sugar is added to this to adjust the pH value to a range of 4.0 to 7.0. Inoculated with lactic acid bacteria having the ability to produce nisin belonging to Lactococcus lactis subsp. Lactis and subjected to batch culture, nisin concentration in the obtained fermentation product was cultured using conventional MRS medium It became clear that it increased remarkably compared with the case.
[0022]
In the same manner as in the case of the liquid (A) obtained by solid-liquid separation of the barley shochu distillation residue, the rice shochu distillation residue is solid-liquid separated. As a result, the same results as in the case of the liquid (A) obtained by solid-liquid separation of the barley shochu distillation residual liquid described above were obtained. That is, the Brix concentration of the liquid component (B) obtained by solid-liquid separation of the rice shochu distillation residue is adjusted to a range of 0.5 to 6.0, and sugar is added to this to adjust the pH value to a range of 4.0 to 7.0. Inoculated with lactic acid bacteria having the ability to produce nisin belonging to Lactococcus lactis subsp. Lactis and subjected to batch culture, nisin concentration in the obtained fermentation product was cultured using conventional MRS medium It became clear that it increased remarkably compared with the case.
[0023]
Next, nisin production by a continuous culture method was examined through experiments using a medium composed of the liquid. That is, the Brix concentration of the liquid is adjusted to 4.0, 3% by weight of glucose is added thereto to adjust the pH value to 5.5, and a liquid medium is obtained. The nisin belonging to Lactococcus lactis subsp. Inoculated with lactic acid bacteria having the ability to produce, using a continuous culture apparatus consisting of a jar fermenter, maintaining the pH value at 5.5 with a pH controller, and further, the filtration culture method described in Reference 1, that is, without microbial cells, Nisin The liquid containing was extracted, and continuous culture was performed in which the fresh liquid medium having the same volume as the extracted liquid was supplied to the jar fermenter. As a control, continuous culture was performed in the same manner except that an MRS medium supplemented with 3% by weight of glucose was used instead of using the fresh liquid medium in the filtration culture. As a result, by using the liquid medium as a fresh medium to be supplied to the jar fermenter in filtration culture, the concentration of nisin in the obtained fermentation product is significantly higher than when the MRS medium is used as the fresh medium. It turned out to increase.
[0024]
Further, Table 1 of Document 1 states that the higher the glucose content of the MRS medium used as a fresh medium, the higher the nisin concentration in the culture solution. From FIG. 4 and FIG. As is apparent, the glucose concentration in the culture solution after the transition to the continuous culture is substantially infinitely close to 0 g / L regardless of the glucose concentration of the supplied MRS medium. Therefore, the present inventors presumed that the glucose concentration in the culture medium during continuous culture might affect nisin productivity, and examined it through experiments.
[0025]
That is, the Brix concentration of the above liquid is adjusted to 4.0, and 3% by weight of glucose is added thereto to adjust the pH value to 5.5, thereby obtaining a liquid medium, and nisin belonging to Lactococcus lactis subsp. Inoculate a lactic acid bacterium having a production ability, use a continuous culture apparatus consisting of a jar fermenter, maintain the pH value at 5.5 by a pH controller, and further, filtration culture method described in Reference 1, that is, without microbial cells, nisin The liquid containing was extracted and the continuous culture | cultivation which supplies the said fresh liquid culture medium of the same volume as the extracted liquid quantity to a jar fermenter was performed. At that time, the glucose concentration of the culture solution was measured using an on-line biochemical controller with a glucose electrode, and after shifting to continuous culture, the liquid medium was supplied to the jar fermenter to adjust the glucose concentration of the culture solution to 0.01, 0.1. , 1.0, 5.0, and 10.0 g / L, and each was continuously cultured separately. As a result, when the glucose concentration in the culture solution after transition to continuous culture was maintained at 0.1, 1.0, 5.0, and 10.0 g / L, the glucose concentration in the culture solution was maintained at 0.01 g / L. In comparison, it was revealed that the concentration of nisin in the culture broth was dramatically increased. From this, it is possible to continuously produce a culture solution containing an extremely high concentration of nisin by maintaining the glucose concentration in the culture solution after transition to continuous culture at 0.1 g / L or more. There was found.
[0026]
Accordingly, a liquid component (A) obtained by solid-liquid separation of the barley shochu distillation residue is used as a medium in which sugar is added to a liquid in which the Brix concentration is adjusted to a range of 0.5 to 6.0, Culturing by adding a lactic acid bacterium having nisin-producing ability to the culture solution, controlling the sugar concentration of the culture solution to at least 0.1 g / L or more and controlling the pH value in the range of 4.0 to 7.0. A fermented product was obtained. When the nisin concentration of the obtained fermentation product was examined, it was found that it was significantly higher than the nisin concentration of the fermentation product obtained by using the conventional MRS medium. When these fermented products were subjected to a sensory test by 10 panelists, the latter had a miscellaneous taste and an irritating taste and had a taste problem, whereas the former was mild and harmonious. It has been found that it has a very excellent taste consisting of excellent flavor and rich umami, and is extremely suitable as a food material.
[0027]
In addition, when the liquid content (B) of the rice shochu distillation residue was used and the experiment described in the above-mentioned method was performed, as in the case where the liquid content of the barley shochu distillation residue was used, It was found that a culture solution containing an extremely high concentration of nisin can be continuously produced by maintaining the glucose concentration in the culture solution after shifting to culture at 0.1 g / L or more. Therefore, a liquid component (B) obtained by solid-liquid separation of the rice shochu distillation residue is used as a medium with a sugar added to a liquid adjusted to a Brix concentration in the range of 0.5 to 6.0. Culturing by adding a lactic acid bacterium having nisin-producing ability to the culture solution, controlling the sugar concentration of the culture solution to at least 0.1 g / L or more and controlling the pH value in the range of 4.0 to 7.0. A fermented product was obtained. When the nisin concentration of the obtained fermentation product was examined, it was found that it was significantly higher than the nisin concentration of the fermentation product obtained by using the conventional MRS medium. When these fermented products were subjected to a sensory test by 10 panelists, the latter had a miscellaneous taste and an irritating taste and had a taste problem, whereas the former was mild and harmonious. It has been found that it has a very excellent taste consisting of excellent flavor and rich umami, and is extremely suitable as a food material.
[0028]
Further, as the medium, a part of the liquid (A) obtained by solid-liquid separation of the barley shochu distillation residue is a liquid (B) obtained by solid-liquid separation of the rice shochu distillation residue. A liquid prepared by adding a sugar to a liquid whose Brix concentration is adjusted to a range of 0.5 to 6.0 is used, and culturing is performed by adding lactic acid bacteria capable of producing nisin to the medium. The fermentation product was obtained by a method carried out while controlling the sugar concentration of at least 0.1 g / L or more and controlling the pH value in the range of 4.0 to 7.0. As a result, it has been clarified that the nisin concentration of the fermentation product is remarkably increased as compared with the nisin concentration of the fermentation product obtained by using the conventional MRS medium. When these fermented products were subjected to a sensory test by 10 panelists, the latter had a miscellaneous taste and an irritating taste and had a taste problem, whereas the former was mild and harmonious. It has been found that it has a very excellent taste consisting of excellent flavor and rich umami, and is extremely suitable as a food material.
