METHOD FOR PRODUCTION OF LACTOBACILLUS PLANTARUM

Abstract

This application provides a method for culturing Lactobacillus plantarum, which comprises culturing Lactobacillus plantarum in a medium comprising molasses, a yeast extract and sucrose at a temperature from 32° C. to 37° C.

Claims

1. A method for production of Lactobacillus plantarum, comprising a step of culturing Lactobacillus plantarum in a medium comprising molasses, a yeast extract, and sucrose, at a temperature from 32° C. to 37° C.

2. The method according to claim 1, wherein the Lactobacillus plantarum is a Lactobacillus plantarum strain (International Accession No. NITE BP-03418).

3. The method according to claim 1, wherein in the medium, (i) the molasses is present at a concentration in a range from 6.0% (w/w) to 13% (w/w) or more in terms of total sugar content, (ii) the yeast extract is present at a concentration in a range from 0.50% (w/w) to 1.2% (w/w) or more in terms of total nitrogen, and (iii) the sucrose is present at a concentration in a range from 3.0% (w/w) to 10% (w/w), wherein the medium during neutralization culture has a pH in a range from 5.5 to 6.5, and wherein a culture time is 16 hours or longer.

4. The method according to claim 3, wherein the molasses is present at a concentration in a range from 6.7% (w/w) to 10% (w/w) or more in terms of total sugar content.

5. The method according to claim 3, wherein the yeast extract is present at a concentration in a range from 0.54% (w/w) to 1.0% (w/w) or more in terms of total nitrogen.

6. The method according to claim 3, wherein the concentration of the sucrose is from 4.0% (w/w) to 8.0% (w/w).

7. The method according to claim 3, wherein the pH of the medium during neutralization culture is from 5.8 to 5.9.

8. The method according to any one of claims 1 to 3, wherein the temperature is 34° C.

9. The method according to claim 3, wherein the culture time is from 20 hours to 30 hours.

10. The method according to any one of claims 1 to 3, wherein the medium further comprises a trace element.

11. The method according to claim 10, wherein the trace element comprises magnesium.

12. The method according to any one of claims 1 to 3, wherein the medium further comprises vitamins.

13. The method according to any one of claims 1 to 3, further comprising a step of collecting a culture product comprising the Lactobacillus plantarum from a culture solution after the culture.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows the relationship between the culture time and the cell count when the lactic acid bacterial strain of the present invention is cultured under conditions in Table 13 (Example).

DETAILED DESCRIPTION

[0021] The present invention will be described in more detail.

[0022] The present invention provides a method for production of Lactobacillus plantarum comprising a step of culturing Lactobacillus plantarum in a medium comprising molasses, a yeast extract and sucrose at a temperature from 32° C. to 37° C.

[0023] The above Lactobacillus plantarum may be any strain. Preferably, Lactobacillus plantarum is a Lactobacillus plantarum strain (International Accession No. NITE BP-03418).

[0024] This strain was internationally deposited at an international depositary institution under the terms of the Budapest Treaty, the NITE (National Institute of Technology and Evaluation) Patent Microorganism Depositary (#122, 2-5-8, Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan), on Feb. 19, 2021. The present inventors have demonstrated that this strain is useful in prevention or suppression (or improvement), etc., of necrotic enteritis in chickens.

[0025] The culture conditions for lactic acid bacteria belonging to the genus Lactobacillus should be optimized for each of the bacterial species, preferably, strains, since bacterial species and strains differ in their auxotrophic requirements.

[0026] In general, examples of essential nutrients for culturing lactic acid bacteria include carbon sources, nitrogen sources, lipids, and trace elements. However, when the culture product of Lactobacillus plantarum is utilized for birds such as chickens, a medium component derived from an animal (for example, a meat extract) cannot be used.

[0027] Under such circumstances, in the method of the present invention, molasses and sucrose are appropriate as carbon sources and a yeast extract is appropriate as a nitrogen source in a medium. The use of molasses provides an advantage that the yield (cell count; for example, about 4.0×10.sup.11 or more cells/g) and the medium cost can be significantly improved.

