JPH08191686A - Medium for producing polysaccharide and production of polysaccharide - Google Patents

Abstract

PURPOSE: To obtain a medium for producing a polysaccharide of lactic acid bacteria, capable of producing a high-purity polysaccharide in high yield by culturing lactic acid bacteria, comprising a complete synthetic medium containing a glucide, a liberated amino acid, a base, a vitamin and a mineral but not a peptone and a peptide. CONSTITUTION: This medium for producing a polysaccharide of lactic acid bacteria comprises a complete synthetic medium obtained by dissolving 2-10% (W/V) of one or more glucides such as lactose, glucose, galactose, fructose, mannose and N-acetylglucosamine, 0.3-3% (W/V) of a liberated amino acid, a base such as adenine, uracil, xanthine and guanine, a vitamin and a mineral in purified water and substantially containing neither peptone nor peptide. Then, the complete synthetic medium is filtered, sterilized and inoculated with a lactic acid bacterium [e.g. Lactococcus lactis ssp cremoris SBT0495 (FERM P-10,053)], the bacterium is cultured at pH6.0 to pH6.5 for about 55 hours and a cultured product is collected from the culture solution to give a high-purity polysaccharide in a high yield.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a lactic acid bacterium polysaccharide production medium comprising a completely synthetic medium. The present invention also relates to a method for producing a polysaccharide by culturing lactic acid bacteria in such a completely synthetic medium. By producing the polysaccharide according to the present invention, the yield of the polysaccharide produced by lactic acid bacteria and the purity thereof can be increased. Polysaccharides produced by lactic acid bacteria are known to have various physiological activities, and are useful as materials for medicines, functional foods and the like.

[0002]

2. Description of the Related Art In recent years, it has been revealed that polysaccharides produced by microorganisms such as lactic acid bacteria have an action of strengthening immune function in the living body and exhibit antitumor activity and the like. Regarding the polysaccharide produced by lactic acid bacteria, Lactobacillus bulgarix
lgaricus ) has a novel high molecular weight polysaccharide substance having immunostimulatory activity (Japanese Patent Laid-Open No. 58-198495), Lactococcus lactis subspecies cremoris.
(Lactococcus lactis ssp.cremoris ) and Lactococcus lactis subspecies lactis (Lactococc
us lactis ssp. lactis ) has a polysaccharide-containing culture having antitumor activity [JP-A-1-277484
Gazette], Streptococc
us lactis ) and Streptococcus cremoris (Strep
tococcus cremoris ) has a novel structure of a polysaccharide having an antitumor activity (JP-A-3-229702), Bifidobacteri breve (Bifidobacteri
um breve) and Lactobacillus
The polysaccharide contained in the culture obtained by culturing casei) has anti-ulcer activity (Japanese Patent Laid-Open No. 4-5236), and a polysaccharide obtained by culturing lactic acid bacteria belonging to the genus Lactococcus It has been found that the culture containing contains an inhibitory effect on serum cholesterol elevation [JP-A-5-65229], and dextrin produced by a microorganism belonging to the genus Leuconostoc is a blood flow improver or a plasma substitute. It is expected that the polysaccharides will be applied to medicines, functional foods, etc., such as industrially produced as medicines such as foods.

Further, in the field of the food industry, it is expected to be applied as a food additive replacing the vegetable gum which has been used as a thickener or stabilizer by utilizing the unique physical properties of polysaccharides. .

On the other hand, the production of polysaccharides using lactic acid bacteria is carried out using a medium mainly composed of skim milk, whey, or an enzymatic hydrolyzate thereof. For example,
Lactococcus lactis subspecies cremoris (Lactococcus lact)
is ssp. cremoris ) at 18 ° C. for 24 hours at pH 5.5 by a constant pH culture method to obtain a polysaccharide [J. Dairy Sci., vo
l.73, pp.1472-1477, 1990], skim milk or permeate obtained by ultrafiltration treatment of skim milk (filtrate) with casamino acid added to the medium as Lactobacillus bulgaricus (Lactobacillus bulgaricus ) and Streptococcus thermophilus
us ) at 37 ° C and Lactobacillus casei (Lacto
bacillus casei) , Lactococcus lactis ssp. cr .
Emoris) and Lactococcus lactis subsp. lactis (Lactococcus lactis ssp. lactis) at 18 to 25 ° C. for a method of obtaining a polysaccharide with each stationary culture [FEMS Microbiol.Review, vol.87, pp. 113-130,
1990], Lactobacillus helveticus was prepared by enzymatically decomposing whey or brewer's yeast extract.
(Lactobacillus helveticus ) for 24 hours at 37 ℃, to obtain a polysaccharide by static culture (Agri.Biol.Chem., Vol.47, p
p.1623-1625, 1983], non-fat milk as a medium, and Streptococcus thermophilus (Streptococcus thermophilu
s ) to obtain polysaccharides by static culture at 43 ° C [Car
bohydr.Res., vol.198, pp.313-321, 1990], using skim milk as a medium, and Lactobacillus delbrueckii (Lactobacillus delbrueckii).
ssp.bulgaricus ) at 37 ° C for 20 hours to obtain a polysaccharide [Carbohydr. Res., vol.239, pp.209-226, 1993]
Etc. are known.