[0029]
In addition to this, a non-adsorbed fraction obtained by subjecting at least a part of the liquid component (A) obtained by solid-liquid separation of the barley shochu distillation residue to an adsorption treatment using a synthetic adsorbent, the liquid component A synthetic adsorbent comprising at least part of a liquid component (A ′) containing (A) the non-adsorbed fraction and a liquid component (B) obtained by solid-liquid separation of rice shochu distillation residue A non-adsorbed fraction obtained by subjecting to an adsorption treatment using a liquid component (B ') containing the non-adsorbed fraction as the liquid component (B), and preparing the liquid component (A' ) And a liquid in which the Brix concentration is adjusted to a range of 0.5 to 6.0 and a sugar added, and the Brix concentration in the range of 0.5 to 6.0 using the liquid component (A ′) and the liquid component (B ′). A liquid prepared by adding sugar to a liquid prepared as above is used as a medium, and lactic acid bacteria capable of producing nisin are added to the medium and cultured. The fermentation product was obtained by a method of culturing while controlling the sugar concentration of the culture solution to at least 0.1 g / L or more and controlling the pH value in the range of 4.0 to 7.0. When the nisin concentration of the obtained fermentation product was examined, the nisin concentration of any fermentation product was significantly higher than the nisin concentration of the fermentation product obtained by using the conventional MRS medium. understood. When these fermented products were subjected to a sensory test by 10 panelists, the latter had a miscellaneous taste and an irritating taste and had a taste problem, whereas the former was mild and harmonious. It has been found that it has a very excellent taste consisting of excellent flavor and rich umami, and is extremely suitable as a food material.
[0030]
Embodiment Example
Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited by these embodiments. The present invention includes a step (a) of obtaining a liquid component (A) by solid-liquid separation of a barley shochu distillation residue, and diluting the liquid component (A) with water so that the Brix concentration is in the range of 0.5 to 6.0. Step (b) of adjusting, step (c) of adding a saccharide to the product obtained in the step (b) to obtain a medium, and adding lactic acid bacteria capable of producing nisin to the medium and culturing, A fermentation production containing a significant amount of nisin, characterized in that it comprises a step (d) of controlling the sugar concentration of the culture solution to at least 0.1 g / L or more and controlling the pH value in the range of 4.0 to 7.0. A method for manufacturing a product is provided. In step (a), a portion of the liquid (A) obtained by solid-liquid separation of the barley shochu distillation residue is used to solidify the rice shochu distillation residue as a by-product in the production of rice shochu. The liquid component (B) obtained by liquid separation may be replaced. Alternatively, at least a part of the liquid component (A) and / or the liquid component (B) may be a non-adsorbed fraction obtained by subjecting it to an adsorption separation process using a synthetic adsorbent. The culture in the step (d) can be performed by a batch culture method and / or a continuous culture method.
[0031]
The barley shochu distillation residual liquid used in the present invention produces barley koji and steamed barley using barley or refined barley as a raw material, koji starch obtained from the barley koji and steamed barley, and / or an enzyme agent. And saccharified using an alcoholic fermentation with yeast to obtain a ripened mash, which is by-produced as a distillation residue when the ripened mash is distilled using a distillation apparatus such as vacuum distillation or atmospheric distillation, This means the distillation residue of barley shochu. Also, in the production of rice shochu, sweet potato shochu, and buckwheat shochu, shochu distillate residue by-produced when barley is used as part of the raw material in the production of shochu is also used as the barley shochu distillate used in the present invention. Is included.
When the barley shochu distillation residue is obtained, the barley koji used for the production of barley shochu may be produced under the koji-making conditions used in normal barley shochu production. Aspergillus kawachii used in the production is preferred. Alternatively, strains of the genus Aspergillus such as Aspergillus awamori used in awamori production and Aspergillus oryzae used in sake production and the like can also be used. Moreover, the yeast used for manufacture of barley shochu can use various yeast for shochu brewing generally used in shochu manufacture.
[0032]
The rice shochu distillation residue used in the present invention is produced by producing rice bran and steamed rice using rice as a raw material, saccharifying the obtained rice bran and starch contained in the steamed rice, and / or using an enzyme agent. In addition, it is subjected to alcohol fermentation with yeast to obtain a mature mash, which is produced as a by-product as a distillation residue when the mature mash is distilled using a distillation apparatus such as vacuum distillation or atmospheric distillation, that is, rice shochu Means distillation residue. Also, in the production of barley shochu, sweet potato shochu, and buckwheat shochu, shochu distillate residue by-produced when using rice as part of the raw material in the production of shochu is also used as the rice shochu distillate used in the present invention. Is included.
In obtaining the rice shochu distillation residue, the rice bran used for the production of the rice shochu may be produced under the koji-making conditions used in normal rice shochu production. Aspergillus kawachii used in the production is preferred. Alternatively, strains of the genus Aspergillus such as Aspergillus awamori used in awamori production and Aspergillus oryzae used in sake production and the like can also be used. Moreover, the yeast used for manufacture of rice shochu can use various shochu liquor yeast generally used in the case of shochu manufacture.
[0033]
In the step (a), the barley shochu distillation residue is separated into solid and liquid to obtain a liquid component. The barley shochu distillation residue is obtained by removing raw barley and water-insoluble fermentation residue derived from barley koji and only the liquid component. Is to use. The solid-liquid separation is performed through a solid-liquid separation method such as a screw press method or a roller press method, or a pre-separation is performed using a filter-press-type solid-liquid separator, and then a centrifuge, a diatomaceous earth filter device, a ceramic A solid-liquid separation process using a filtration device or a filtration press is performed to obtain the liquid component.
[0034]
In the step (I), the reason why the rice shochu distillation residue is obtained by solid-liquid separation is to obtain a liquid component by removing raw rice and rice bran-derived water-insoluble fermentation residue from the rice shochu distillation residue. Is to use. The solid-liquid separation is performed through a solid-liquid separation method such as a screw press method or a roller press method, or a pre-separation is performed using a filter-press-type solid-liquid separator, and then a centrifuge, a diatomaceous earth filter device, a ceramic A solid-liquid separation process using a filtration device or a filtration press is performed to obtain the liquid component.
