JPH0763358B2 - Lactic acid bacteria starter for silage preparation - Google Patents

Description

TECHNICAL FIELD The present invention relates to a lactic acid bacterium starter for silage preparation.

More specifically, the Lactobacillus casei C-1631 of the present invention
Alone or Streptococcus lactis C-6513
The present invention relates to a lactic acid bacterium starter for silage preparation in which

When the lactic acid bacterium starter of the present invention is applied at the time of silage preparation, it is possible to obtain a silage having a good flavor and a homogeneity which cattle prefer to eat, which is of great benefit to the dairy industry.

Prior Art In order to prepare good quality silage, it is essential to sufficiently carry out lactic acid fermentation in the initial stage to rapidly lower the pH. However, in reality, depending on the type of plant to be silage, its water content, temperature, etc., sufficient fermentation does not always occur. For this reason, it is recommended to perform stable lactic acid fermentation by artificially adding live lactic acid bacteria suitable for silage fermentation, not relying only on lactic acid bacteria adhering to plants. The lactic acid bacterium viable cells of the above have been investigated and some effects have been observed, but a sufficient lactic acid bacterium starter has not been found yet.

Problems to be solved by the invention Cows prefer to eat flavorful silage, and it is the goal of dairy farmers to prepare this flavorful silage, but at the same time, this flavorful silage is in a homogeneous state. Must be prepared in. Good silage conditions are low pH (4.2 or less). High lactic acid content. Low butyric acid content. Low ammonia content. High dry matter recovery rate. The digestive absorption rate (feeding rate) is high. Silage satisfying these various conditions is evaluated as having high flavor, high palatability and high quality.

Means for Solving the Problems The present inventors rapidly produce lactic acid to lower the pH,
By suppressing the production of butyric acid and ammonia due to abnormal fermentation, and selecting the lactic acid bacteria necessary for preparing highly flavorful silage in a homogeneous state, and conducting diligent research, newly isolated Lactobacillus Casei C-1631
(Lactobacillus casei C-1631) alone or Streptococcus lactis C-6513 (Streptococcus lact
It was found that by using two strains of is C-6513) at the same time, silage with a remarkable flavor can be prepared in a homogeneous state.

The present invention is Lactobacillus casei C-1631 alone, or
The present invention also relates to a lactic acid bacterium starter for silage preparation containing Streptococcus lactis C-6513 as an effective bacterium.

The present invention relates to Lactobacillus casei C-1631 (Lactobacillus
s casei C-1631) Streptococcus lactis C-651 (Streptococcus lactis C-651)
3) The purpose of the present invention is to provide a viable cell agent that enables stable preparation of high-quality silage by adding a viable cell agent together when preparing silage.

Two effective strains of the present invention are Lactobacillus casei C-1631 and Streptococcus lactis C-6513, which were newly isolated by the present inventors from the natural world.

Lactobacillus casei C-1631 has been deposited at the Institute of Microbial Science and Technology, Institute of Industrial Science and Technology as Microorganism Research Institute No. 9809. The mycological properties of this bacterium are as follows.

A. Morphological properties (1) Bacilli with a width of 0.3 to 0.6 μm and a length of 1.5 to 2.5 μm (2) Gram positive (3) Spore formation: none (4) Motility: none (5) Facultative anaerobic B . Growth conditions on various media (1) APT liquid media (Difco): Very good growth.

(2) Colony shape on BL agar medium (30 ° C, 3 days, anaerobic culture): Rough gray colony with orange in the center and yellowish rim around the center C. Physiological properties (1) CO from glucose ₂ production: - (2) production of catalase: - (3) reduction of nitrate resistance: - (4) liquefaction of gelatin: - (5) H ₂ S production: - (6) indole production: - (7) Aerobic growth: + (8) Growth at 15 ° C: + (9) Growth at 45 ° C: + (10) Optical activity of lactic acid: L (+) (11) Fermentability of sugar Arabinose:-Xylose: -Rhamnose:-Glucose: + Mannose: + Fructose: + Galactose: + Sucrose: + Maltose: + Cellobiose: + Lactose:-Trehalose: + Mellibiose:-Raffinose:-Starch:-Mannitol: + Sorbitol: + Esk Phosphorus: + Salicin: + Streptococcus lactis C-6513 has been deposited with the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology as Microindustry Research Institute No. 9810. The mycological properties of this bacterium are as follows. Is.