However, when the lactic acid bacterium is statically cultivated to produce a polysaccharide, the production amount of the polysaccharide is about 150 mg / liter, which is compared with the production efficiency of substance production by microorganisms that is generally performed. It is not always a satisfactory value. Further, as a method of recovering a polysaccharide from a culture solution in which lactic acid bacteria are cultured to produce a polysaccharide, a method of removing bacterial cells by centrifugation, a method of concentrating and recovering a polysaccharide by ultrafiltration of a supernatant, Alternatively, there is a method of precipitating and recovering polysaccharides in the presence of alcohol at a concentration of 50% or more, but lactose and amino acids and peptides contained in skim milk used for culturing lactic acid bacteria are sterilized during medium preparation. Since a browning reaction is caused by heat treatment such as to form a complex of lactose with an amino acid or a peptide, the complex may be recovered together with the polysaccharide and the purity may be lowered.

Furthermore, when a medium mainly composed of skim milk or whey is used, the phosphate group contained in the polysaccharide produced by lactic acid bacteria is ion-bonded with the protein or peptide contained in the medium, and It forms a complex of saccharide and protein or peptide, which causes a decrease in the purity of the recovered polysaccharide. By the way, when a lactic acid bacterium produces a polysaccharide using a medium mainly composed of skim milk and whey, and the polysaccharide is precipitated with 50% alcohol, the content of the polysaccharide in the precipitate is about It is about 35% by weight, which is not a satisfactory value.

Therefore, skim milk or whey used as a medium for culturing lactic acid bacteria to produce a polysaccharide is hydrolyzed with an enzyme in advance and subjected to ultrafiltration to remove proteins and high-molecular peptides. And a method of removing a protein or peptide forming a complex with a polysaccharide produced by lactic acid bacteria by hydrolyzing with an enzyme. However, in such a method, complicated treatment such as enzyme treatment or ultrafiltration treatment is required, and since such treatment requires a long time, it is not suitable for industrial production such as producing a large amount of polysaccharides. Not suitable.

[0008]

In view of the above-mentioned problems, the present inventors have conducted diligent research on a medium suitable for culturing lactic acid bacteria to produce a large amount of polysaccharides.
It was found that the purity can be increased together with the amount of polysaccharide produced by lactic acid bacteria by using a completely synthetic medium prepared by mixing the components of each medium as a basic medium and using the nitrogen component as a free amino acid mixture. Then, while selecting a suitable carbohydrate as a medium component, changing the sterilization method of the completely synthetic medium from heat sterilization to filtration sterilization, and further culturing the lactic acid bacterium by a constant pH culturing method, the lactic acid bacterium was used The inventors have found the optimum conditions for producing sugars and completed the present invention.

Therefore, it is an object of the present invention to provide a medium suitable for culturing lactic acid bacteria and producing a large amount of polysaccharide with high purity. Moreover, this invention makes it a subject to provide the manufacturing method of the polysaccharide which cultures a lactic acid bacterium and mass-produces a polysaccharide with high purity. Examples of polysaccharides in the present invention include glucose, galactose or phosphorylated polysaccharides composed of rhamnose and a phosphate group, but the present invention can also be used for production of other polysaccharides. .

[0010]

[Means for Solving the Problems] The present inventors examined the following items in order to find a medium and culture conditions suitable for culturing lactic acid bacteria to produce a large amount of polysaccharides. . Sterilization method of medium 10% (W / V) Reduced skim milk After sterilizing at 115 ° C for 30 minutes, enzyme actinase E (Kaken Pharmaceutical Co., Ltd.) was added at a rate of 50 mg / liter and treated at 37 ° C for 16 hours. The protein was decomposed. After deactivating the enzyme by heat treatment at 100 ° C. for 30 minutes, the enzyme was applied to an ultrafiltration device having a membrane with a molecular weight cut off of 3,000 to obtain about 8 liters of permeate (filtrate). Of this permeate, 4 liters was the medium sterilized by autoclave and the remaining 4 liters
The medium was filtered and sterilized with a 0.2 μm membrane.