[0035]
The liquid component (A) and / or the liquid component (B) is subjected to an adsorption separation process using a synthetic adsorbent to make a non-adsorbed fraction, which removes components that present miscellaneous taste contained in the liquid component. It is done for the purpose of doing. Specific examples of the synthetic adsorbent include Amberlite XAD-4, Amberlite XAD-16, Amberlite XAD-1180 and Amberlite XAD-2000 manufactured by Organo Corporation, and Sepa beads manufactured by Mitsubishi Chemical Corporation. Aromatic (or styrene) synthetic adsorbents such as SP850 and Diaion HP20, Amberlite XAD-7 from Organo Corporation, and Methacrylic (or Diaion HP2MG from Mitsubishi Chemical Corporation) (or Synthetic adsorbents may also be mentioned. In addition to these, aromatic modified synthetic adsorbents such as Sephapez SP207 manufactured by Mitsubishi Chemical Corporation may be used in some cases. The non-adsorbed fraction obtained in this manner can be used in place of at least a part of the liquid content.
[0036]
In the step (b), the liquid component (A) supplied from the step (b) [including the non-adsorbed fraction of the liquid component (A)] and the liquid component (B) [the liquid component (B) In order to adjust the concentration of each Brix to 0.5 to 6.0, the medium component concentration suitable for nisin productivity of lactic acid bacteria having nisin-producing ability is included. This can increase the productivity of nisin. The Brix concentration is more preferably adjusted to a range of 2.0 to 4.0. That is, in this case, the nisin productivity can be significantly improved.
[0037]
Preferable specific examples of the sugar added in the step (c) include glucose, xylose, sucrose, fructose, maltose, ribose, galactose, mannose, mannitol, cellobiose, saccharose, and trehalose. The sugar concentration of the liquid medium varies depending on the type of sugar used, but is generally in the range of 5 to 70 g / L. Among these sugars, glucose is particularly preferable, and the glucose concentration in this case is preferably set in the range of 5 to 70 g / L.
[0038]
The lactic acid bacterium having nisin-producing ability used in the step (d) may be any strain as long as it has nisin-producing ability, and lactic acid bacteria belonging to Lactococcus lactis subsp. Lactis are particularly preferable. Specifically, preferred examples of strains to be produced when nisin is nisin A include Lactococcus lactis NCDO497, Lactococcus lactis NIZO R5, Lactococcus lactis ATCC 7962, and Lactococcus lactis ATCC11454. In addition, as a strain when nisin to be produced is Nisin Z, Lactococcus lactis NIZO 22186, Lactococcus lactis NRRL-B-18583, Lactococcus lactis NCFB2118, Lactococcus lactis NCFB2054, Lactococcus lactis NIZO N9, Lactococcus lactis NIZO 221186, Lactoc 1 (JCM7638), Lactococcus lactis subsp.Lactis A. Ishizaki Chizuka (JCM11180), Lactococcus lactis subsp.Lactis A. Ishizaki Yasaka 5B (JCM11181), Lactococcus lactis subsp. Lactis A. Ishizaki Yasaka 8B (JCM11183) and Lactococcus lactis subsp. Lactis A. Ishizaki Yasaka 9B (JCM11184) can be mentioned as preferable examples.
[0039]
The step of culturing in step (d) while controlling the sugar concentration of the culture solution to at least 0.1 g / L and controlling the pH value of the culture solution in the range of 4.0 to 7.0 is a batch culture method or a continuous culture method. Can be done. In addition, a fed-batch culture method can be employed. When controlling the pH value of the culture solution in the range of 4.0 to 7.0, as described above, a pH measurement composite glass electrode is attached to the culture tank and measured with a pH meter, and the actual pH value is set to the set pH value. When the pH is lower than that, it can be maintained at a set pH value by supplying an alkaline solution such as a sodium hydroxide solution to the culture tank using a pH controller or the like, and preferably within a pH value range of 5.0 to 6.5. adjust. Note that the fluctuation range of the predetermined pH value is preferably 0.1 or less. In the step (d), when the sugar concentration (glucose concentration) of the culture solution is controlled to at least 0.1 g / L or more, when the culture is performed in a continuous culture method or a fed-batch culture method, a glucose analyzer or the like is used. Can be controlled using. In this step, in addition to glucose, a substrate solution containing a nitrogen source consisting of yeast extract or polypeptone or a substrate solution consisting of the above liquid medium can be supplied to the culture tank, whereby the liquid in the culture step The glucose concentration of the medium can be maintained at a desired value. The glucose concentration is at least 0.1 g / L or more, but preferably by maintaining it at 1.0 g / L or more, the activity of lactic acid bacteria having nisin production ability and the specific nisin production rate can be maintained at high values. The culture temperature during the culture may be set to an optimum temperature for the growth of the lactic acid bacteria to be used, and is generally in the range of 20 to 40 ° C, preferably in the range of 25 to 37 ° C. The stirring speed is 10 to 1000 rpm, preferably 50 to 300 rpm.
[0040]
When the culture in the step (d) is performed in a continuous culture mode, the culture is started in accordance with the above-described culture conditions, and the bacterial cell concentration is increased, and the process shifts to continuous culture at the stage where the latter stage of the logarithmic growth phase is reached. In the continuous culture method, a pH measurement composite glass electrode is attached to the culture tank, measured with a pH meter, and an alkaline solution such as a sodium hydroxide solution is cultured when the actual pH value is lower than the set pH value. By supplying to the tank, the set pH value can be maintained. At this time, the same amount of the sterilization solution as the liquid medium or alkaline solution supplied to the culture tank is withdrawn from the culture tank via an ultrafiltration membrane or a microfiltration membrane, so that the liquid volume of the culture tank is kept constant. Can be held. In addition to the pH control, a process online turbidimeter probe is attached to the culture tank, the output is input to the DDC controller, and the same amount of culture solution (bacteria) as the sterilized water or liquid medium supplied to the culture tank Is extracted from the culture tank, the turbidity of the culture tank can be controlled, and the cell concentration and the amount of the culture solution in the culture solution can be kept constant. The bacterial cell concentration is set in the range of 4.0 to 18.0 g / L, preferably in the range of 8.0 to 15.0 g / L. The dilution rate of the medium can be set to an arbitrary value depending on the cell concentration of the culture solution and the nisin production rate, and generally 0.1 to 2.0 hr. ^-1 The range is preferably 0.2 to 1.2 hr. ^-1 The range.
[0041]
【Example】
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
[0042]
Barley shochu was produced for the purpose of providing the following examples. Barley (70% refined) was used as a raw material.
[Manufacture of barley straw]
After absorbing 40% (w / w) of barley and steaming for 40 minutes, it is allowed to cool to 40 ° C, inoculated with 1 kg of seed meal (birch) per ton of barley, 38 ° C, RH 95% for 24 hours, 32 hours Barley straw was produced by maintaining at ℃ 92% RH for 20 hours.
[Manufacture of steamed barley]
Barley was absorbed by 40% (w / w), steamed for 40 minutes, and then allowed to cool to 40 ° C. to produce steamed barley.
