JPH01211487A - Lactic acid bacteria starter for preparing silage - Google Patents

Abstract

PURPOSE:To obtain a homogeneous silage, willingly edible by cattle and having excellent flavor, by using a specific microorganism of the genus Lactobacillus or further a microorganism of the genus Streptococcus as an effective microorganism. CONSTITUTION:A viable microbial pharmaceutical of Lactobacillus.casei C-1631 (FERM P-9809) alone or combination thereof with a viable microbial pharmaceutical Streptococcus.lactis C-6513 (FERM P-9810) in combination as a lactio acid bacteria starter is added to a material in preparing a silage.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a lactic acid bacteria starter for silage preparation.

More specifically, Lactobacillus casei C-1 of the present invention
This invention relates to a lactic acid bacteria starter for silage preparation using Streptococcus lactis C-631 alone or in combination with Streptococcus lactis C-6513.

If the lactic acid bacteria starter of the present invention is applied during silage preparation, it will be possible to obtain homogeneous silage with good flavor that cows like to eat, which will greatly benefit the dairy industry.

In order to prepare high-quality silage, it is important to carry out sufficient lactic acid fermentation at the initial stage to quickly lower the pH. However, depending on the type of plant to be used as silage, its moisture content, temperature, etc., sufficient fermentation does not always occur. It is recommended that stable lactic acid fermentation be achieved by artificially adding live lactic acid bacteria suitable for fermentation, and a number of live lactic acid bacteria have been studied for this purpose, and although some effects have been seen. Sufficient lactic acid bacteria starter has not been found yet.

What cows like to eat is flavorful silage, and the goal of dairy farmers is to prepare this flavorful silage. It must be prepared in

The present inventors selected the lactic acid bacteria necessary for preparing flavorful silage in a homogeneous state and conducted extensive research, and found that the newly isolated Lactobacillus casei C-163
1 (Lactobacillus casei C-1
631) alone or with Streptococcus lactis C-6513 (Streptococcus 1ac
It has been found that by using two strains of S. tis C-6513 at the same time, it is possible to homogeneously prepare silage with a significantly richer flavor.

The present invention provides Lactobacillus casei C-1631 alone,
or, and Streptococcus lactis C-6513
The present invention relates to a lactic acid bacteria starter for silage preparation using the effective bacteria.

The present invention relates to Lactobacillus casei C-1631 (Lac
tobacillus casai C-1631) Probiotic agent alone?

Or Streptococcus lactis C-6513 (
Streptococcus 1actis C-65
13) The purpose of the present invention is to provide a viable bacterial agent that makes it possible to stably prepare high-quality silage by adding it in combination with a viable bacterial agent during silage preparation.

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

Lactobacillus casei C-1631 has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology as FAIKEN Bacteria No. 9809, and the mycological properties of this bacterium are as follows.

A. Morphological properties (1) Width 0.3-0.6μ, length 1.5-2.5μ■
Bacillus (2) Gram-positive (3) Spore formation: None 4) Motility: None 5) Facultative anaerobic B, growth status on various media (1) APT liquid medium (Difco): Very good Grow.

(2) Colo on BL agar medium (anaerobic culture at 30°C for 3 days): - Shape: orange in the center, rough yellowish-greenish colo on the periphery: - C0 physiological properties (1) Glucose C02 production from: - (2) Production of catalase: - (3) Reducibility of nitrate: - (4) Liquefaction of gelatin: - (s) ) Production of I, S: - (6) Production of indole: - (7) Aerobic growth: + (8) Growth at 15°C: + (9) Growth at 45°C: + (10) Optical rotation of lactic acid: L (+) (11) Fermentability of sugar arabinose di Monoxylose: Monorhamnose: Monoglucose: + Mannose: + Fructose: + Galactose: + Sucrose: + Maltose: + Cellobiose: + Lactose: - Trehalose: + Melibiose 21 Raffinose: 1 Starch: 1 Mannitol: + Sorbitol: + Aesculin: + Salicin: + In addition, Streptococcus lactis C-6513 was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiology Research Institute No. 9810.
The mycological properties of this bacterium are as follows.

A, Morphological properties (1) Coccus with a diameter of 1.0 to 1.5 μ- (2) Gram-positive (3) Spore formation: None 4) Motility: None 5) Facultative anaerobic B, Various media Growth status (1) Trypticase Say liquid medium (BB
L): Grows very well. .