The two culture media having different sterilization methods were transferred to a 5 liter jar fermenter maintained at 18 ° C., and about 200 ml of a preculture of lactic acid bacteria in the logarithmic growth phase was added to start the culture. During the growth of the bacterial cells, the pH was maintained at 6 while neutralizing lactic acid by automatic titration with 3N potassium hydroxide, and the turbidity of the culture solution was periodically measured to examine the bacterial growth. 10-15 more after growth reaches stationary phase
Culturing was continued for about 50 hours, and the cultivation was finished after about 50 hours.

The culture solution recovered from the jar fermenter was centrifuged (20,000 × g, 60 minutes) to remove the bacterial cells, and 4 L of ethanol was mixed with the supernatant to precipitate the polysaccharide, which was then centrifuged. The precipitated polysaccharide was recovered by separation (5,000 xg, 20 minutes). Further, the operation of dissolving the recovered polysaccharide in 200 ml of distilled water, mixing an equal amount of ethanol and precipitating again was repeated twice to recover the polysaccharide. The polysaccharide recovered in this way is lyophilized,
It was used for measurement of dry weight and analysis of chemical composition.

In order to purify the polysaccharide, the freeze-dried product was treated with a phosphate buffer solution (p
It was dissolved in H 7.5) and treated with the enzyme Pronase (Boehringer) at 30 ° C. for 16 hours to decompose the protein, and precipitation with ethanol and dissolution with distilled water were repeated in the same manner as above to purify the polysaccharide. Table 1 shows the results.

[0014]

[Table 1]

The dry weight of the ethanol precipitate of the culture broth in which lactic acid bacteria were cultivated in the heat-sterilized medium was higher than that in the case of using the filter-sterilized medium, but its chemical composition had a high protein content and a high content. The sugar purity was low. Since there is no significant difference between the dry weights of the purified polysaccharides, the complex of lactose and peptides produced by the browning reaction by heat sterilization increases the dry weight of the ethanol precipitate. it was thought. Therefore, by changing the sterilization method of the medium used for culturing the lactic acid bacteria from heat sterilization to filtration sterilization, the purity of the polysaccharide recovered as the ethanol precipitate can be improved.

Nitrogen content of medium A completely synthetic medium having the composition shown in Table 2 was used as a basic medium.

[Table 2]

Amino acid mixture of complete synthetic medium, casamino acid (Difco
, Kashiton (Difco), Tryptone (Difco) and Tryptone L-42 (Oxoid), and a medium containing lactose as a sugar source at 5% (W / V). Was prepared. Then, lactic acid bacteria were cultured using this medium to produce a polysaccharide. The medium was sterilized by filtration, and the culture method, the polysaccharide collection method and the purification method were the same as those used when the sterilization method of the medium was examined.

The results are shown in Table 3.

[Table 3]

The dry weight of the recovered ethanol precipitate was higher in the medium containing peptides such as peptone and tryptone as the nitrogen component than in the medium containing amino acids as the nitrogen component, but the protein composition was higher in the chemical composition. High and the purity of the polysaccharide was low. On the other hand, the purity of the polysaccharide is as high as about 68% in the medium containing amino acid as a nitrogen component and substantially not containing peptone or peptide, and there is no great difference in dry weight of the purified polysaccharide between the two.
It was found that the purity of the polysaccharide recovered as an ethanol precipitate can be improved by using a medium containing a peptide-free amino acid as a nitrogen component.
Therefore, it can be said that the amino acid mixture and casamino acid of the completely synthetic medium having the composition shown in Table 2 are suitable as the nitrogen component of the medium for producing lactic acid bacterium polysaccharide.

Glucose components of medium Using a completely synthetic medium having the composition shown in Table 2 as a basic medium, all the carbohydrate components contained in the completely synthetic medium are lactose, glucose, galactose, fructose, mannose and N. -A medium was prepared by substituting each of the six sugar components of acetylglucosamine for a concentration of 150 mM. Then, lactic acid bacteria were cultured using this medium to produce a polysaccharide. The medium was sterilized by filtration, and the culture method, the polysaccharide collection method and the purification method were the same as those used when the sterilization method of the medium was examined.

The results are shown in Table 4.

[Table 4]

When fructose or mannose was used as the sugar component, the production amount of the polysaccharide tended to be low, but when lactose, glucose, galactose and N-acetylglucosamine were used as the sugar component, the polysaccharide was produced. It was found that all of the four types of sugars were suitable as sugar components of the medium for producing lactic acid bacterium polysaccharides. It should be noted that fructose and mannose can also be sufficiently used by using them in combination with any of lactose, glucose, galactose, or N-acetylglucosamine.