A. Morphological properties (1) Coccus with a diameter of 1.0 to 1.5 μm (2) Gram-positive (3) Spore formation: None (4) Motility: None (5) Facultative anaerobic B. Growth condition on various media (1) Trypticase Soy liquid medium (BLL): grows very well.

(2) Colony shape in BL agar medium (30 ° C, 3 days, anaerobic culture): Gray smooth colony C. Physiological properties (1) CO ₂ production from glucose:-(2) Catalase production:- (3) Growth in 0.1% methylene blue milk: + (4) Growth in 6.5% NaCl:-(5) Growth in 40% bile: + (6) Growth at pH 9.6:-(7) 60 ° C , Survival after treatment for 30 minutes:-(8) Hemolytic properties of horse blood:-(9) Liquefaction of gelatin:-(10) Hydrolysis of starch:-(11) Degradation of sodium hippurate:-(12 ) Esculin hydrolysis :-( 13) NH ₃ formation from arginine: + (14) Growth at 10 ° C: + (15) Growth at 45 ° C:-(16) Fermentability of sugar Arabinose: + Xylose : + Rhamnose:-Glucose: + Mannose: + Fructose: + Galactose: + Sucrose: + Ma Lactose: + Cellobiose: + Lactose: + Trehalose: + Meribiose:-Raffinose:-Mannitol: + Sorbitol:-Salicin: + Next, Lactobacillus casei C-16 which is an effective bacterium of the present invention.
31 and the method for culturing Streptococcus lactis C-6513 will be described.

As the medium, a medium containing a carbon source, a nitrogen source, an inorganic substance, vitamins, amino acids and the like, which is generally used for culturing microorganisms, is widely used. As the carbon source, any assimilable carbon compound may be used, and for example, glucose, sucrose, maltose or the like is used.

Any available nitrogen compound may be used as the nitrogen source, and for example, peptone, meat extract, caseinic acid hydrolyzate and the like are used. In addition, salts of phosphates, magnesium, sodium, potassium, calcium, iron, manganese, etc., vitamins, amino acids, antifoaming agents, and surfactants are used as necessary.

As the medium, either a liquid medium or a solid medium can be used. The initial pH of the medium is pH 5 to 8, preferably pH 6 to 8, the culture temperature is 20 to 42 ° C, preferably 25 to 40 ° C, and the culture time is
It is 12 hours to 3 days, preferably 15 to 24 hours.

These cultures may be carried out for each strain alone, or both strains may be mixed and carried out simultaneously.

The culture thus obtained can be used as it is or as washed isolated bacterial cells. In addition, the culture or the isolated microbial cell can be used as it is or after adding an additive, drying, or after being formulated, it can be used. The probiotic agents Lactobacillus casei C-1631 and Streptococcus lactis C-6513 of the present invention are each 1 g of silage plant.
It may be added in an amount of 10 ² to 10 ⁸ , preferably 10 ⁴ to 10 ⁷ .

Next, production examples and examples of the present invention will be shown.

Production Example 1 200 g of casein peptone, 30 g of yeast extract, 20 g of potassium phosphate 2 g, and 50 g of glucose were dissolved in 10 liters of demineralized water, and a medium adjusted to pH 7.5 with IN sodium hydroxide solution was placed in a jar fermenter. Lactobacillus casei C-1631, FERM pre-incubated with sterilization for 15 minutes, cooling
The culture solution of P-9809 was inoculated and statically cultured at 35 ° C. for 20 hours.

The culture broth thus obtained was centrifuged to collect the bacterial cells, which was lyophilized using a 10% skim milk 1% sodium glutamate solution that had been sterilized in advance as a dispersion medium, and a Lactobacillus casei C-1631 viable agent. Obtained 450 g. The viable cell count contained in this viable cell agent was 1 × 10 ¹⁰ cells / g.

Production Example 2 A medium was prepared in the same manner as in Production Example 1, pre-cultured beforehand, inoculated with a culture solution of Streptococcus lactis C-6513, FERM P-9810, and statically cultured at 30 ° C. for 18 hours.

The thus-obtained culture solution was centrifuged to collect the cells, and the cells were freeze-dried using 10% skim milk as a dispersion medium to obtain 410 g of Streptococcus lactis C-6513 viable cell agent. The viable cell count contained in this viable cell agent was 1 × 10 ¹⁰ cells / g.