[Manufacture of barley shochu and barley shochu distillation residue]
In the first preparation, barley koji (3 tons as barley) produced by the above method was added with 3.6 kiloliters of water and 1 kg (wet weight) of cultured cells of shochu yeast as yeast to obtain primary mash. The first moromi was subjected to fermentation for 5 days (first stage fermentation). Next, in the secondary charge, 11.4 kiloliters of water and steamed barley (7 tons as barley) produced by the method described above were added to the primary mash after the first stage fermentation for 11 days (2nd stage fermentation). (Fermentation of the eyes). The fermentation temperature was set to 25 ° C. for both the primary charge and the secondary charge. The secondary mash after the second stage fermentation was subjected to simple distillation by a conventional method to obtain 10 kiloliters of barley shochu and 15 kiloliters of barley shochu distillation residue. The obtained barley shochu distillation residue was used in the following examples.
[0043]
Rice shochu was produced for the purpose of providing the following examples. As a raw material, polished rice (70% polished) was used.
[Manufacture of rice bran]
Rice bran is made by absorbing 35% (w / w) water of rice, steaming for 40 minutes, allowing to cool to 40 ° C, inoculating 1g of white koji mold per kg of rice, 38 ° C, RH95% for 24 hours , 32 ° C., RH 92% for 20 hours.
[Manufacture of steamed rice]
Steamed rice was produced by absorbing 35% (w / w) of rice, steaming for 40 minutes, and allowing to cool to 40 ° C.
[Manufacture of rice shochu and rice shochu distillation residue]
In the first preparation, the rice bran (3 tons as rice) produced by the method described above was added with 3.6 kiloliters of water and 1 kg (wet weight) of cultured cells of shochu yeast as yeast to obtain primary mash. The first moromi was subjected to fermentation for 5 days (first stage fermentation). Next, in the secondary charging, 11.4 kiloliters of water and 7 tons of steamed rice (7 tons of rice) produced by the method described above were added to the primary mash after the first stage fermentation for 15 days (second stage fermentation). Of fermentation). The fermentation temperature was set to 25 ° C. for both the primary charge and the secondary charge. The secondary mash after the second stage fermentation was subjected to single distillation by a conventional method to obtain 10 kiloliters of rice shochu and 15 kiloliters of rice shochu distillation residue. The obtained rice shochu distillation residue was used in the following examples.
[0044]
[Example 1]
1． Preparation of lactic acid bacteria culture medium from barley shochu distillation residue
The barley shochu distillation residue obtained in [Production of barley shochu and barley shochu distillation residue] as described in [0042] above was centrifuged at 8000 rpm for 10 min to obtain a liquid content of the barley shochu distillation residue. The liquid was diluted with water to adjust its Brix concentration to 4, added with 3.6% by weight of glucose, adjusted to pH 5.5 with sodium hydroxide, and then sterilized at 121 ° C for 15 minutes. A lactic acid bacteria culture medium was obtained.
2． Pre-culture of lactic acid bacteria capable of producing nisin
50 μl of a stock strain of Lactococcus lactis IO-1 was inoculated into 10 ml of TGC medium and cultured at 37 ° C. for 18 hours to obtain a culture solution. 10 ml of the culture solution was inoculated into 100 ml of CMG medium, and 3 ml at 37 ° C. A pre-culture solution of lactic acid bacteria was obtained by culturing with shaking at 100 rpm for an hour.
3． Main culture of lactic acid bacteria capable of producing nisin
Into a 2 L jar fermenter, 500 ml of the lactic acid bacteria culture medium obtained in 1 above and 25 ml of the lactic acid bacteria preculture solution obtained in 2 above were introduced, stirring speed 250 rpm, culture temperature 30 ° C., culture time 24 hours, pH 5. Batch culture was performed under the conditions of 5.
[0045]
[Comparative Example 1]
1． Preparation of MRS medium
MRS medium (1% by weight of peptone, 1% by weight of meat extract, 0.5% by weight of yeast extract, K ₂ HPO _Four 0.2 wt%, diammonium citrate 0.2 wt%, glucose 3.6 wt%, Tween 80 0.1 wt%, sodium acetate 0.5 wt%, MGSO _Four ・ 7H ₂ O 0.058 wt%, MnSO _Four ・ 4H ₂ O 0.028 wt%) was adjusted to pH 5.5 with sodium hydroxide, and then sterilized at 121 ° C. for 15 minutes to obtain a medium for lactic acid bacteria culture.
2． Pre-culture of lactic acid bacteria capable of producing nisin
50 μl of a stock strain of Lactococcus lactis IO-1 is inoculated into 10 ml of TGC medium and cultured by standing at 37 ° C. for 18 hours to obtain a culture medium. A pre-culture solution of lactic acid bacteria was obtained by culturing with shaking at 100 rpm for an hour.
3． Main culture of lactic acid bacteria capable of producing nisin
Into a 2 L jar fermenter, introduce 500 ml of the MRS medium prepared in 1 above and 25 ml of the lactic acid bacteria preculture solution obtained in 2 above, stirring speed 250 rpm, culture temperature 30 ° C., culture time 24 hours, pH 5.5 Batch culture was performed under conditions.
[0046]
[Example 2]
1． Preparation of lactic acid bacteria culture medium from barley shochu distillation residue
The barley shochu distillation residue obtained in [Production of barley shochu and barley shochu distillation residue] as described in [0042] above was centrifuged at 8000 rpm for 10 min to obtain a liquid content of the barley shochu distillation residue. Then, dilute the liquid with water to adjust its Brix concentration to 4, add 3.6% glucose, adjust pH to 5.5 with sodium hydroxide, and then sterilize at 121 ° C for 15 minutes To obtain a culture medium for lactic acid bacteria culture.
2． Preparation of substrate solution for glucose concentration adjustment from barley shochu distillation residue
The barley shochu distillation residue obtained in [Production of barley shochu and barley shochu distillation residue] as described in [0042] above was centrifuged at 8000 rpm for 10 min to obtain a liquid content of the barley shochu distillation residue. Then, dilute the liquid with water to adjust its Brix concentration to 4, add 1.0 wt% glucose, adjust to pH 5.5 with sodium hydroxide, and then sterilize at 121 ° C for 15 minutes To obtain a glucose concentration adjusting substrate solution.
[0047]
3． Pre-culture of lactic acid bacteria capable of producing nisin
50 μl of a stock strain of Lactococcus lactis IO-1 is inoculated into 10 ml of TGC medium and cultured by standing at 37 ° C. for 18 hours to obtain a culture medium. A pre-culture solution of lactic acid bacteria was obtained by culturing with shaking at 100 rpm for an hour. Four. Main culture of lactic acid bacteria capable of producing nisin
The main culture of lactic acid bacteria having the ability to produce nisin was performed in a continuous culture system using a continuous culture apparatus consisting of a 3 L jar fermenter.
That is, 1800 ml of the lactic acid bacteria culture medium obtained in 1 above and 90 ml of the lactic acid bacteria preculture solution obtained in 3 above were introduced into the 3 L jar fermenter, and batched under the conditions of stirring speed 250 rpm, culture temperature 30 ° C., pH 5.5. When the glucose concentration of the culture solution reaches 4 g / L 8 hours after the start of culture, the culture solution is transferred to continuous culture with pH control, glucose concentration control, and turbidity control. The concentration was maintained at 4 g / L, and the continuous culture was performed until 42 hours after the start of the culture.
