JP5346764B2 - Production method of hyaluronic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing hyaluronic acid formulated in various kinds of foods and drinks in high yield. <P>SOLUTION: The method for producing hyaluronic acid includes inoculating a lactic acid bacterium belonging to Streptococcus thermophilus on a medium, and culturing the lactic acid bacterium under conditions of pH 6-8 at 37-40&deg;C. Hyaluronic acid is obtained by the method. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a method for producing hyaluronic acid by lactic acid bacteria.

Hyaluronic acid is an expensive glucosaminoglucan together with chondroitin sulfate, heparin and the like, and is known to exist widely in microorganisms even though it is a trace amount in mammalian connective tissue. And high purity hyaluronic acid extracted and purified from these has high moisturizing properties, high viscosity, wound healing properties, etc., so paying attention to its functions, cosmetic compounding materials, ophthalmic surgery Widely used as a protective agent, an arthritis therapeutic agent, and the like.
In recent years, consumer interest in added value such as health benefits and functionality has increased in the food field, and hyaluronic acid can be expected to have various effects depending on its functionality. It is also attracting attention as one.

  Conventionally, hyaluronic acid has been produced by an industrial process using extraction from chicken crowns, cattle joints or whale cartilage. However, there is only a very small amount of hyaluronic acid in the living body, and in the tissue. Because it forms a complex with proteins and other mucopolysaccharides, it requires complicated steps to separate and purify the proteins and mucopolysaccharides, making it difficult to produce in large quantities. In general, a method for producing hyaluronic acid according to JP-A No. 2004-259266 is carried out (see Patent Documents 1 to 4).

  Among microorganisms of the genus Streptococcus, hyaluronic acid-producing microorganisms include Lancefield serogroups A, C and D, specifically Streptococcus pyogenes, Streptococcus zooepidemicus (Streptococcus zooepidemicus). ), Streptococcus equi (Streptococcus equi), Streptococcus equisimilis, Streptococcus dysgalactiae, and Pasturera multocida (patteurella multocida) have been well known for a long time. See -8).

However, among these microorganisms, Lancefield serogroup A type bacteria and Pasteurella are known as pathogens for humans, and all these bacteria produce streptridine (soluble hemolysin), and β -Because of its hemolytic properties, industrial hyaluronic acid production methods using these microorganisms are harmful from hyaluronic acid produced by the microorganisms in addition to using detoxified strains to ensure safety. There is a need to take another means such as purification in the process of removing substances, and the workability is not very good in terms of manufacturing.
Further, since it has been pointed out that the production method of hyaluronic acid by a culture method using microorganisms is generally low in productivity, a means for improving or improving the productivity is important. Many reports have been made for the purpose of improving the rate (see Patent Documents 5-9).
Among these, a method for producing hyaluronic acid using lactic acid bacteria belonging to Streptococcus thermophilus has been reported (see Patent Document 9).

Japanese Patent Publication No. 4-55675 Japanese Patent Publication No. 4-4868 Japanese Patent Publication No. 4-43637 Japanese Examined Patent Publication No. 4-39998 Japanese Examined Patent Publication No. 4-12960 Japanese Patent Publication No. 4-18839 Japanese Patent Publication No. 7-2117 JP 2000-189185 A JP 2009-112260 A

F.E.Kendall et al., J. Biol.chem., 118,61,1937 W.A.Pierce et al., J.Baact, 63,301,1952 A.P.MacLennan, J.Gen.Microbiol., 14,134-142,1956 B. Holmstrom et al., Appl. Microbiol., 15,1409-1413, 1967 J.B.Woolcock., J.Gen.Microbiol., 85,372-375,1974 E. kjems et al., Acta Path.Microbiol.Scand.Sect.B, 84,162-164,1976 T. Bergan et al., Acta Path.Microbiol.Scand, 75,97-103,1969 J.A. Cifonelli., Carbohyd.Res., 14,272-276,1970

  An object of the present invention is to provide a method for easily and efficiently producing hyaluronic acid which has functionality that can be expected to have effects such as beauty and can be blended in various foods and drinks.