Production Example 3 A medium was prepared in the same manner as in Production Example 1 and pre-cultured in advance to inoculate a culture solution of Lactobacillus casei C-1631, FERM P-9809 and Streptococcus lactis C-6513, FERM P-9810. Then, the mixture was allowed to stand at 30 ° C. for 18 hours for mixed culture.

The culture broth thus obtained was centrifuged to collect the bacterial cells, and 10% skim milk / 1% sodium glutamate solution was lyophilized as a dispersion to obtain Lactobacillus casei C-1631 and Streptococcus lactis C-. Mixed microbial agent with 6513 490g
Got The number of viable bacteria contained in this probiotic agent is 1 × 10 ¹⁰ cells / g of Lactobacillus casei C-1631, Streptococcus
Lactis C-6513 was 6 × 10 ⁹ pieces / g.

Production Example 4 To 1 part of the dried bacterial cell obtained in Production Example 1, 9 parts of bran was added and mixed well to obtain a viable cell agent. The viable cell count contained in this viable cell agent was 8 × 10 ⁸ cells / g.

Production Example 5 To 1 part of the dried cells obtained in Production Example 2, 9 parts of calcium carbonate was added and mixed well to obtain a viable cell agent. The viable cell count contained in this viable cell agent was 7 × 10 ⁸ cells / g.

Production Example 6 To 1 part of the dried bacterial cell obtained in Production Example 3, 9 parts of corn starch was added and mixed well to obtain a viable cell agent. The number of viable bacteria contained in this probiotic agent is Lactobacillus casei C-1631
8 × 10 ⁸ pieces / g, Streptococcus lactis C-6513
Was 5 × 10 ⁸ pieces / g.

Example 1 Mowed grass (orchard, reed canary, Italian, alfalfa) was dried in the sun for half a day, then shredded and mixed to obtain 700 g of the combined use group in Production Example 4 and Production Example 5. 0.5 g each of the probiotic agent was added, pressed into a plastic container with a lid, filled and aged at 30 ° C for 22 days to prepare silage, and subsequently aged at 30 ° C for 22 days,
Further, it was aged at 37 ° C for 10 days. In addition, a group in which the Lactobacillus casei C-1631 biocide obtained in Production Example 4 was added alone at the same ratio was used as a single group, and a group in which no biocide was added was used as a control group.

The results are shown in Table 1.

The pH of the sample was a filtrate of 10% (w / v%) extract (5 ° C., 24 hr.) Of shredded silage. The organic acid was quantified by gas chromatographic analysis of the above sample, and the ammonia nitrogen was quantified by the semi-micro vacuum distillation method.

As is clear from Table 1, both the pH value and the amount of lactic acid were excellent in the viable cell addition group after aging at 30 ° C for 22 days. In addition, among the probiotic agent-added groups, the combined group in which Streptococcus lactis C-6513 was further added was superior to the Lactobacillus casei C-1631-added single group. Further, a considerable deterioration was observed in the control group after aging at 37 ° C for 10 days, whereas it was hardly observed in the single group and the combined group. A small amount of butyric acid is newly produced in the single plot and the combined plot, but the combined plot shows almost no deterioration and good quality silage should be prepared even when the temperature is high. You can see that In addition, in a sensory test conducted by four panel members, the single group and the combined group were excellent in flavor and color tone under any aging condition. Among the single and combined plots, the combined plot was the best. Thus, it was possible to prepare silage of excellent quality under both aging conditions.

Example 2 The cut orchard grass was dried in the sun for half a day and then shredded, and 0.75 g of each of the probiotic agents obtained in Production Example 4 and Production Example 5 was added to 1.5 kg of the combined use group, It was pressed into a thick polyethylene bag, packed, and aged at 25 ° C for 23 days to prepare silage, and further aged at 37 ° C for 14 days. As a single section, the live bacterial agent obtained in Production Example 4 was used.
0.75 g was added and aged in the same manner as the combined use group. In addition, a control group was one in which no viable cell agent was added.

The results are shown in Table 2.

The sample preparation and analysis methods are the same as in Example 1.

As is clear from Table 2, the pH value was lower in the probiotic agent-added group under both aging conditions, and the probiotic agent-added group was superior in terms of flavor and color tone. In addition, among the probiotic agent-added groups, the combined use group in which Streptococcus lactis C-6513 was further added has a pH value of 0.1 lower than the Lactobacillus casei C-1631 single-added group, and also in flavor and color tone. It was a little better.