[0028] Specific examples of the culture conditions are such that, in a medium, the compounding amount of molasses (for example, total sugar content of about 50% (w/w) to 80% (w/w)) is from about 9% (w/w) to 20% (w/w) or more, preferably about 12% (w/w) to 16% (w/w) or more, and the total sugar content is from about 4.5% (w/w) to about 16% (w/w) or more, preferably about 6.0% (w/w) to 13% (w/w) or more, the compounding amount of a yeast extract is from 0.50% (w/w) to 1.2% (w/w) or more in terms of total nitrogen, and the concentration of sucrose is from 3.0% (w/w) to 10% (w/w), and the pH of the medium in neutralization culture is from 5.5 to 6.5 and the culture time is 16 hours or longer.

[0029] Preferably, the compounding amount of molasses is from 6.7% (w/w) to 10% (w/w) or more in terms of total sugar content, the compounding amount of a yeast extract is from 0.54% (w/w) to 1.0% (w/w) or more in terms of total nitrogen, and the concentration of sucrose is from 4.0% (w/w) to 8.0% (w/w).

[0030] Further, the above medium may also contain a surfactant such as polysorbate (POLYSORBATE)™, e.g., polysorbate 80, or Tween and the like. The medium may also contain as other medium components, sodium acetate, trace elements, for example, metal salts of magnesium, manganese, zinc and the like (for example, magnesium sulfate, manganese sulfate, and zinc chloride), and others, vitamins (for example, thiamine and biotin).

[0031] Preferably, the medium contains at least polysorbate 80 in an amount of 0.3% (w/w), sodium acetate trihydrate in an amount of 0.5% (w/w), magnesium sulfateheptahydrate in an amount of 0.1% (w/w).

[0032] Regarding trace elements, a metal component(s) contained in molasses is thought to effectively act on the culturing of Lactobacillus plantarum. The reason for this is that if trace elements and vitamins are deficient, bacterial growth can be suppressed.

[0033] At the time of culturing, the pH of the medium will become acidic. Hence, an aqueous sodium hydroxide (NaOH) solution is preferably added to maintain the pH at from 5.8 to 5.9.

[0034] The culture temperature is from 28° C. to 40° C., and the preferable culture temperature is 34° C. The culture time is from 20 to 30 hours.

[0035] More preferable culture conditions are also shown in Table 13 shown later.

[0036] Culturing is performed under aerobic conditions and can be performed while stirring and maintaining the temperature and the pH at fixed levels.

[0037] Inoculation with Lactobacillus plantarum can be performed by introducing it in a proportion of, for example, 2%(v/v), into the above medium in an incubator.

[0038] During culturing, the time point of completion of culturing (for example, 20 to 30 hours) may be determined by monitoring sodium hydroxide consumption, which is proportional to lactic acid bacterial growth, and sampling the culture solution as needed over time to determine a cell count.

[0039] After completion of culturing, solid-liquid separation is performed by centrifugation to collect a fraction containing Lactobacillus plantarum as a culture product, and then the culture product is washed with water and a buffer as necessary. Furthermore, the culture product fraction can be subjected to a dry step to produce a final product. Drying can be performed by a method such as vacuum drying, lyophilization, and spray drying. With such a technique, for example, 4.0×10.sup.11 or more cells/g of the cells of Lactobacillus plantarum with a water content of less than 10% can be obtained.

[0040] If further needed, dead cells of Lactobacillus plantarum may also be prepared. Dead cells can be obtained by subjecting viable cells to physical or chemical treatment such as heat treatment, formalin treatment, acid treatment, gamma ray sterilization, and disruption treatment (using a homogenizer, a blender, glass spheres, ultrasonic wave or the like).

EXAMPLES

[0041] The present invention will be further described in detail with the following Examples, but the scope of the present invention is not limited by the Examples.

Example 1

[0042] <Determining Optimal Conditions for Culturing Lactobacillus plantarum Strain>

(1) Culture Method

[0043] In an incubator of a fermentor (Type: Desktop, Marubishi Bioengineering Co., Ltd., Tokyo, Japan), the Lactobacillus plantarum strain (International Accession No. NITE BP-03418) was cultured using culture conditions of different medium compositions, pH, temperatures, and times set forth below.

(2) The Presence or the Absence of Molasses

[0044] Effects of the presence or the absence of molasses and the contents of additives on cell yield (based on NaOH consumption) are shown in Table 1, Table 2 and Table 3.