The present inventor has disclosed a method for producing hyaluronic acid safely using lactic acid bacteria belonging to Streptococcus thermophilus that can also be used for producing yogurt, and in particular, by adding soybean peptide to a medium containing milk components, the productivity of hyaluronic acid Has already been reported (see Patent Document 9).
However, in this method of adding soy peptide to the medium, the number of steps for adding the soy peptide to the medium is increased in the step of preparing the medium; since the added soy peptide is precipitated in the hyaluronic acid purification step, the viscosity of the soy peptide is increased. It is difficult to easily and efficiently produce hyaluronic acid due to the time and process required to remove it from certain hyaluronic acid. For this reason, this method has a demerit that the production cost of hyaluronic acid increases because it is difficult to improve working efficiency and the price of soybean raw materials is unstable, especially at the industrial level. There has been a demand for a method that is not used or a reduction in the amount used.
In culturing lactic acid bacteria, it is preferable to include lactose, which is one of the main energy sources of lactic acid bacteria, in the medium composition. However, if a large amount of lactose remains in the culture solution after completion of the culture, Lactose will be mixed in. For this reason, lactose is usually removed by dialysis etc. in the purification process of hyaluronic acid, but it is possible to reduce the lactose concentration in the culture solution at the end of the culture in advance while maintaining the productivity of hyaluronic acid. It is required from the viewpoint of improving efficiency and improving the quality of hyaluronic acid.

  Therefore, as a result of intensive studies conducted by the present inventor to solve the above-mentioned problems, a lactic acid bacterium belonging to Streptococcus thermophilus has a specific temperature of 37 ° C. to 40 ° C. while adjusting the culture solution to pH 6-8. The inventors have found that the production amount of hyaluronic acid can be increased efficiently by culturing in a temperature zone, and the present invention has been completed.

  That is, the present invention provides a method for producing hyaluronic acid, which comprises inoculating a lactic acid bacterium belonging to Streptococcus thermophilus into a medium and culturing at 37 to 40 ° C. under a pH of 6 to 8, and the method. Provided hyaluronic acid.

  According to the production method of the present invention, lactic acid bacteria belonging to Streptococcus thermophilus can be produced at 37 to 40 ° C. under conditions of pH 6 to 8 to produce hyaluronic acid easily and efficiently at a lower cost. It becomes possible. Since the lactic acid bacterium is a bacterium that can be used for the production of yogurt and the like, the hyaluronic acid obtained using the lactic acid bacterium is highly safe and can be incorporated into various foods, cosmetics, and pharmaceuticals.

It is a figure which shows the relationship between the culture temperature of this invention, a hyaluronic acid yield, and (mu) max.

  In the method of the present invention, lactic acid bacteria belonging to Streptococcus thermophilus are inoculated on a medium, and cultured at a temperature of 37 to 40 ° C. under a pH of 6 to 8. As a result, hyaluronic acid can be obtained which is cheaper, simple and efficient and can be blended in food and drink.

  The lactic acid bacterium belonging to Streptococcus thermophilus used in the method of the present invention (hereinafter also referred to as “lactic acid bacterium of the present invention”) is not particularly limited as long as it has the ability to produce hyaluronic acid. Specifically, Streptococcus thermophilus YIT2084 strain (FERM BP-10879) can be mentioned.