The pH control was performed by supplying a 5N sodium hydroxide solution to the culture tank and maintaining the pH at 5.5 using a pH sensor. The glucose concentration control is performed by measuring the glucose concentration of the culture solution using an on-line biochemical controller BF-410 (manufactured by Able Co., Ltd.) with a glucose electrode. The adjustment substrate solution was supplied to the culture tank, and the glucose concentration of the culture solution was maintained at 4 g / L. In the pH control and the glucose concentration control, a sterilization solution having the same amount as the total amount of the 5N sodium hydroxide solution and the glucose concentration adjusting substrate solution supplied to the culture tank is microfiltered. By pulling out from the culture tank through the membrane, the liquid volume of the culture tank was kept constant. The turbidity control uses a laser turbidimeter, and the same amount of culture solution (containing bacterial cells) as the turbidity control supply solution (0.5% by weight of yeast extract, 0.5% by weight of polypeptone) supplied to the culture tank. Was extracted from the culture tank, thereby maintaining the cell concentration and liquid volume of the culture tank at a constant level.
[0048]
[Comparative Example 2]
1． Preparation of MRS medium
MRS medium (1% by weight of peptone, 1% by weight of meat extract, 0.5% by weight of yeast extract, K ₂ HPO _Four 0.2 wt%, diammonium citrate 0.2 wt%, glucose 3.6 wt%, Tween 80 0.1 wt%, sodium acetate 0.5 wt%, MGSO _Four ・ 7H ₂ O 0.058 wt%, MnSO _Four ・ 4H ₂ O 0.028 wt%) was adjusted to pH 5.5 with sodium hydroxide and then sterilized at 121 ° C. for 15 minutes to obtain an MRS medium for lactic acid bacteria culture.
2． Pre-culture of lactic acid bacteria capable of producing nisin
50 μl of a stock strain of Lactococcus lactis IO-1 is inoculated into 10 ml of TGC medium and cultured by standing at 37 ° C. for 18 hours to obtain a culture medium. A pre-culture solution of lactic acid bacteria was obtained by culturing with shaking at 100 rpm for an hour.
3． Main culture of lactic acid bacteria capable of producing nisin
The main culture of lactic acid bacteria having the ability to produce nisin was performed in a continuous culture system using a continuous culture apparatus consisting of a 3 L jar fermenter.
That is, 1800 ml of the lactic acid bacteria culture medium obtained in 1 above and 90 ml of the lactic acid bacteria preculture solution obtained in 3 above were introduced into the 3 L jar fermenter, and the stirring speed was 250 rpm, the culture temperature was 30 ° C., and the pH was 5.5. At the time when the glucose concentration of the culture solution reached less than 0.1 g / L at 12 hours after the start of culture, it was transferred to continuous culture with pH control, glucose concentration control, and turbidity control. The glucose concentration of the liquid was kept below 0.1 g / L, and the continuous culture was performed until 42 hours after the start of the culture.
The pH control was performed by supplying a 5N sodium hydroxide solution to the culture tank and maintaining the pH at 5.5 using a pH sensor. The glucose concentration control is performed by measuring the glucose concentration of the culture solution using an on-line biochemical controller BF-410 (manufactured by Able Co., Ltd.) with a glucose electrode. Yeast extract 0.5% by weight, polypeptone 0.5% by weight, NaCl 0.5% by weight) was supplied to the culture tank to keep the glucose concentration of the culture solution below 0.1 g / L. Further, in the pH control and the glucose concentration control, by removing the same amount of sterilization solution as the 5N sodium hydroxide solution and the substrate solution supplied to the culture tank from the culture tank through the microfiltration membrane, The liquid volume of the culture tank was kept constant. The turbidity control uses a laser turbidimeter, and the same amount of culture solution (containing bacterial cells) as the turbidity control supply solution (0.5% by weight of yeast extract, 0.5% by weight of polypeptone) supplied to the culture tank. Was extracted from the culture tank, thereby maintaining the cell concentration and liquid volume of the culture tank at a constant level.
[0049]
[Example 3]
Instead of Lactococcus lactis IO-1 used in Example 2, Lactococcus lactis NIZO 22186, Lactococcus lactis NRRL-B-18583, Lactococcus lactis NCFB2118, Lactococcus lactis NCFB2054, Lactococcus lactis NIZO N9, Lactococcus lactis subactocius lactis NIZOoc221 Lactis A. Ishizaki Chizuka (JCM11180), Lactococcus lactis subsp.Lactis A. Ishizaki Yasaka 5B (JCM11181), Lactococcus lactis subsp. Lactis A. Ishizaki Yasaka 7B (JCM11182), Lactococcus lactis subsp. And Lactococcus lactis subsp. Lactis A. Ishizaki Yasaka 9B (JCM11184) were used for continuous culture in the same manner as in Example 2.
[0050]
[Comparative Example 3]
Instead of Lactococcus lactis IO-1 used in Comparative Example 2, Lactococcus lactis NIZO 22186, Lactococcus lactis NRRL-B-18583, Lactococcus lactis NCFB2118, Lactococcus lactis NCFB2054, Lactococcus lactis NIZO N9, Lactococcus lactis NIZO 221cusLact. Lactis A. Ishizaki Chizuka (JCM11180), Lactococcus lactis subsp.Lactis A. Ishizaki Yasaka 5B (JCM11181), Lactococcus lactis subsp. Lactis A. Ishizaki Yasaka 7B (JCM11182), Lactococcus lactis subsp. And Lactococcus lactis subsp. Lactis A. Ishizaki Yasaka 9B (JCM11184) were used for continuous culture in the same manner as in Comparative Example 2.
[0051]
[Example 4]
1． Preparation of lactic acid bacteria culture medium from barley shochu distillation residue
The barley shochu distillation residue obtained in [Production of barley shochu and barley shochu distillation residue] as described in [0042] above was centrifuged at 8000 rpm for 10 min to obtain a liquid content of the barley shochu distillation residue. The liquid was subjected to a ceramic filtration device to obtain a clarified liquid, and the clarified liquid was subjected to a vacuum evaporator to concentrate it to about 3 times to obtain a concentrated liquid, which was filled with a synthetic adsorbent. Contact the column to obtain a non-adsorbable fraction solution that is non-adsorbable to the synthetic adsorbent eluted from the packed column, dilute the non-adsorbable fraction solution with water, and adjust its Brix concentration to 4. Then, 3.6% by weight of glucose was added, and then adjusted to pH 5.5 with sodium hydroxide, and then sterilized at 121 ° C. for 15 minutes to obtain a culture medium for lactic acid bacteria culture.