TABLE-US-00001 TABLE 1 Medium composition Low High concen- concen- No No tration tration Medium molasses molasses molasses molasses component (w/w, %) (w/w/, %) (w/w, %) (w/w, %) Molasses 0.0 0.0 6.0 12 Yeast extract 8.1 10 8.1 8.1 Sucrose 8.5 8.5 8.5 8.5 POLYSORBATE ® 0.3 0.3 0.3 0.3 80 CH.sub.3COONa•3H.sub.2O 0.5 0.5 0.5 0.5 MgSO.sub.4•7H.sub.2O 0.1 0.1 0.1 0.1 Water 83 83 81 77

TABLE-US-00002 TABLE 2 Effects of the presence or the absence of molasses and the amount of molasses added on cell yield (NaOH consumption) Total nitrogen NaOH consumption Medium component (%) (g) No molasses 1 0.84 254 No molasses 2 1.20 256 Low concentration 0.84 313 molasses High concentration 0.84 321 molasses

[0045] As shown in Table 2, addition of molasses resulted in a sharp increase in NaOH consumption. It was considered that a trace component in molasses contributed to the yield.

TABLE-US-00003 TABLE 3 Effects of the amount of molasses added on cell yield (NaOH consumption) Molasses Total nitrogen NaOH consumption (%) (%)* (g) 6.0 0.84 304 9.3 0.84 313 12 0.84 321 14 0.84 306 *Total nitrogen was fixed at 0.84% to conduct test.

[0046] As shown in Table 3, the maximum amount (%) of molasses added is 12%.

[0047] In old production methods, molasses is not used as a medium component. However, in the production methods of this invention, addition of molasses resulted in increased cell counts (yields) (Table 4).

TABLE-US-00004 TABLE 4 Comparison of cell count based on the presence or the absence of molasses Yielded cell Medium NaOH Total count amount consumption* cell count (cells/mL) (kg) (kg) (cells/batch) Old 6.65 × 10.sup.9  2.3 0.2067 1.67 × 10.sup.13 production method 1 Old 7.25 × 10.sup.9  2.3 0.1972 1.81 × 10.sup.13 production method 2 Method 1 of 2.03 × 10.sup.10 2.3 0.3056 5.29 × 10.sup.13 this invention Method 2 of 1.67 × 10.sup.10 2.3 0.3147 4.37 × 10.sup.13 this invention *NaOH consumption: Can be used for evaluation of cell amount at the time of neutralization culture. Yields are compared based on NaOH consumption and cell count in neutralization culture.
The test results in the production line are as follows (Table 5).

TABLE-US-00005 TABLE 5 Comparison of cell count in production line Amount Total cell Dry cell Dry cell added count/batch powder concentration Method (kg) (cells) (kg) (cells/g) Old production 2800 1.89 × 10.sup.16 34.7 5.44 × 10.sup.11 method (measured value) Method of this 2800 5.20 × 10.sup.16 53.5 9.92 × 10.sup.11 invention (measured value)

(3) Amount of Yeast Extract Added

[0048] With the medium composition shown in Table 6, a culture temperature of 34° C., stirring at 100 rpm, a medium capacity of 2.3 kg, and pH 5.9, culturing was performed under conditions of neutralization culture and the culture time of 27 hours.

TABLE-US-00006 TABLE 6 Medium composition Low amount Moderate amount High amount added added added Medium component (w/w, %) (w/w, %) (w/w, %) Yeast extract 7.0 10 12 Sucrose 8.5 8.5 8.5 POLYSORBATE ® 80 0.3 0.3 0.3 CH.sub.3COONa•3H.sub.2O 0.5 0.5 0.5 MgSO.sub.4•7H.sub.2O 0.1 0.1 0.1 Water 84 81 79

[0049] The results are shown in Table 7.

TABLE-US-00007 TABLE 7 Effects of the amount of yeast extract added on cell yield (NaOH consumption) Yeast extract Total nitrogen NaOH consumption (w/w, %) (%) (g) 7.0 0.84 239 10 1.20 256 12 1.44 252

[0050] When the amount of the carbon source was sufficient and a yeast extract supplied a nitrogen source and the trace amounts of minerals, the total nitrogen was 1.2% and the growth of the lactic acid bacterial strain of this invention reached a plateau. The results indicate that the trace components may be a growth rate-limiting factor.

