JPH06271415A - Novel antifungal produced by lactobacillus and its compound - Google Patents

Abstract

PURPOSE:To obtain an antimicrobial substance which has high antifungal activity and can be used against food deterioration and decomposition. CONSTITUTION:An antifungal substance which is produced by Lactobacillus helveticus K-4 (FERM-12249), namely a peptide having the following sequence: H-Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-OH and its complex.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel antibacterial substance produced by lactic acid bacteria, and more specifically, a lactic acid bacterium strain Lactobacillus
Helvetics K-4 (Lactobacillus helveticus K-
4. The present invention relates to antibacterial substances produced by Microtechnology Research Institute, Inc. 12249) and their compounding substances.

[0002]

2. Description of the Related Art Organic acids such as lactic acid, hydrogen peroxide, and low molecular weight compounds such as diacetyl are already known as antibacterial substances produced by lactic acid bacteria, but the existence of protein antibacterial substances is also known. There is. For example, Nisin produced by Lactococcus lactis
["Nature", 3913, 551, 19
44], and Lact. Cremoris (Lact. Cremor
is) production diplococcin report [Biochem. J., 3
8, 178, 1944]. However, at present, the structures of substances other than nisin have not been clarified.

Lactobacillus helveticus is a useful lactic acid bacterium used as a yogurt starter and a cheese starter. However, it has not yet been known that the fermented product produces a macromolecular antibacterial substance such as a peptide or a complex thereof according to the present invention.

[0004]

As described above, obtaining an antibacterial substance produced by lactic acid bacteria can be an important technique as an antibacterial means for preventing spoilage and spoilage of foods. From this point of view, the present inventors have conducted extensive research on proteinaceous antibacterial substances produced by lactic acid bacteria, and have purified and identified antibacterial substances in the lactic acid bacterium strain Lactobacillus helveticus K-4.
And its primary structure was successfully elucidated and the present invention was completed. Therefore, an object of the present invention is to provide an antibacterial substance produced by a novel lactic acid bacterium, a compounded substance of the antibacterial substance, and the antibacterial substance and the compounded substance thereof as means for preventing deterioration and spoilage of food.

[0005]

[Means for Solving the Problems] That is, the present invention is an antibacterial substance produced by the lactic acid bacterium strain Lactobacillus helveticus K-4, H-Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-OH. A peptide having the structure of and a complex thereof, and a compounded substance obtained by mixing these antibacterial substances are included. Incidentally, the complex means a peptide having a peptide having the above structure as a part of the structure and having an antibacterial function, and the compounding substance is dispersed for the purpose of increasing the amount of the antibacterial peptide in dextrin, starch or the like. Or a substance in which an antibacterial peptide is mixed with other food additives having antibacterial activity such as polylysine and glycine.

The antibacterial substance of the present invention can be separated by the following method, but is not limited thereto.

[0007]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited thereto. Example 1 1% of Lactobacillus helveticus K-4 was added to 10% (W / W) reduced skim milk medium sterilized at 110 ° C. for 15 minutes.
Inoculated and cultured for 24 hours and allowed to solidify. This was repeated twice and used as an inoculum, and 2% was inoculated into the same medium and cultured for 72 hours to obtain fermented milk. Then, using a Hitachi high-speed centrifuge, 8
The cells were removed from the fermented milk by centrifugation at 000 rpm for 20 min to obtain a culture supernatant. Hereinafter, the active fraction was determined by the antibacterial activity measurement method (nephelometry) shown in FIG. 1, and the purification was carried out by the antibacterial substance purification method shown in FIG. The purification conditions are shown in Tables 1 to 3.

[0008]

[Table 1]

[0009]

[Table 2]

[0010]

[Table 3]

That is, 50 ml of the culture supernatant was separated by gel filtration using Sephadex G-25 as a carrier to obtain an active fraction. For further purification, separation was carried out by cation exchange using S-Sepharose FF as a carrier, and a peak showing high antibacterial activity was obtained. When this active fraction was analyzed by SDS-PAGE, a single band was obtained. Further, when this component was subjected to reverse phase HPLC for desalting and purification, a single peak and a single band were confirmed to have high antibacterial activity. Furthermore, in order to determine the primary structure of this antibacterial substance, as a result of analysis by a peptide sequencer (Applied Biosys), the primary structure was H-Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-OH. The structure was determined. The molecular weight was 1248 daltons. The antibacterial activity in the purification process is shown in Table 4. The term "unit" used herein means 1 unit of antibacterial activity that suppresses an increase in turbidity due to the growth of test bacteria by 1%.

