JPH0463674B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a growth promoter for lactic acid bacteria, and more particularly to a growth promoter for lactic acid bacteria made of chitosan oligosaccharide obtained by decomposing chitosan with chitosanase produced by a microorganism belonging to the genus Bacillus. The lactic acid bacteria growth promoter of the present invention can promote the growth of lactic acid bacteria by adding it, and can be used for the growth of lactic acid bacteria, and can also be used in dairy products such as cheese, butter, yogurt, and lactic acid bacteria drinks, pickles, and silage. Alternatively, it can be used for the growth of lactic acid bacteria in the use of lactic acid bacteria, such as in the production of lactic acid. [Technical Background and Description of Prior Art] Lactic acid bacteria are bacteria that produce large amounts of lactic acid by fermenting sugars. Things are known (Comprehensive food encyclopedia, edited by Yoshito Sakurai)
"Lactic acid bacteria"), lactic acid bacteria are used in many fields such as dairy products such as cheese, butter, yogurt or lactic acid drinks, pickles, silage or the production of lactic acid. So far, yeast extracts, protein hydrolysis products, fruit and vegetable extracts, etc. have been known as substances that promote the growth of lactic acid bacteria. (Miyamoto et al.: Japanese Society of Agricultural Chemistry 1986 Conference Abstracts No. 576)
page). The chitosan oligosaccharide in the present invention is a decomposition product of chitosan, but is not decomposed to D-glucosamine, which is a monosaccharide that is a constituent unit of chitosan, and has a low degree of polymerization of D-glucosamine in chitosan. It is an oligosaccharide whose constituent unit is D-glucosamine. On the other hand, chitosan is a polysaccharide obtained by deacetylating chitin contained in the shells of crustaceans such as shrimp and crabs.
Chitosan is a polysaccharide linked in a linear chain by bonds, and chitosan with a low degree of polymerization obtained by decomposing chitosan is also known. Methods for decomposing chitosan include chemical methods such as hydrolysis with hydrochloric acid, oxidative decomposition with nitrous acid, and oxidative decomposition with chlorine;
and enzyme (chitosanase) methods.
As a microorganism that produces chitosanase, Bacillus sp. R-4 [Tominaga et al.: Y. Tominaga et al.
al: Biochimica et Biophysica Acta, Vol. 410, pp. 145-155 (1957)], Penicillium islandicum (DMFenton et al.: Journal of General Microbiology)
al Journal of General Microbiology), Vol. 126, pp. 151-165 (1981)], Bacillus
sp.) 99-5 (Horiuchi: Japanese Society of Agricultural Chemistry, Abstracts of the 1981 Conference, p. 550), Streptomyces No. 6 [G.S. Price et al.: Journal of Bacteriology (JS)
Price et al: Journal of Bacteriology, Vol. 124, pp. 1574-1584 (1975)], Streptomyces griseus [A. Ohtakara et al.: Chitin, Chitosan and Related Enzymes (A. Ohtakara et al. :
Chitin, Chitosan and Related Enzymes) No.
147-160 (1985) Academic Press] and Bacillus sp. No. 7-M (Patent Application
60-120673) and that chitosan affects the growth of plant pathogenic fungi [P.S. Stoessel et al.
Phytopathologische Zeitschrift) Volume 111, Volume 82
−89 pages (1984), CRAllan et al.: Experimental Mycology (CRAllan et al.
al: Experimental Mycology) Volume 3, No. 285-
Page 287 (1979): and that chitosan decomposition products affect the inhibition of mold growth in peas [DFKendra et al. Experimental Mycology (DFKendra et al.
Mycology) Volume 8, pp. 276-281 (1984)]
It is known that chitosan and mildly decomposed products of chitosan inhibit the growth of bacteria (patent application filed in 1983).
No. 223749) and controlling plant diseases using chitosan and chitosan decomposition products (Special Application No. 223749)
61-40400) is proposed. In addition, chitosan is decomposed by chitosanase, which is produced by microorganisms belonging to the Pacillus genus and is stable in the pH range of 5 to 11, to produce chitosan oligosaccharides that do not contain the monosaccharide of D-glucosamine, but mainly have a degree of polymerization of 2 to 8. A method of manufacturing has been proposed (Japanese Patent Application No. 167427-1981). The present inventors have been conducting research on chitosan for many years, and in their research, they discovered that chitosan oligosaccharide is effective in promoting the growth of lactic acid bacteria, and based on that knowledge, they have arrived at the present invention. [Object of the Invention and Summary of the Invention] An object of the present invention is to provide a lactic acid bacteria growth promoter, and more specifically, to provide a lactic acid bacteria growth promoter that is not harmful to the human body and can be used safely. It's about doing. The present invention is a lactic acid bacteria growth promoter whose active ingredient is chitosan oligosaccharide obtained by decomposing chitosan with chitosanase produced by a microorganism belonging to the genus Bacillus. The chitosan oligosaccharide of the lactic acid bacteria growth promoter of the present invention is a chitosan oligosaccharide in which the degree of polymerization of D-glucosamine is 2 to 8. It can be obtained by decomposing it with chitosanase, which is stable in the pH range of 5 to 11, but such chitosanase is