2． Pre-culture of lactic acid bacteria
50 μl of a stock strain of Lactococcus lactis IO-1 is inoculated into 10 ml of TGC medium and cultured by standing at 37 ° C. for 18 hours to obtain a culture medium. A pre-culture solution of lactic acid bacteria was obtained by culturing with shaking at 100 rpm for an hour.
3． Main culture of lactic acid bacteria
Into a 2L jar fermenter, introduce 500 ml of the lactic acid bacteria culture medium obtained in 1 above and 25 ml of the lactic acid bacteria preculture medium obtained in 2 above, stirring speed 250 rpm, culture temperature 30 ° C., culture time 24 hours, pH 5.5 Batch culture was performed under conditions.
[0052]
[Example 5]
1． Preparation of lactic acid bacteria culture medium from barley shochu distillation residue
The barley shochu distillation residue obtained in [Production of barley shochu and barley shochu distillation residue] as described in [0042] above is centrifuged under the conditions of 8000 rpm and 10 min to obtain a liquid, and the liquid is A clarified liquid is obtained by attaching to a filtration apparatus, and the clarified liquid is subjected to a vacuum evaporator to concentrate it to about 3 times to obtain a concentrated liquid, and the concentrated liquid is contacted with a column filled with a synthetic adsorbent, A non-adsorbable fraction solution showing non-adsorbability with respect to the synthetic adsorbent eluted from the column was obtained, the non-adsorbable fraction solution was diluted with water to adjust its Brix concentration to 4, and glucose was adjusted to 3.6. After adjusting the pH to 5.5 with sodium hydroxide, sterilization was performed at 121 ° C. for 15 minutes to obtain a medium for culturing lactic acid bacteria.
2． Preparation of substrate solution for glucose concentration adjustment from barley shochu distillation residue
The barley shochu distillation residue obtained in [Production of barley shochu and barley shochu distillation residue] as described in [0042] above is centrifuged under the conditions of 8000 rpm and 10 min to obtain a liquid, and the liquid is A clarified liquid is obtained by attaching to a filtration apparatus, and the clarified liquid is subjected to a vacuum evaporator to concentrate it to about 3 times to obtain a concentrated liquid, and the concentrated liquid is contacted with a column filled with a synthetic adsorbent, A non-adsorbable fraction solution showing non-adsorbability to the synthetic adsorbent eluted from the column is obtained, the non-adsorbable fraction solution is diluted with water to adjust its Brix concentration to 4, and glucose is adjusted to 1.0. % By weight, adjusted to pH 5.5 with sodium hydroxide, and then sterilized at 121 ° C. for 15 minutes to obtain a substrate solution for adjusting glucose concentration.
3． Pre-culture of lactic acid bacteria capable of producing nisin
50 μl of a stock strain of Lactococcus lactis IO-1 is inoculated into 10 ml of TGC medium, and left to stand at 37 ° C. for 18 hours to obtain a culture medium. A pre-culture solution of lactic acid bacteria was obtained by culturing with shaking at 100 rpm for an hour.
[0053]
Four. Main culture of lactic acid bacteria capable of producing nisin
The main culture of lactic acid bacteria having the ability to produce nisin was carried out in a continuous culture system using a continuous culture apparatus consisting of a 3L jar fermenter.
That is, 1800 ml of the lactic acid bacteria culture medium obtained in 1 above and 90 ml of the lactic acid bacteria preculture solution obtained in 3 above were introduced into the 3 L jar fermenter, and the stirring speed was 250 rpm, the culture temperature was 30 ° C., and the pH was 5.5. At the time when the glucose concentration of the culture solution reached 4 g / L at 8 hours after the start of the culture, it was transferred to continuous culture with pH control, glucose concentration control, and turbidity control. The continuous culture was performed up to 42 hours after the start of culture while maintaining the glucose concentration at 4 g / L.
The pH control was performed by supplying a 5N sodium hydroxide solution to the culture tank and maintaining the pH at 5.5 using a pH sensor. The glucose concentration control is performed by measuring the glucose concentration of the culture solution using an on-line biochemical controller BF-410 (manufactured by Able Co., Ltd.) with a glucose electrode. The adjustment substrate solution was supplied to the culture tank, and the glucose concentration of the culture solution was maintained at 4 g / L. Further, in the pH control and the glucose concentration control, the same amount of the sterilization solution as the 5N sodium hydroxide solution and the glucose concentration adjusting substrate solution supplied to the culture tank is removed from the culture tank through the microfiltration membrane. By extracting, the liquid volume of the culture tank was kept constant. The turbidity control uses a laser turbidimeter, and the same amount of culture solution (containing bacterial cells) as the turbidity control supply solution (0.5% by weight of yeast extract, 0.5% by weight of polypeptone) supplied to the culture tank. Was extracted from the culture tank, thereby maintaining the cell concentration and liquid volume of the culture tank at a constant level.
[0054]
[Example 6]
1． Preparation of lactic acid bacteria culture medium from barley shochu distillation residue
The barley shochu distillation residue obtained in [Production of barley shochu and barley shochu distillation residue] as described in [0042] above was centrifuged at 8000 rpm for 10 min to obtain a liquid content of the barley shochu distillation residue. Then, dilute the liquid with water to adjust its Brix concentration to 4, add 3.6% glucose, adjust pH to 5.5 with sodium hydroxide, and then sterilize at 121 ° C for 15 minutes To obtain a culture medium for lactic acid bacteria culture.
2． Preparation of lactic acid bacteria culture medium from rice shochu distillation residue
The rice shochu distillation residue obtained in [Production of rice shochu and rice shochu distillation residue] described in [0043] above is centrifuged at 8000 rpm for 10 min to obtain a liquid, and the liquid is washed with water. Adjust the Brix concentration to 4 and add 3.6% by weight of glucose, adjust to pH 5.5 with sodium hydroxide, sterilize at 121 ° C for 15 minutes, and culture medium for lactic acid bacteria Got.
3． Pre-culture of lactic acid bacteria capable of producing nisin
50 μl of a stock strain of Lactococcus lactis IO-1 was inoculated into 10 ml of TGC medium and cultured at 37 ° C. for 18 hours to obtain a culture solution. 10 ml of the culture solution was inoculated into 100 ml of CMG medium, and 3 ml at 37 ° C. A pre-culture solution of lactic acid bacteria was obtained by culturing with shaking at 100 rpm for an hour.
Four. Main culture of lactic acid bacteria capable of producing nisin
Into a 2L jar fermenter, introduce 250 ml of the lactic acid bacteria culture medium obtained in 1 above, 250 ml of the lactic acid bacteria culture medium obtained in 2 above, and 25 ml of the lactic acid bacteria preculture solution obtained in 3 above, stirring speed 250 rpm, culture temperature Batch culture was performed under the conditions of 30 ° C., culture time of 24 hours, and pH 5.5.