In the method of the present invention, hyaluronic acid is produced by inoculating a medium with lactic acid bacteria belonging to Streptococcus thermophilus and culturing at a temperature of 37 ° C. to 40 ° C. while adjusting the culture solution to pH 6-8. .
By adjusting and culturing so that it may become this specific temperature range of 37-40 degreeC, it becomes possible to increase the production amount of a hyaluronic acid simply.
The difference in the production amount of hyaluronic acid due to changes in the culture temperature of lactic acid bacteria belonging to Streptococcus thermophilus has not been known so far, but the optimum culture temperature of lactic acid bacteria belonging to Streptococcus thermophilus is generally around 42 ° C. (See L. De Vuyst et al., J. Appl. Microbiol., 84: 1059-1068, 1998; B. Zisu and NP Shah, J. Dairy Sci., 86: 3405-3415, 2003), At this temperature of 42 ° C., the maximum specific growth rate (μmax) is the highest. It has also been reported that the amount of metabolites produced increases with increasing μmax in the logarithmic growth phase (L. De Vuyst et al., J. Appl. Microbiol., 84: 1059-1068, 1998). However, as shown in Example 3 below, in hyaluronic acid production, there was no positive correlation between μmax in the logarithmic growth phase and the amount of hyaluronic acid produced. That is, as is apparent from FIG. 1, the growth rate of Streptococcus thermophilus is not increased under the condition of 37 to 40 ° C. compared to 42 ° C. Nevertheless, hyaluronic acid production is significantly increased compared to 42 ° C. This indicates that the production capacity of hyaluronic acid is improved in the range of 37 to 40 ° C. regardless of the growth rate, and such productivity improvement was completely unexpected. In addition, in the range of 37 to 40 ° C., no or little lactose was observed in the culture solution at the end of the culture, so that high-quality hyaluronic acid can be obtained efficiently.
Moreover, as shown in Example 1 to be described later, when the culture is carried out by adjusting to an acidic region having a pH of less than 6, the amount of hyaluronic acid produced decreases. For this reason, since the pH of a culture solution falls with the growth of the lactic acid bacteria of this invention, it is necessary to control the culture solution in culture to pH 6-8 using various pH adjusters, such as caustic soda, caustic potash, and ammonia. It is. The pH is preferably 6.0 to 7.5, more preferably 6.3 to 7.3, and more preferably 6.8 to 7.3, because the production amount of hyaluronic acid increases.

  In the method of the present invention, culture of lactic acid bacteria belonging to Streptococcus thermophilus is performed by stirring culture, aeration stirring culture, etc., except that the culture solution is adjusted to pH 6-8 and the culture temperature is adjusted to 37 ° C.-40 ° C. What is necessary is just to perform by a well-known method.

In the method of the present invention, the medium used for culturing the lactic acid bacteria of the present invention is not particularly limited, and a normal medium can be used.
For example, carbon sources such as glucose, fructose, galactose, sucrose, inorganic salts such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium sulfite, sodium thiosulfate, ammonium phosphate, soy peptide, polypeptone, yeast extract In addition to organic nutrient sources such as corn steep liquor, as well as nutrient media usually used for the growth of lactic acid bacteria to which various amino acids and vitamins are added as required, media containing animal or plant-derived milk, A milk medium can be used.

In particular, it is preferable to produce fermented milk using a medium containing a milk component in that the hyaluronic acid produced by the lactic acid bacterium of the present invention can be used as it is as a milk component that can be consumed.
In the present specification, the milk component means raw milk of animal milk such as cow milk and goat milk, heated milk, skim milk powder, whole milk powder or fresh cream; or vegetable milk such as soy milk, almond milk, coconut milk and the like. Means a product containing at least various milk proteins.

The hyaluronic acid produced by the lactic acid bacterium of the present invention (hereinafter also referred to as “the hyaluronic acid of the present invention”) may be separated and collected from the culture solution according to a normal method for separating and collecting polysaccharides. For example, after separating insoluble matter such as cells in the culture solution by filtration or centrifugation, purified hyaluronic acid can be separated from this solution using a solvent precipitating agent such as ethanol.
When a milk medium is used, it may be used as it is as a milk component containing hyaluronic acid produced by the lactic acid bacterium of the present invention, but hyaluronic acid can be fractionated and used according to a conventional method. is there.

  The hyaluronic acid of the present invention thus obtained has a molecular weight distribution different from that of conventional general hyaluronic acid. Specifically, the average molecular weight of the hyaluronic acid of the present invention is about 300,000 to 1,500,000, and further about 500,000 to 1,000,000. Moreover, the molecular weight distribution is in the range of about 100,000 to 3 million, and the maximum peak is around 1 million. Since the maximum peak of conventional general hyaluronic acid is around 2 million to 3 million, the hyaluronic acid of the present invention has a lower molecular weight than conventional general hyaluronic acid and improved skin permeability. it seems to do.