[0012]

[Table 4]

Example 2 Udon was prepared according to the production method and the formulation shown in Table 5, and the antibacterial substance obtained in Example 1 was added to this, and a storage test was carried out at 10 ° C. The pH was measured after uniformly mixing udon and 9 times the amount of distilled water. Regarding the number of general viable cells, the number of colonies was counted by pouring into a standard agar medium and culturing at 35 ° C. for 72 hours. The results obtained are shown in Table 6, and the addition of 1 ppm of the purified peptide showed a sufficiently high antibacterial activity.

[0014]

[Table 5]

[0015]

[Table 6] Degradation and spoilage of food differ depending on the type or form thereof, and the amount of peptide added is appropriately determined depending on the type or form.

Example 3, Comparative Examples 1 and 2 In evaluating the heat resistance, the antibacterial substance obtained in Example 1 was added to 0.01 M phosphate buffer (pH = 4.5) at 10%.
It melt | dissolved so that it might become 0 ppm, and it was set as the evaluation sample. Comparative Example 1 was prepared by inoculating 10 ³ cells / g of E. coli (IFO 1649) in a standard liquid medium. Similarly, E. col was added to standard liquid medium.
Comparative Example 2 was prepared by inoculating 10 ³ cells / g of i (IFO 1649) and adding 1% of phosphate buffer. E. standard liquid medium.
Example 3 (A) was prepared by inoculating 10 ^{3 of} coli (IFO 1649) / g and adding 1% of the evaluation sample. E. coli (IFO 1649) was inoculated into the standard liquid medium at a rate of 10 ³ / g, and 12
Example 3 (B) was prepared by adding 1% of the evaluation sample heated at 1 ° C. for 15 minutes. Each was cultured at 35 ° C. for 48 hours, diluted and added to a standard agar medium, and cultured at 35 ° C. for 72 hours, and then the number of bacteria was measured. As shown in Table 7, heat resistance was confirmed.

[0017]

[Table 7]

Example 4 In evaluating the pH characteristics of antibacterial activity, the antibacterial substance obtained in Example 1 was dissolved in water to 100 ppm and then p
H was adjusted to 3.5, 4.5, 6.5, and 8.5 to obtain evaluation samples. Add E. coli (IFO 1649) to standard liquid medium
After inoculating 0 ³ cells / g, adding 1% of each of the evaluation samples, culturing each at 35 ° C. for 48 hours, diluting and adding to standard agar medium, and culturing at 35 ° C. for 72 hours, the number of bacteria was measured. The antibacterial property was evaluated. As shown in Table 8, the results showed that the antibacterial property was observed in a wide pH range.

[0019]

[Table 8]

Example 5, Comparative Example 3 In evaluating the characteristics of the compounded substance obtained by adding polylysine to the antibacterial substance obtained in Example 1, E. c was added to a standard liquid medium.
oli (IFO 1649) was 10 ³ U / g inoculated, polylysine (manufactured by Chisso Corporation) test group was added 1% 1% solution as in Example 5 (A), polylysine 1% and antimicrobials 0. 5pp
The test section in which 1% of the m-added solution was added was designated as Example 5 (B), and the one without the compounded substance was designated as Comparative Example 3. 3 each
After culturing at 5 ° C for 48 hours, it was diluted and added to a standard agar medium, and after culturing at 35 ° C for 72 hours, the number of bacteria was measured. The results are shown in Table 9.
As shown in, it was confirmed that the antibacterial properties of food additives having antibacterial activity were enhanced.

[0021]

[Table 9]

Example 6 Peptides having the same sequence as the peptides separated and identified in Example 1 were synthesized and their homology was compared.
Peptide synthesis was carried out by the in-phase synthesis method [P. B. Merrifie
ld), "Journal of the American Chemical Society (J.Am.Chem.Soc.)" Vol.85, p149 (19
63)]. 1. Evaluation of heat resistance of synthetic products When evaluating the heat resistance of synthetic products, 0.01
It was dissolved in M phosphate buffer (pH = 4.5) at 100 ppm to obtain an evaluation sample. E. standard liquid medium.
coli (IFO 1649) control group and those 10 ³ U / g inoculated, the standard liquid media E. coli (IFO 1649) 10 ³ U / g
After inoculation and adding 1% of the evaluation sample, addition group 1, E. coli (IFO 1649) was inoculated into the standard liquid medium at 10 ³ / g, and 1% of the evaluation sample heated at 121 ° C for 15 minutes was added. Addition group 2 was added. Each was cultured at 35 ° C. for 48 hours, diluted and added to a standard agar medium, and cultured at 35 ° C. for 72 hours, and then the number of bacteria was measured. The results are as shown in Table 10.
Heat resistance was recognized.