[0055]
[Example 7]
1． Preparation of lactic acid bacteria culture medium from barley shochu distillation residue
The barley shochu distillation residue obtained in [Manufacture of barley shochu and barley shochu distillation residue] described in [0042] above was centrifuged at 8000 rpm for 10 min to obtain a liquid component, and the liquid component was Adjust the Brix concentration to 4 and add 3.6% by weight of glucose, adjust to pH 5.5 with sodium hydroxide, sterilize at 121 ° C for 15 minutes, and culture medium for lactic acid bacteria Got.
2． Preparation of substrate solution for glucose concentration adjustment from barley shochu distillation residue
The barley shochu distillation residue obtained in [Manufacture of barley shochu and barley shochu distillation residue] described in [0042] above was centrifuged at 8000 rpm for 10 min to obtain a liquid component, and the liquid component was Adjust the Brix concentration to 4 and add 1.0% by weight of glucose, adjust to pH 5.5 with sodium hydroxide, and sterilize at 121 ° C for 15 minutes to adjust the glucose concentration A substrate solution was obtained.
3． Preparation of lactic acid bacteria culture medium from rice shochu distillation residue
The rice shochu distillation residue obtained in the distillation step of rice shochu production was centrifuged at 8000 rpm for 10 minutes to obtain a liquid content of the rice shochu distillation residue, the liquid content was adjusted to Brix 4, and glucose was 3.6. After adjusting the pH to 5.5 with sodium hydroxide, sterilization was performed at 121 ° C. for 15 minutes to obtain a medium for culturing lactic acid bacteria.
Four. Preparation of substrate solution for adjusting glucose concentration from rice shochu distillation residue
The rice shochu distillation residue obtained in [Production of rice shochu and rice shochu distillation residue] described in [0043] above is centrifuged at 8000 rpm for 10 min to obtain a liquid, and the liquid is washed with water. Adjust the Brix concentration to 4 and add 1.0% by weight of glucose, adjust to pH 5.5 with sodium hydroxide, and sterilize at 121 ° C for 15 minutes to adjust the glucose concentration A substrate solution was obtained.
[0056]
Five. Pre-culture of lactic acid bacteria capable of producing nisin
50 μl of a stock strain of Lactococcus lactis IO-1 was inoculated into 10 ml of TGC medium and cultured at 37 ° C. for 18 hours to obtain a culture solution. 10 ml of the culture solution was inoculated into 100 ml of CMG medium, and 3 ml at 37 ° C. A pre-culture solution of lactic acid bacteria was obtained by culturing with shaking at 100 rpm for an hour.
6． Main culture of lactic acid bacteria capable of producing nisin
The main culture of lactic acid bacteria having the ability to produce nisin was carried out in a continuous culture system using a continuous culture apparatus consisting of a 3L jar fermenter.
Specifically, 900 ml of the lactic acid bacteria culture medium obtained in 1 above, 900 ml of the lactic acid bacteria culture medium obtained in 3 above, and 90 ml of the lactic acid bacteria preculture solution obtained in 5 above were introduced into the 3 L jar fermenter, and the stirring speed was 250 rpm. , When subjected to batch culture under conditions of culture temperature 30 ° C, pH 5.5, pH control, glucose concentration control, and turbidity when the glucose concentration of the culture solution reaches 4g / L 8 hours after the start of culture The system was shifted to continuous culture with control, and the continuous culture was performed up to 42 hours after the start of culture while maintaining the glucose concentration of the culture solution at 4 g / L.
The pH control was performed by supplying a 5N sodium hydroxide solution to the culture tank and maintaining the pH at 5.5 using a pH sensor. The glucose concentration control is performed by measuring the glucose concentration of the culture solution using an on-line biochemical controller BF-410 (manufactured by Able Co., Ltd.) with a glucose electrode. Supply the mixed solution obtained by mixing the substrate solution for adjustment and the substrate solution for glucose concentration adjustment obtained in the above 4 at a ratio of 1: 1 to the culture tank to keep the glucose concentration of the culture solution at 4 g / L Was done. Further, in the pH control and the glucose concentration control, the same amount of the sterilization solution as the 5N sodium hydroxide solution and the glucose concentration adjusting substrate solution supplied to the culture tank is removed from the culture tank through the microfiltration membrane. By extracting, the liquid volume of the culture tank was kept constant. The turbidity control uses a laser turbidimeter, and the same amount of culture solution (containing bacterial cells) as the turbidity control supply solution (0.5% by weight of yeast extract, 0.5% by weight of polypeptone) supplied to the culture tank. Was extracted from the culture tank, and thereby the cell concentration and the amount of liquid in the culture tank were kept constant.
[0057]
Each of the fermented products obtained in Example 1, Example 2, Example 4 to Example 7, Comparative Example 1 and Comparative Example 2 was subjected to the following Test Example 1, and the nisin concentration of the fermented product was measured. .
[0058]
[Test Example 1]
Each fermentation product obtained in Example 1, Example 2, Example 4 to Example 7, Comparative Example 1 and Comparative Example 2 was subjected to centrifugation at 9000 rpm for 30 minutes to obtain a top of the fermentation product. After adding the concentrated hydrochloric acid to the supernatant 1 of each fermentation product to adjust the pH to 3.0, leave it overnight at 4 ° C, and again centrifuge at 9000 rpm for 30 min. The supernatant 2 of the fermentation product was obtained, and the nisin concentration of the culture solution was measured according to the following method using the obtained supernatant 2 of each fermentation product as a sample. That is, Micrococcus luteus (IFO N0.3333) was used as an indicator bacterium, and nisin (ICN) dissolved in 0.01N HCl to 3000 IU / ml was used as a nisin A standard solution. Inoculate 10 ml of LB liquid medium with the above indicator bacteria in a platinum loop, shake culture under conditions of a culture temperature of 30 ° C., a stirring speed of 100 rpm, and a culture time of 18 hours to obtain a culture solution, and 100 μl of the culture solution was cooled to an appropriate temperature Suspend in 2.0 ml of soft agar to obtain a suspension. The suspension is layered on LB agar medium and allowed to stand for 1 hour, and then the fermentation product supernatant 2 diluted to an appropriate concentration with 0.01 N HCl is used. Spot 10 μl each on the LB agar medium, incubate at 30 ° C. for 24 hours, confirm the presence or absence of growth-inhibiting circles that have inhibited growth around the spot, and the maximum dilution rate of the sample when the growth-inhibiting circles appear D was determined, and the nisin antibacterial activity value was calculated from the relational expression of nisin antibacterial activity (AU / ml) = 1 / D × sample addition amount (ml).
[0059]
[Evaluation 1]
Table 1 shows the nisin antibacterial activity values of the fermented products obtained in Example 1 and Comparative Example 1 24 hours after the start of culture. The results shown in Table 1 revealed the following. That is, the nisin antibacterial activity value 24 hours after the start of the culture was as low as 1200 IU / ml for the fermented product obtained in Comparative Example 1, whereas the fermented product obtained in Example 1 was 4500 IU. The value was extremely high at / ml. That is, the nisin antibacterial activity value of the fermented product obtained by the nisin production method of the present invention using the culture medium for lactic acid bacteria obtained from the barley shochu distillation residue is determined by the conventional nisin production method using the MRS medium. It was found that the fermentation product obtained reached about 3.8 times the nisin antibacterial activity value.