  The hyaluronic acid of the present invention obtained by the method of the present invention can be used in the form of pharmaceuticals, cosmetics, etc. as in the prior art, and is also produced by lactic acid bacteria belonging to Streptococcus thermophilus that can be used for the production of yogurt etc. Therefore, it can be administered in the form of food. For example, if it is used in the form of pharmaceuticals and nutritional supplements that promote effects such as beauty and anti-aging, it is orally administered as a solid preparation such as a capsule, granule, tablet, powder, or a liquid preparation such as a syrup. Can be administered.

  During the production of these preparations, excipients such as lactose, starch, crystalline cellulose, calcium lactate, magnesium aluminate metasilicate, and silicic anhydride; binders such as sucrose, hydroxypropylcellulose, and polyvinylpyrrolidone; carboxymethylcellulose, carboxymethylcellulose Disintegrants such as calcium; lubricants such as magnesium stearate, talc, monoglyceride and sucrose fatty acid ester, and other components acceptable as pharmaceuticals and foods can be appropriately blended.

  In the case of a general food form with the expectation of the same physiological effect, a product obtained by subjecting the hyaluronic acid obtained by the method of the present invention as it is or appropriately purified to oil, fat, tablet confectionery, fermented milk, koji, seasoning It can be added to foods and drinks such as sprinkles and manufactured by conventional methods.

When making fermented food, especially fermented milk, it can manufacture by making it ferment (cultivate) with the lactic acid bacteria which belong to the above-mentioned Streptococcus thermophilus in a fermentation raw material.
At this time, it is not always necessary to maintain the pH of the culture solution at 6 to 8 and the culture temperature at 37 ° C. to 40 ° C. When these are not retained, in the production of the fermentation product, the optimal culture temperature of the lactic acid bacteria is appropriately selected except for securing the pH conditions and culture temperature conditions of the present invention so that the amount of hyaluronic acid produced is a desired concentration. It can be set to a temperature such as pH or refrigeration temperature.
Fermented milk refers to fermented milk specified by a ministerial ordinance, milk-containing beverages such as dairy lactic acid bacteria beverages, dairy drinks containing sterilized fermented milk, and kefir Etc.
During fermentation, other bacteria such as Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus zeae, Lactobacillus zeae, Lactobacillus Lactobacillus genus bacteria such as John Sony, Lactobacillus delbrukki Subspecies Bulgaricus, Lactobacillus delbrukki Subspices delbrukki and Lactococcus lactis Subspecies lactis, Lactococcus lactis Lactococcus bacteria such as Talam, Lactococcus raffinolactis, Leuconostoc bacterium such as Leuconostoc mesenteloites, Leuconostoc lactis, et Enterococcus bacteria such as Enterococcus faecalis and Enterococcus faecium can be used.

  In addition, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium animaris and other Bifidobacterium genus bacteria, yeast and other microorganisms are used. Also good. These can also be used 1 type or in combination of 2 or more types.

  In addition, other food materials, that is, various sugars, emulsifiers, thickeners, sweeteners, acidulants, fruit juices, and the like may be appropriately mixed with these foods. Specifically, sugars such as sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose, xylose, sorbitol, xylitol, erythritol, lactitol, palatinit, reduced starch syrup, reduced maltose starch syrup, sucrose fatty acid Examples include emulsifiers such as esters, glycerin fatty acid esters, and lecithin, and thickening (stabilizing) agents such as carrageenan, guar gum, xanthan gum, pectin, and locust bean gum. In addition, various vitamins such as vitamin A and vitamin B, and minerals such as calcium, iron, manganese, and zinc can be blended.

  In addition, the hyaluronic acid obtained by the method of the present invention can be in the form of external preparations such as cosmetics such as milky lotion, cosmetic liquid, foundation, face cream, hand cream, lotion, essence, shampoo and rinse. These external preparations may be produced according to conventional methods, and the hyaluronic acid obtained by the method of the present invention is also isolated and purified from the medium, or as a milk component containing hyaluronic acid at any stage of the production. What is necessary is just to mix | blend suitably. In addition, these external preparations include components usually used in cosmetic production, for example, surfactants, oils, alcohols, thickeners, preservatives, antioxidants, chelating agents, pH adjusters, if necessary. Agents, fragrances, pigments, vitamins, amino acids and the like can be blended.