Example 3 The cut alfalfa was dried in the sun for half a day and then shredded into 115 kg of the living bacterial agent alone addition zone.
After adding 57.5 g of the probiotic agent obtained in step 1, the mixture was pressed into a 200-liter sealed drum and then aged for 28 days to prepare silage. Further, 57.5 g of the viable cell agent obtained in Production Example 5 was further added to the group to which the viable cell agent was added alone as a combined group, and the same preparation was carried out. The moisture content of the stuffed alfalfa is 74.9%,
The average temperature was about 20 ℃ during the aging period. In addition, a control group was one in which no viable cell agent was added.

The results are shown in Table 3.

The sample preparation and analysis methods are the same as in Example 1. In addition, the evaluation of grass silage was performed by a sensory test with a maximum score of 40, a pH of 60, and an organic acid of 100 (based on Freig's rating method), for a total of 200.

As is clear from Table 3, silage having a better quality was able to be prepared in the group containing the probiotic agent. In addition, among the live-bacterial agent-added groups, Streptococcus lactis C-65 was added more than the Lactobacillus casei C-1631-added group.
The combined use group in which 13 was added was superior.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1:46) (72) Inventor Shoji Kawabe 4-9-2 Ginza, Chuo-ku, Tokyo Livestock Association Inside the National Dairy Agricultural Cooperative Federation (72) Inventor Kenbun Iwanami 2-4-1, Ebisu Minami, Shibuya-ku, Tokyo Calpis Food Industry Co., Ltd. R & D Center (72) Inventor Yoshiyoshi Maruta Ebisu, Shibuya-ku, Tokyo Minami 2-4-1 Calpis Food Industry Co., Ltd. R & D Center (72) Inventor Kazuya Murota 2-4-1 Ebisu, Shibuya-ku, Tokyo Calpis Food Industry R & D Center (72) Inventor Hiroshi Miyazaki 2-4-1, Ebisu Minami, Shibuya-ku, Tokyo Calpis Food Industry Co., Ltd. Research & Development Center (56) Bibliography JP Akira 60-9453 (JP, A) JP Akira 61-209554 (JP, A) JP Akira 56-160949 (JP, A) JP Akira 64-10981 (JP, A)

Claims (1)

[Claims] 1. Lactobacillus casei C-1631 and Streptococcus lactis C-65 having the following mycological properties.
A lactic acid bacterium starter for silage preparation that uses 13 as an effective bacterium. (A) Mycological properties of Lactobacillus casei C-1631. (1) Gram positive (2) Spore formation:-(3) Bacillus (4) CO ₂ production from glucose:-(5) Catalase production:-(6) Nitrate reduction:-(7) Gelatin liquefaction: - (8) H ₂ S production: - (9) indole production: - (10) aerobic growth: + (11) growth at 15 ° C.: + (12) growth at 45 ° C.: + (13) Optical rotation of lactic acid: L (+) (14) Fermentability of sugar Arabinose:-Xylose:-Rhamnose:-Glucose: + Mannose: + Fructose: + Galactose: + Sucrose: + Maltose: + Cellobiose: + Lactose: -trehalose: + melibiose:-raffinose:-starch:-mannitol: + sorbitol: + esculin: + salicin: + (B) Mycological properties of Streptococcus lactis C-6513. (1) Gram positive (2) Formation of spores: - (3) CO ₂ production from aureus (4) Glucose: - (5) Production of Catalase: - (6) Growth in 0.1% methylene blue milk: + (7 ) Growth with 6.5% NaCl:-(8) Growth with 40% bile: + (9) Growth with pH 9.6:-(10) Survivability after treatment at 60 ° C for 30 minutes:-(11) Hemolytic properties of horse blood: − (12) Liquefaction of gelatin: − (13) Hydrolysis of starch: − (14) Degradation of sodium hippurate: − (15) Hydrolysis of esculin: − (16) NH from arginine Generation of ₃ : + (17) Growth at 10 ° C: + (18) Growth at 45 ° C:-(19) Fermentability of sugar Arabinose: + Xylose: + Rhamnose:-Glucose: + Mannose: + Fructose: + Galactose: + Sucrose: + Maltose: + Cellobiose: + Lactose: + Tre Loin: + melibiose: - raffinose: - mannitol: + sorbitol: - salicin: +