The nisin antibacterial activity values in the continuous culture of Example 2 and Comparative Example 2 are shown in Table 2. The results shown in Table 2 revealed the following. That is, in the comparative example 2, the nisin antibacterial activity value gradually decreased after showing the maximum value at the 12th hour after the start of the culture, which was transferred from the batch culture to the continuous culture, and 1000 IU / ml at the 42th hour after the start of the culture. In contrast, in Example 2, after showing a maximum value at 12 hours after the start of culture from batch culture to continuous culture, it decreased until 24 hours, but after 24 hours, It rose again and reached 6300 IU / ml 42 hours after the start of culture. That is, it is obtained by the method for producing nisin of the present invention, wherein the culture medium for lactic acid bacteria obtained from the barley shochu distillation residue is used, and the culture is maintained while maintaining the glucose concentration of the culture solution at 4 g / L during continuous culture. The nisin antibacterial activity value of the fermented product is a fermentation obtained by a conventionally known nisin production method using MRS medium and culturing while maintaining the glucose concentration of the culture solution at less than 0.1 g / L during continuous culture. It was found to reach 6.3 times the nisin antibacterial activity value of the product.
The results of Example 3 and Comparative Example 3 revealed the following. That is, the nisin antibacterial activity value of each fermentation product obtained by subjecting various lactic acid bacteria strains having nisin-producing ability to continuous culture in Example 3, even when any strain is used, Comparative Example 3 The value was significantly higher than when the conventionally known nisin production method shown in FIG.
[0060]
The results of Example 4 and Example 5 revealed the following. That is, instead of the liquid obtained by solid-liquid separation of the barley shochu distillation residue used in the present invention, the liquid is filtered to obtain a clarified liquid, and the clarified liquid is concentrated to obtain a concentrated liquid. When the solution comprising the non-adsorbable fraction described in the above-mentioned Document 6 obtained by subjecting the concentrated solution to an adsorption treatment using a synthetic adsorbent is used, the fermentation product obtained in Example 4 and Example 5 is used. The nisin antibacterial activity value was found to be equivalent to the nisin antibacterial activity value of the fermented products obtained in Example 1 and Example 2, respectively.
[0061]
The results of Example 6 and Example 7 revealed the following. That is, Example 6 and Example in the case where a liquid mixture obtained by solid-liquid separation of barley shochu distillation residue and a liquid component obtained by solid-liquid separation of rice shochu distillation residue is used in the medium It was found that the nisin antibacterial activity value of the fermented product obtained in 7 was equivalent to the nisin antibacterial activity value of the fermented product obtained in each of Example 1 and Example 2.
[0062]
[Test Example 2]
Each of the fermented products obtained in Example 1, Example 2, Example 4 to Example 7, Comparative Example 1 and Comparative Example 2 was subjected to a sensory test for taste. That is, each of the fermentation products obtained in Example 1, Example 2, Examples 4 to 7, Comparative Example 1 and Comparative Example 2 was subjected to a sensory test by 10 panelists, and the fermentation products The taste was evaluated.
[0063]
[Evaluation 2]
Table 3 shows the sensory test results of the respective fermentation products obtained in Example 1, Example 2, Example 4 to Example 7, Comparative Example 1 and Comparative Example 2. The results shown in Table 3 revealed the following. That is, each fermentation product obtained in Comparative Example 1 and Comparative Example 2 had a very monotonous flavor and an unfavorable miscellaneous taste, so the value of sensory evaluation was extremely low, whereas Example 1 and Example 2, and the fermented products obtained in Examples 4 to 7 both have a mellow, harmonious flavor and rich umami, so the sensory evaluation value is remarkably high. As a result, it turned out to be suitable. And from the comments of the above panelers, among the fermented products obtained in Example 1, Example 2, Example 4 to Example 7, all the fermented products obtained in Example 4 and Example 5, The fermentation product obtained in Example 6 and Example 7 has a characteristic of exhibiting an outstanding mellow flavor, and both the fermentation products obtained in Example 1 and Example 2 have a further taste. It became clear that it has the characteristic of exhibiting rich umami.
[0064]
From the above results, it is understood that according to the present invention, it is possible to dramatically increase nisin productivity as compared with a conventionally known nisin production method. In addition, the fermented product obtained by the present invention contains a significant amount of nisin and, in addition to excellent antibacterial activity, exhibits a mellow and harmonious flavor and rich umami, so it can be used very suitably as a food. .
[0065]
[Table 1]

[0066]
[Table 2]

[0067]
[Table 3]

[0068]
【The invention's effect】
As described above in detail, the method for producing a fermented product containing nisin of the present invention comprises a liquid component (A) obtained by solid-liquid separation of a barley shochu distillation residue, and a Brix concentration of 0.5 to 6.0. A medium prepared by adding sugar to a liquid adjusted to the above range is used as a medium, and culturing is performed by adding lactic acid bacteria capable of producing nisin to the medium. The culture is performed at a sugar concentration of at least 0.1 g / L or more. And the pH value is controlled in the range of 4.0 to 7.0, and enables efficient production of a fermented product containing a significant amount of nisin. The fermented product containing a significant amount of nisin thus obtained has excellent taste and can be suitably used as a food.

Claims (6)

  A method of culturing microorganisms to produce a nisin-containing fermented product, using lactic acid bacteria having nisin-producing ability as microorganisms, and solid-liquid separation of barley shochu distillation residue as a medium for culturing the lactic acid bacteria. The method for producing a nisin-containing fermented product, characterized in that the liquid component itself is used, and when the liquid component itself is used, the Brix concentration of the liquid component is adjusted to a range of 0.5 to 6.0 and sugar is added thereto. .   In culturing the microorganism, the lactic acid bacterium is added to the medium to which the sugar is added, and the sugar concentration of the culture solution is controlled to at least 0.1 g / L and the pH value is controlled within the range of 4.0 to 7.0. The method for producing a nisin-containing fermented product according to claim 1, wherein the method is performed.   The method for producing a nisin-containing fermented product according to claim 1 or 2, wherein the Brix concentration of the liquid is adjusted by diluting the liquid with water.   The nisin-containing fermentation according to claim 1, 2 or 3, wherein when the liquid component itself is used, at least a part of the liquid component is subjected to an adsorption treatment using a synthetic adsorbent to form a non-adsorbed fraction, and the Brix concentration is adjusted. Production method of the product.   The method for producing a nisin-containing fermentation product according to claim 4, wherein the synthetic adsorbent is selected from the group consisting of aromatic, aromatic modified, and methacrylic synthetic adsorbents. The method for producing a nisin- containing fermented product according to claim 1 or 2, wherein the culture is performed by a batch culture method and / or a continuous culture method.