[0051] The effects on the growth of the lactic acid bacterial strain of this invention were examined when the concentration of molasses was fixed at 12% and the amount of a yeast extract added was decreased. The results are shown in Table 8.

TABLE-US-00008 TABLE 8 Effects of the amount of yeast extract added on cell yield (NaOH consumption) Total nitrogen NaOH consumption Yeast extract (%) (g) High amount added 0.84 323 Moderate amount added 0.54 302 Low amount added 0.24 229

[0052] Table 8 shows that since a significant improvement in yield cannot be expected even when the total nitrogen exceeds 0.54%, efficiency is excellent with the total nitrogen of a yeast extract of 0.54%. However, since molasses is a natural raw material, the composition of micronutrients may vary depending on lots. Since the mineral composition of molasses changes with the season and other factors, the amount (%) of molasses added should be increased by about 2%. This means that the amount of Molasses added to achieve stable production is 14%.

(4) Effects of the Amount of Sucrose Added

[0053] The amount of sucrose needed was examined by fixing the amount (%) of molasses added at 14%. The results are shown in Table 9.

TABLE-US-00009 TABLE 9 Effects of the presence or the absence of sucrose and the amount of sucrose added on cell yield (NaOH consumption) Sucrose NaOH consumption Cell count (w/w, %) (g) (cells/mL) 0 291 1.21 × 10.sup.10 4 327 1.79 × 10.sup.10 8 330 1.78 × 10.sup.10

[0054] Table 9 shows that the amount (%) of sucrose added of 4% is appropriate.

(5) Effects of Temperatures

[0055] The effects of culture temperatures of 28° C., 34° C., and 40° C. on the growth of the lactic acid bacterial strain of this invention are examined. The results are shown in Table 10.

TABLE-US-00010 TABLE 10 Effects of temperature on cell yield (NaOH consumption) Culture temperature NaOH consumption (° C.) (g) 28 88.72 34 114.0 40 94.81

[0056] The culture temperature of 34° C. is appropriate.

(6) Effects of pH

[0057] The effects of pH 5.0, 5.8, and 6.5 in neutralization culture on the growth of the lactic acid bacterial strain of this invention were examined. The results are shown in Table 11.

TABLE-US-00011 TABLE 11 Effects of pH on cell yield (NaOH consumption) NaOH consumption pH (g) 5.0 98.66 5.8 176.7 6.5 148.1

[0058] As shown in Table 11, the optimal pH for neutralization culture is around 5.8.

(7) Effects Due to Culture Time

[0059] The relationship between culture time and cell count was examined when the lactic acid bacterial strain of this invention was cultured under the conditions in Table 13 (described below). As a result, it was revealed that even 20 hours or longer of culturing did not result in a remarkable change in cell count (Table 12, FIG. 1).

TABLE-US-00012 TABLE 12 Effects of culture time on cell count Culture time Cell count (hr) (cells/mL) 20 1.55 × 10.sup.10 23.5 1.54 × 10.sup.10 27.2 1.56 × 10.sup.10

(8) Optimal Conditions

[0060] Based on the above results, optimal conditions are shown in Table 13.

TABLE-US-00013 TABLE 13 Optimal conditions of medium components Optimal conditions Medium component (w/w, %) Molasses 14*   Yeast extract  5.19 (Total nitrogen)  (0.54) Sucrose 4.0 POLYSORBATE ® 80 0.3 CH.sub.3COONa•3H.sub.2O 0.5 MgSO.sub.4•7H.sub.2O 0.1 Water 76   *Total sugar content of 6.7%(w/w)

[0061] Furthermore, the optimal culture temperature, the optimal pHs and the optimal culture time were as follows.

[0062] The optimal culture temperature is 34° C.

[0063] Regarding the optimal pH, neutralization culture is performed at a pH from 5.8 to 5.9.

[0064] The optimal culture time is 20 hours or longer.

INDUSTRIAL APPLICABILITY

[0065] According to this invention, optimal culture conditions for one of lactic acid bacteria, Lactobacillus plantarum, were determined, and thus the yield of the above lactic acid bacteria can be efficiently increased in the production of the lactic acid bacteria.