Method for culturing lactic acid bacterium A completely synthetic medium having the composition shown in Table 2 was used as a basic medium, and lactose was added only to the sugar component so as to be 5% (W / V),
A 2 liter medium (pH 6) was prepared by filter sterilization. About 1 liter of this is transferred to a pre-sterilized glass container and used for static culture, and the remaining 1 liter is transferred to a 2 liter jar fermenter and fixed.
Used for pH culture. Then, 50 ml of the preculture in the logarithmic growth phase was added to each of the same medium to start the culture, and the static culture was left as it was.The constant pH culture was performed by automatic titration of a 3N potassium hydroxide solution. Culturing was carried out with the inside pH kept constant at 6. After culturing for about 45 hours, both culture solutions were recovered, and the polysaccharide was recovered by the same method as used when examining the sterilization method of the medium.

The results are shown in Table 5.

[Table 5]

The amount of ethanol precipitates in the culture broth obtained by the constant pH culture at pH 6 was about three times as large as that in the static culture, and the lactic acid bacterium was cultured by the constant pH culture method to produce polysaccharides. It was found that the production efficiency would be greatly improved.

Optimum pH of culture A completely synthetic medium containing 5% (W / V) lactose was used to prepare mediums having different pHs, and the pH of each medium was maintained by automatic titration of 3N potassium hydroxide. pH culture was performed. After culturing for about 50 hours, the culture solution was recovered, and the polysaccharide was recovered by the same method as that used when the sterilization method of the medium was examined. The production amount of the obtained polysaccharide depended on the sugar content in ethanol precipitation.

The results are shown in FIG.

The production amount of polysaccharide showed the maximum value in the vicinity of pH 6.3, and the amount of the polysaccharide recovered by ethanol precipitation of the culture solution in which the lactic acid bacterium was cultured under this condition was about 930 mg / liter. The amount of polysaccharides recovered in static culture is about
Since it was 150 mg / liter, it was found that by adjusting the pH to 6.3, the production amount of polysaccharide could be increased about 6 times under the usual culture conditions of lactic acid bacteria.

As described above, as a medium used for culturing lactic acid bacteria to produce a polysaccharide, a conventionally known completely synthetic medium is used as a basic medium, and a free amino acid mixture or casamino acid is added as a nitrogen component, As the sugar component, a mixture of one or more selected from lactose, glucose, galactose, fructose, mannose and N-acetylglucosamine is suitable. The medium is preferably sterilized by filtration. Further, when culturing lactic acid bacteria using this medium, it is preferable to carry out constant pH culture in the range of pH 6.0 to 6.5. The lactic acid bacterium polysaccharide production medium of the present invention and the polysaccharide production method of the present invention can be applied to any lactic acid bacterium species that produces a polysaccharide.

Next, the present invention will be described in detail by showing examples.

Example 1 Otto et al. [FEMS Microbiol. Lett., V
ol. 16, pp.69-74, 1990], 4 liters of a complete synthetic medium having the composition shown in Table 2 was prepared. The medium is sterilized by filtration, and a 5-liter jar fermenter (Appl
ikon) to adjust the pH by automatic titration of 3N potassium hydroxide.
A constant pH culture maintained at 6.3 was performed. Lactococcus lactis subspecies cremoris (L
actococcus lactis ssp. cremoris ) SBT 0495 (FERM P
-10053) was cultured for about 55 hours, and then about 4.7 liters of the culture solution was collected. Centrifuge this culture (20,000 xg,
Cells for 60 minutes to completely remove the cells, and the resulting supernatant was about 4.5.
The polysaccharide was precipitated by mixing an equal amount of ethanol with 1 liter. The precipitate was collected by centrifugation (4,000 xg, 10 minutes), fully dissolved in 500 ml of pure water, and then ethanol 5 again.
The operation of precipitating the polysaccharide with 00 ml was repeated twice to obtain a crudely purified polysaccharide. The dry weight of this polysaccharide crude product was about 3.8 g, and its sugar content and phosphoric acid content were each about 6 g.
It was 5.5% and about 7.8%. A part of this is decomposed into proteins with the enzyme Pronase (Boehringer), and purified polysaccharides are recovered again by ethanol precipitation to prepare dried products. As a result of calculation, the purity of the crude polysaccharide purified product is about
It was 72%.

[0031]

Example 2 Instead of the free amino acid mixture used in preparing the completely synthetic medium of Example 1, casamino acid (Difco
0.5% (w / v) and 7% of lactose which is a sugar component.
60 liters of medium (w / v) was prepared. Polysaccharides were produced by repeating the culture 15 times in the same manner as in Example 1 except that the culture time for one culture was 70 hours. The dried product thus obtained weighed about 79 g, and the purity of the polysaccharide calculated in the same manner as in Example 1 was about 68%.