It can be produced by -M (Feikoken Bibori No. 8139). [Specific Description of the Invention] Chitosan oligosaccharide, which is the active ingredient of the lactic acid bacteria growth promoter of the present invention, can be used regardless of its degree of deacetylation.
Preferably, those with a degree of deacetylation of 50 to 100% are used. Chitosan oligosaccharide can be used in any form, such as alone or as a salt, as long as it promotes the growth of lactic acid bacteria. Preference is given to using the salts of , nitric acid, formic acid, acetic acid, lactic acid, glutamic acid, adipic acid or ascorbic acid. Furthermore, the polymerization degree of D-glucosamine in the chitosan oligosaccharide used is in the range of 2 to 8. Chitosan oligosaccharide can be used in any amount within the range that promotes the growth of lactic acid bacteria, but it is preferably used at a concentration in the medium of 0.1 to 0.4%. Chitosan oligosaccharides can also be used alone or in compositions with solid, pulverulent or liquid inert carriers. For the chitosan oligosaccharide used in the lactic acid bacteria growth promoter of the present invention, it is preferable to use a method that does not degrade chitosan into monosaccharides. Therefore, it is preferable to decompose chitosan. Such chitosanase is produced by microorganisms belonging to the genus Bacillus, and has a pH of 5 to 11.
It is preferable to use a chitosanase that is stable in the range of , and it is even more preferable to use a chitosanase produced by Bacillus No. 7-M (Feikoken Bacteria No. 8139). Bacillus sp. No. 7-M is a bacterium that can grow in a medium containing chitin or chitosan as the sole carbon source, and was isolated from the soil of a virgin swamp in Unzen, Obama-cho, Minamitakagi-gun, Nagasaki Prefecture. This parent strain was treated with N-methyl-N'-nitroso-N-nitrosoguanidine (NTG) to induce mutations, and among the streptomycin-resistant mutants obtained, highly active chitosanase was selected. This is a mutant strain that was isolated as one capable of producing , and has been deposited with the National Institute of Microbial Technology, Ministry of International Trade and Industry as FERM P-8139. The mycological properties of Bacillus No. 7-M are shown below. A Cell morphology (1) Cell shape and size: Short bacilli, (broth and broth agar slant culture, 37℃, 24~
(72 hour culture) (2) Presence or absence of cell pleomorphism: None, (3) Presence or absence of motility: Yes (Meat juice agar semi-fluid high-layer puncture culture) (4) Presence or absence of spores: Yes, endospores and Naked spores, spherical, [Dorner's staining method and modified Witz method] (5) Gram staining: positive, [broth agar slant culture, 37°C, 18 hours, modified Hucker method] B. Growth status in each medium (1) Broth agar plate culture (37°C, 24-168 hours): Forms round, raised, milky-white colonies with thread-like peripheries. The surface of the colony is uneven, slightly shiny, and translucent. It will get better as time passes. Does not produce pigment. (2) Broth agar slant culture (37°C, 24-168 hours): Forms a milky white colony that rises in a spread shape. Colonies have convex circular ridges and are shiny.
It grows well and will spread over time. Does not produce pigment. (3) Meat juice liquid culture (37℃, 24-168 hours): No film is formed on the surface. The whole thing becomes cloudy over time. Granular sediments are formed at the bottom and gradually increase in number. (4) Meat juice gelatin puncture culture (25°C, 24-168 hours): Grows along the puncture line and liquefies. The surface and interior grow funnel-shaped and liquefy. The liquefied part becomes cloudy. (5) Litmus milk (37°C, 24-168 hours): After two days, the upper part gradually liquefied, and on the fourth day, the color completely changed and became acidic. It does not coagulate. As time passed, liquefaction progressed and it became translucent. C Physiological properties (1) Nitrate reduction: - (Nitrate broth medium, 37℃, 24-120 hours) (2) Denitrification reaction: - (Komagata et al. method, using fermentation tube, 37℃, 24-120 hours)
(120 hours) (3) MR test: + (37℃, 24-168 hours) (4) VP test (acetylmethylcarbinol formation test: + (37℃, 24-168 hours) (5) Indole formation: - (37℃, 24-168 hours) (6) Hydrogen sulfide generation: - (TSI agar method, 37℃, 24-168 hours) (7) Starch hydrolysis: + (37℃, 24-168 hours) (8) Utilization of citric acid (Koser's medium, 37℃, 24-168 hours): - (Christensen's medium, 37℃, 24-168 hours): + (9) Utilization of inorganic nitrogen source (37℃, 24 hours) ~168 hours) Nitrate: undetermined, ammonium salt: undetermined, (10) Pigment production (mannitrate/yeast extract agar slant): - [King A agar slant]: - (11) Fluorescence: None (12) Urease: + (Christensen-urea agar medium, 37℃, 24
~168 hours) (13) Oxidase: + (gravy agar, 37℃, 24-48 hours) (14) Catalase: + broth agar, 37℃, 24-48 hours) (15) Range of growth: (gravy agar, 37℃, 24-48 hours) Agar medium) Temperature: undetermined, PH: 5-10, added salt concentration: undetermined, (16) Attitude towards oxygen: aerobic (1% glucose broth high-rise agar medium, 37℃, 24
~72 hours) (17) 0-F test (Hugh-Leifson method, 37°C, D-glucose): Acid is produced fermentatively. (fermentative) (18) Presence or absence of acid and gas generation from sugars (37℃, 24-168 hours)