  What is necessary is just to determine suitably the compounding quantity of the hyaluronic acid obtained by the method of this invention in the said pharmaceutical, cosmetics, or foodstuffs so that the effect is acquired and problems, such as an overdose, do not arise.

  EXAMPLES Hereinafter, although an Example and a test example are given and this invention is demonstrated still in detail, this invention is not restrict | limited at all by these Examples.

Example 1 (Effect by pH adjustment)
(1) Strain and culture Streptococcus thermophilus YIT2084 strain (FERM BP-10879) was cultured according to the following method.
A 10% (w / v) skim milk (hereinafter referred to as “10% skim milk”) solution medium (1 L) was sterilized in an autoclave at 121 ° C. for 15 minutes, and this was further treated with Streptococcus thermophilus YIT2084 strain (FERM BP-10879). The starter was inoculated with 0.1% (v / v) and cultured at a culture temperature of 35 ° C. for 24 hours. In this culture, the pH of the medium is adjusted to around 7.8, 7.3, 6.8, 6.3, 5.8 or 5.3 using 8N aqueous sodium hydroxide solution, Stirring was performed at 100 rpm.
The composition of the 10% skim milk solution medium was 100 g of skim milk in 1 L of the medium. Further, a 10% skim milk solution medium was used as a starter medium.

(2) Extraction and preparation of hyaluronic acid Each culture solution (1 L) obtained above was cooled in ice, and trichloroacetic acid (hereinafter referred to as “TCA”) was added so that the final concentration was 10% (w / v). In addition, the mixture was allowed to stand at 4 ° C. for 2 hours. This was centrifuged at 18,700 G for 30 minutes, and the resulting precipitate was removed. Then, an equal amount of 99% cold ethanol was added to the supernatant, and the mixture was allowed to stand at 4 ° C. overnight. After standing, the precipitate formed by centrifuging at 18,700 G for 30 minutes is separated, dissolved by adding pure water to it, dialyzed using Spectra / Por Membrane (MWCO3500), and adjusted to pH 7 Thereafter, it was freeze-dried to obtain hyaluronic acid.

  As shown in Table 1, it was revealed that the amount of hyaluronic acid produced was higher when the pH range was adjusted to neutral conditions than acidic or alkaline conditions.

Example 2
(1) Strain and culture Streptococcus thermophilus YIT2084 strain (FERM BP-10879) was cultured according to the following method.
Sterilize 10% skim milk solution medium (1 L) in an autoclave at 121 ° C. for 15 minutes, inoculate 0.1% (v / v) starter of Streptococcus thermophilus YIT2084 strain (FERM BP-10879), and culture temperature The culture was performed at 37 ° C. for 28 hours. Cultivation at this time was carried out with stirring at a rotational speed of 100 rpm while adjusting the pH of the medium to about 6.8 using an 8N aqueous sodium hydroxide solution. Further, the culture was performed in the same manner except that the culture temperature “37 ° C.” was changed to “40 ° C.”.
The composition of the 10% skim milk solution medium was 100 g of skim milk in 1 L of the medium. Further, a 10% skim milk solution medium was used as a starter medium.