[0023]

[Table 10]

2. PH dependence test of synthetic products In evaluating the pH characteristics of the antibacterial activity of synthetic products, the synthetic products were dissolved in water to 100 ppm and the pH was adjusted to 3.5.
The samples were adjusted to 4.5, 6.5 and 8.5 and used as evaluation samples. E. coli (IFO 1649) was added to the standard liquid medium at 10 ³ cells / g.
Inoculate, add 1% of each evaluation sample,
After culturing at ℃ for 48 hours, dilute and add to standard agar medium,
After culturing at 72 ° C for 72 hours, the number of bacteria was measured and the antibacterial property was evaluated. As a result, as shown in Table 11, antibacterial properties were observed in a wide pH range.

[0025]

[Table 11]

3. Antibacterial Effect of Synthetic Products Against Gram-Yin Positive Bacteria To evaluate the antibacterial effect of Synthetic products against Gram-negative positive bacteria, after dissolving the synthetic products in water to 100 ppm,
The pH was adjusted to 4.5 to give an evaluation sample. E. coli (IFO 1649) and S. aureus (IAM 1011) were inoculated into the standard liquid medium at 10 ³ cells / g each, and 1% of each evaluation sample was added.
After culturing at 35 ° C for 48 hours, dilute and add to standard agar medium,
After culturing at 35 ° C. for 72 hours, the number of bacteria was measured and evaluated.
As a result, as shown in Table 12, antibacterial properties were recognized against both bacterial species.

[0027]

[Table 12]

As described above, it was confirmed that the synthetic peptide has the same properties as the peptide produced by lactic acid bacteria, and that the peptide has the sequence of the above primary structure.

[0029]

The antibacterial substance of the present invention exhibits a high antibacterial activity and is effective as an antibacterial means for preventing deterioration and spoilage of food.

[Brief description of drawings]

FIG. 1 is an explanatory diagram of a method for measuring antibacterial activity.

FIG. 2 is an explanatory diagram of a method for purifying an antibacterial substance.

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[Procedure amendment]

[Submission date] December 13, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0016

[Correction method] Change

[Correction content]

Example 3, Comparative Examples 1 and 2 In evaluating the heat resistance, the antibacterial substance obtained in Example 1 was added to 0.01 M phosphate buffer ( _PH = 4.5) at 10%.
It melt | dissolved so that it might become 0 ppm, and it was set as the evaluation sample. E. coli was added to standard liquid medium. coli (JCM 1649) 10
Comparative Example 1 was prepared by inoculating ³ cells / g. Similarly, standard liquid medium was added to E. coli. E. coli (JCM 1649) 10 ³
Comparative Example 2 was prepared by inoculating co / g and adding 1% of phosphate buffer. E. coli was added to standard liquid medium. coli (JCM
1649) was inoculated with 10 ³ cells / g and the evaluation sample was 1%.
The one added was designated as Example 3 (A). E. coli was added to standard liquid medium. Example 3 (B) was prepared by inoculating 10 ³ cells / g of E. coli (JCM 1649) and adding 1% of the evaluation sample heated at 121 ° C. for 15 minutes. Each 35 ℃, 48
After culturing for a period of time, dilute and add to standard agar medium at 35 ° C for 7
After culturing for 2 hours, the number of bacteria was measured. As shown in Table 7, heat resistance was confirmed.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction content]

Example 4 In evaluating the pH characteristics of antibacterial activity, the antibacterial substance obtained in Example 1 was dissolved in water to 100 ppm,
The pH was adjusted to 3.5, 4.5, 6.5, 8.5 and used as an evaluation sample. E. coli was added to standard liquid medium. coli (JCM
1649) was inoculated at a rate of 10 ³ / g, 1% of the evaluation sample was added to each, and each was cultured at 35 ° C. for 48 hours, then diluted and added to a standard agar medium, and cultured at 35 ° C. for 72 hours to determine the number of bacteria. The measurement was performed to evaluate the antibacterial property. As shown in Table 8, the results showed that the antibacterial property was observed in a wide pH range.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction content]