[0032]

[Example 3] 3% (w / v) of glucose was used in place of lactose which is a sugar component used in the preparation of the completely synthetic medium of Example 1.
4 liters of the mixed medium was prepared. The same lactic acid bacterium as in Example 1 was cultured for about 60 hours, then about 5 liters of the culture solution was recovered, and the polysaccharide was recovered by precipitation with 50% ethanol as in Example 1 to obtain a crudely purified product. The dry weight of the crude polysaccharide product was about 5.2 g, and the purity was about 70%.

[0033]

[Example 4] By the same medium and culture conditions as in Example 1, Lactobacillus delbrueckii ssp.
bulgaricus ) NCFB2772 was cultured, and the polysaccharide was recovered in the same manner as in Example 1 to obtain a crudely purified product. The dry weight of the crude polysaccharide product was about 2.3 g, and the purity was about 78%.

[0034]

[Example 5] Lactobacillus lacti (Lactobacillus lacti) was cultured under the same medium and culture conditions as in Example 1.
s ) NCFB860 was cultured, and the polysaccharide was recovered in the same manner as in Example 1 to obtain a crudely purified product. The dry weight of the crude polysaccharide product was about 1.8 g, and the purity was about 83%.

[0035]

[Example 6] By the same medium and culture conditions as in Example 1, Streptococcus thermophilus (Streptococc
us thermophilus ) DSM20259 was cultured, and the polysaccharide was recovered in the same manner as in Example 1 to obtain a crudely purified product. The dry weight of the polysaccharide crude product was about 1.5 g, and the purity was about 80%.

[0036]

INDUSTRIAL APPLICABILITY The present invention relates to a medium used for culturing lactic acid bacteria for producing a polysaccharide expected to be applied to foods, pharmaceuticals and the like, and a method for culturing the medium. By using the method, about 6
Up to 7 times more polysaccharides can be produced, and even if ethanol precipitation, which is a simple method for recovering polysaccharides, is performed, the polysaccharides can be recovered with a purity about twice that of conventional methods. Therefore, according to the present invention, it becomes possible to produce polysaccharides having various physiological functions on an industrial scale. The polysaccharide produced by the method of the present invention is produced by lactic acid bacteria that are traditionally used when producing fermented foods such as cheese and yogurt, and is recognized as safe (GRAS: Gen
erally Recognized As Safe) is well established, so it can be used as a raw material for medicines and foods without any problems.

[Brief description of drawings]

FIG. 1 shows the relationship between pH in constant pH culture and the amount of polysaccharide produced by lactic acid bacteria.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display area C12R 1: 225) (C12N 1/20 C12R 1:46) (C12P 19/04 C12R 1:01) (C12P 19 / 04 C12R 1: 225) (C12P 19/04 C12R 1:46) (72) Inventor Shinichi Takafuji 62-32 Kotsumi, Kawagoe City, Saitama Prefecture (72) Inventor Taisuke Iwasaki, 9752 NAEN, Haren, Hemstelhaus Lahn 17

Claims (9)

[Claims] 1. A lactic acid bacterium polysaccharide production medium comprising a completely synthetic medium containing sugars, free amino acids, bases, vitamins and minerals and substantially free of peptone and peptides. 2. The medium according to claim 1, which contains a carbohydrate component of 2 to 10% (w / v) and an amino acid component of 0.3 to 3% (w / v). 3. The medium according to claim 1, which contains, as a sugar component, one or more sugars selected from the group consisting of lactose, glucose, galactose, fructose, mannose and N-acetylglucosamine. 4. The medium according to claim 1, which has been sterilized by filtration. 5. A lactic acid bacterium is cultured in a completely synthetic medium containing sugar, free amino acid, base, vitamin and mineral and substantially free of peptone and peptide, and the produced polysaccharide is collected. And a method for producing a polysaccharide. 6. The method according to claim 5, wherein a completely synthetic medium containing a sugar component of 2 to 10% (w / v) and an amino acid component of 0.3 to 3% (w / v) is used. 7. The method according to claim 5 or 6, wherein a complete synthetic medium containing at least one sugar selected from the group consisting of lactose, glucose, galactose, fructose, mannose and N-acetylglucosamine is used as the sugar component. Law. 8. The production method according to claim 5, wherein a completely synthetic medium which has been sterilized by filtration is used. 9. The method according to claim 5, wherein the lactic acid bacterium is cultured at pH 6.0 to 6.5.