【table】

[Table] The above mycological properties are described in Bergey's Manual of Determinative Bacteriology.
When searching the 8th edition (1974) of Bacteriology, it was found that strain No. 7-M most likely belongs to the genus Bacillus. The enzymatic chemical properties of chitosanase produced by Bacillus No. 7-M are as shown below. (1) Action: Acts on chitosan and arbitrarily removes β from the internal chain of the molecule.
-1,4 bond is decomposed and mainly chitosan oligosaccharide (GlcN) _o (n=2 to 8) (dimer to octamer)
generate. Chitosan oligosaccharides can be separated from the chitosan decomposition solution using high performance liquid chromatography. The decomposition degree of chitosan in this decomposition solution is about 45%. It also acts on carboxymethyl cellulose (CMC), degrading it to some extent, but has no effect on chitin. (2) Action temperature range and optimal action temperature: When soluble chitosan is used as a substrate, it works up to 80°C, and the optimal action temperature is 50°C. Figure 1 shows the relationship between temperature and specific activity when reacting for 10 minutes at pH 6.0. (3) Action PH range and optimal PH: Works in the PH range of 3 to 9, and the optimal PH is PH 6.
It is. A reaction solution prepared by adding 2 ml of each pH buffer solution and 1 ml of enzyme solution to 1 ml of 1% soluble chitosan was heated at 37°C for 10 min.
Figure 2 shows the relationship between pH and specific activity of the enzyme when reacting for minutes. (4) Thermal stability: Almost stable until kept warm at 50℃ for 15 minutes.
Approximately 40% of the enzyme is removed by heating at 60°C for 15 minutes.
was completely inactivated by heating at 70°C for 15 minutes. Figure 3 shows the relationship between temperature and specific activity. (5) PH stability: The remaining enzyme activity was measured after being left in a 0.1M buffer at 30°C for 2 hours, and it was found to be stable in the PH range of 5 to 11. One of the major characteristics of chitosanase produced by Bacillus No. 7-M is that it is stable at pH 10 to 11. PH
Figure 4 shows the relationship between specific activity and specific activity. (6) Inhibitor: Chitosanase produced by Bacillus No. 7-M was treated with HgCl ₂ , PbCl ₂ , at a final concentration of 1×10 ⁻³ M.
Almost 100% due to the presence of AgNO ₃ and PCMB
was inhibited. (7) Substrate specificity: When various substrates are used and the final concentration of the substrate is 0.25%, the amount of total reducing sugars and hexosamine released after 1 hour by 1 mg of enzyme protein per 4 ml of enzyme reaction solution (mg /mg protein/hour) was measured. The results are shown in Table 1.

〔Effect of the invention〕

Chitosan oligosaccharides promote the growth of lactic acid bacteria.

[Brief explanation of the drawing]

Figure 1 is a chart showing the action temperature range and optimal action temperature range of chitosanase produced by Bacillus No. 7-M, and Figure 2 is a chart showing the action PH range and optimal action temperature range of chitosanase produced by Bacillus No. 7-M. A chart showing the optimum pH, Figure 3 is for Bacillus No.7-M
Figure 4 shows the relationship between PH and specific activity (PH stability) of chitosanase produced by Bacillus No. 7-M. Figure 5 is a diagram showing the molecular weight of chitosanase produced by Bacillus No. 7-M by SDS-polyacrylamide electrophoresis, and Figure 6 is a diagram showing the molecular weight of chitosanase produced by Bacillus No. 7-M.
FIG. 7 is a chart showing the molecular weight of chitosanase produced by gel filtration method, FIG. 7 is a chart showing the degree of polymerization distribution of chitosan oligosaccharide obtained in Reference Example 2, and FIG. It is a diagram showing the growth of Bulgaricus bacteria.

Claims (1)

[Scope of Claims] 1. Chitosan oligosaccharide is produced by a microorganism belonging to the genus Bacillus, and is obtained by decomposing chitosan with chitosanase, which is stable in the pH range of 5 to 11, and has a polymerization degree of D-glucosamine. A lactic acid bacteria growth promoter characterized in that the active ingredient is a chitosan oligosaccharide having 2 to 8. 2 The microorganism belonging to the genus Bacillus is Bacillus No.7.
-M (Feikoken Bibori No. 8139), the lactic acid bacteria growth promoter according to claim 1.