(2) Detection of hyaluronic acid Hyaluronic acid was detected by the following method using biotinylated hyaluronic acid binding protein (b-HABP, Seikagaku Corporation).
A 96-well immunoplate (Nunc) was used for the assay. Streptococcus zooepidemicus-derived sodium hyaluronate (Wako) was dissolved in 20 mM sodium carbonate buffer (pH 9.6) to a concentration of 100 μg / mL, and 200 μL was applied to the well. The inside of the well was coated with hyaluronic acid by incubating at 37 ° C. for 4 hours.
After the incubation, the plate was washed three times with PBS (PBST) containing 0.05% (v / v) Tween 20 (registered trademark). All washing was performed by this operation. Further, 200 μL of bovine serum albumin (Boehringer Mannheim) (1% (v / v)) dissolved in PBST was applied and blocked at 4 ° C. for 18 hours. After washing, 100 μL each of the culture supernatant appropriately diluted with PBST and b-HABP (0.5 μg / ml in PBST) were simultaneously applied and incubated at 37 ° C. for 1 hour.
After washing, 200 μL of ExtraAvidin alkaline phosphatase conjugate (Sigma) diluted 1/70000 with PBST was applied and incubated at 37 ° C. for 30 minutes. After washing, 4-nitrophenyl phosphate disium salt hexahydrate (Sigma) dissolved in 0.1 M glycine buffer (pH 10.4) containing 1 mM MgCl ₂ and 2 mM ZnCl ₂ (1 tablet / 20 mL) was applied in 200 mL.
The hyaluronic acid concentration contained in the culture supernatant was calculated by subtracting the absorbance immediately after application (405 nm) from the absorbance after incubation for 1 hour at 37 ° C. (405 nm). For the calibration curve, hyaluronic acid used for coating was used.

  As shown in Table 2, it was confirmed by the biotinylated hyaluronic acid binding protein that the polysaccharide in the culture supernatant was hyaluronic acid.

Example 3 (Effect by adjusting culture temperature)
(1) Strain and culture Streptococcus thermophilus YIT2084 strain (FERM BP-10879) was cultured according to the following method.
Each culture was performed in the same manner as in Example 2 (around pH 6.8) except that the culture temperature was “35 ° C., 37 ° C., 40 ° C. or 42 ° C.”.
(2) Extraction and preparation of hyaluronic acid In the same manner as in Example 1, each hyaluronic acid was obtained from each culture solution after completion of the culture.

  As shown in FIG. 1 and Table 3, the amount of hyaluronic acid produced markedly increased when cultured at 37 ° C. to 40 ° C. In addition, μmax in the logarithmic growth phase showed low values at 37 ° C. and 40 ° C., and no positive correlation with hyaluronic acid yield was observed.

In addition, under the culture conditions of culture temperatures “37 ° C.” and “40 ° C.”, the detected amount of hyaluronic acid in Example 2 and the yield of the above-mentioned hyaluronic acid were almost the same. Hyaluronic acid when cultured appears to be highly pure.
Further, the lactose concentration of the culture solution at the end of the culture was measured using an F kit (Boehringer Mannheim). As shown in Table 4, it was found that the lactose concentration was very low at 40 ° C and 42 ° C. If the lactose concentration is low, there is no possibility that lactose will remain in the purified hyaluronic acid, and it is considered that high-quality hyaluronic acid can be obtained. Therefore, setting the culture temperature to 40 ° C. is very useful not only for high yield but also for obtaining good quality hyaluronic acid.

(3) Molecular weight analysis The above hyaluronic acid was dissolved in a 50 mM sodium chloride solution so as to be 5 mg / mL, and subjected to HPLC as follows. Moreover, HPLC was also measured for hyaluronic acid standard products (sodium hyaluronate; manufactured by Wako Pure Chemical Industries, Ltd.) under the same conditions.
HPLC analysis Analyzer Waters 600
Detector Differential refractometer RI-980
Column Shodex Sugar KS-804
Column temperature 80 ℃
Elution Buffer 50 mM sodium chloride Elution rate 1.0 mL / min
The hyaluronic acid has a broad molecular weight compared to the standard hyaluronic acid, its molecular weight distribution is in the range of about 100,000 to 3 million, and its maximum peak is around 1 million. . The average molecular weight was about 500,000 to 1,000,000.

Claims (2)

A method for producing hyaluronic acid, comprising inoculating Streptococcus thermophilus strain YIT2084 (FERM BP-10879) into a medium and culturing at 37 to 40 ° C under pH 6 to 8 conditions. Method for producing hyaluronic acid according to claim 1 Symbol placement, wherein the medium is a medium containing milk components.

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