Example 5, Comparative Example 3 In evaluating the characteristics of the compounded substance obtained by adding polylysine to the antibacterial substance obtained in Example 1, E.
E. coli (JCM 1649) was inoculated with 10 ³ cells / g,
A test section in which 1% of polylysine (manufactured by Chisso Corporation) was added as 1% was used as Example 5 (A), and a test section in which 1% of solution containing 1% of polylysine and 0.5 ppm of antibacterial substance was added was used as Example 5. Comparative Example 3 in which (B) is used and no compounded substance is added
And Each was cultured at 35 ° C. for 48 hours, diluted and added to a standard agar medium, cultured at 35 ° C. for 72 hours, and the number of bacteria was measured. The results, as shown in Table 9, were found to enhance the antibacterial properties of food additives having antibacterial activity.

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] 0022

[Correction method] Change

[Correction content]

Example 6 Peptides having the same sequence as the peptides separated and identified in Example 1 were synthesized and their homology was compared.
Peptide synthesis was carried out by the in-phase synthesis method [P. B. Merrifield
ifiel), “Journal of the American Chemical Society (J. Am. Chem. So.
c. ) ”Vo 1.85, p149 (1963)]. 1. Evaluation of heat resistance of synthetic products When evaluating the heat resistance of synthetic products, 0.01
It was dissolved in M phosphate buffer (pH = 4.5) at 100 ppm to obtain an evaluation sample. E. coli was added to standard liquid medium. E. coli (JCM 1649) inoculated with 10 ³ cells / g was used as a control, and E. coli (JC
M 1649) was inoculated at a rate of 10 ³ / g, and 1% of the evaluation sample was added to the addition group 1, and the standard liquid medium was added to E. col
i (JCM 1649) was inoculated with 10 ³ cells / g, 121
Addition section 2 was obtained by adding 1% of the evaluation sample heated at 15 ° C for 15 minutes. Each was cultured at 35 ° C. for 48 hours, diluted and added to a standard agar medium, and cultured at 35 ° C. for 72 hours, and then the number of bacteria was measured. As shown in Table 10, the heat resistance was confirmed.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Name of item to be corrected] 0024

[Correction method] Change

[Correction content]

2. PH Dependence Test of Synthetic Product In evaluating the pH characteristics of the antibacterial activity of the synthetic product, the synthetic product was dissolved in water to 100 ppm and the pH was adjusted to 3.
The samples were adjusted to 5, 4.5, 6.5 and 8.5 and used as evaluation samples. E. coli was added to standard liquid medium. coli (JCM 164
9) was inoculated at a rate of 10 ³ / g, 1% of each evaluation sample was added, and each was incubated at 35 ° C for 48 hours, diluted and added to a standard agar medium, and incubated at 35 ° C for 72 hours to measure the number of bacteria. And
The antibacterial property was evaluated. As shown in Table 11, the results show a wide p
Antibacterial properties were observed in the H range.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

3. Antibacterial Effect of Synthetic Products Against Gram-Yin Positive Bacteria In evaluating the antibacterial effect of Synthetic products against Gram-negative positive bacteria, the synthetic product was dissolved in water to 100 ppm and the pH was adjusted to 4.5 to obtain an evaluation sample. . E. coli was added to standard liquid medium. coli (JCM 1649), S.I. au
Reus (IAM1011) was inoculated at 10 ³ cells / g each, and 1% of the evaluation sample was added to each, and after culturing at 35 ° C. for 48 hours, diluted addition to standard agar medium was performed and culturing at 35 ° C. for 72 hours to determine the number of bacteria. The measurement was performed and evaluated. As a result, as shown in Table 12, antibacterial properties were recognized against both bacterial species.

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Claims (4)

[Claims] 1. Lactobacillus helveticus K-4
(Lactobacillus helveticus K-4, Microtechnology Research Institute
No.) produced antibacterial substances. 2. The antibacterial substance is H-Arg-Pro-Lys-His-Pro-Il.
The antibacterial substance according to claim 1, which is a peptide having a structure of e-Lys-His-Gln-OH and a complex thereof. 3. An antibacterial substance obtained by synthesizing a peptide containing the structure according to claim 2. 4. A compounded substance comprising the antibacterial substance according to any one of claims 1 to 3.