(2) Colo on BL agar medium (anaerobic culture at 30°C for 3 days): - Shape: gray smooth colo: - C0 physiological properties (1) C02 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 in p) 19.6: - (7) Survival after treatment at 60°C for 30 minutes :- (8) Hemolytic properties of horse blood:- (9) Liquefaction of gelatin:- (10) Hydrolysis of starch:- (11) Decomposition of sodium hippurate:- (12) Hydrolysis of esculin:- (13) ) NH2 production from arginine: +(14) 1
Growth at 0℃: + (15) Growth at 45℃: - (16) Fermentability of sugars Arabinose: + Xylose: + Rhamnose: - Glucose: + Mannose: + Fructose: + Galactose: + Sucrose: + Maltose :+ Cellobiose: + Lactose: + Trehalose: + Melibiose: - Raffinose: One Mannitol: + Sorbitol: One Salicin: + Next, Lactobacillus casei C-, which is an effective bacterium of the present invention.
1631 and Streptococcus lactis C-651
2. The culture method will be explained.

As the medium, a medium containing a carbon source, a nitrogen source, inorganic substances, vitamins, amino acids, etc., which is commonly used for culturing microorganisms, is widely used. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, maltose, etc.

Any available nitrogen compound may be used as the nitrogen source, such as peptone, meat extract, caseic acid hydrolyzate, and the like. Other salts such as phosphate, magnesium, sodium, potassium, calcium, iron, and manganese, 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 p) 15-8. Preferably the pH is 6 to 8, and the culture temperature is 20 to 42°C, preferably 25 to 40°C.
The culture time is 12 hours to 3 days, preferably 15 to 3 days.
It is 24 hours.

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

The culture thus obtained can be used as it is or as isolated bacterial cells after washing. In addition, the culture or isolated bacterial cells may be dried as they are or with additives added, or after being formulated into a preparation. 6513 each contains 102 to 101 pieces, preferably 1 piece per gram of silage plant.
What is necessary is just to add so that it may become 04-107 pieces.

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 dipotassium phosphate, and 50 g of glucose were dissolved in 10Q of demineralized water, and the culture medium whose pH was adjusted to 7.5 with IN sodium hydroxide solution was placed in a jar fermentor. 121”C
Lactobacillus casei C-1631, FERM P-980, which was cooled after sterilization for 115 minutes and pre-cultured
9 was inoculated and statically cultured at 35° C. for 20 hours.

The culture fluid thus obtained was centrifuged to collect the bacterial cells, which were freeze-dried using a previously sterilized 10% skim milk/1% sodium glutamate solution as a dispersion medium.

450 g of Lactobacillus casei C-1631 live bacterial agent was obtained. The number of viable bacteria contained in this viable bacteria agent is 1. X 10”
pieces/g.

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

The culture solution thus obtained was centrifuged to collect the bacterial cells, which were freeze-dried using 10% skim milk as a dispersion medium to obtain 410 g of Streptococcus lactis C-6513 live bacterial preparation. The number of viable bacteria contained in this viable bacteria agent was 1X10''/g.

Production Example 3 A medium was prepared in the same manner as in Production Example 1, and Lactobacillus casei C-1631, FERNP-9 was precultured.
809 and Streptococcus lactis C-6513,
Inoculate the culture solution of FERM P-9810 and incubate at 30℃ for 1
A mixed culture was carried out for 8 hours.

The culture fluid thus obtained was centrifuged to collect bacterial cells, which were freeze-dried using 10% skim milk and 1% sodium glutamate solution as a dispersion medium.
490 g of a mixed viable bacterial agent of Streptococcus lactis C-6513 and Streptococcus lactis C-6513 was obtained. The number of viable bacteria contained in this viable bacteria agent is 1 x 101 Lactobacillus casei C-1631.
″ pieces/g, Streptococcus lactis C-6513
was 6×10′ pieces/g.

Production Example 4 To 1 part of the dried bacterial cells obtained in Production Example 1, 9 parts of bran was added and mixed well to obtain a viable bacterial agent.

The number of viable bacteria contained in this viable bacteria agent was 8×10' cells/g.

Production Example 5 9 parts of calcium carbonate was added to 1 part of the dried bacterial cells obtained in Production Example 2, and the mixture was thoroughly mixed to obtain a viable bacterial agent. The number of viable bacteria contained in this viable bacteria agent was 7XIO''/g.

Production Example 6 To 1 part of the dried bacterial cells obtained in Production Example 3, 9 parts of cornstarch was added and mixed well to obtain a viable bacterial agent. The number of viable bacteria contained in this viable bacteria agent is Lactobacillus casei C-1
The amount of Streptococcus lactis C-631 was 8×10′/g, and the amount of Streptococcus lactis C-6513 was 5×IO”/g.

Example 1 After drying cut grasses (Orchard, Reed Canary, Italian, Alfalfa) in the sun for half a day, they are shredded and mixed, and 700g of the grass is mixed with Production Example 4.
Then, 0.5 g of the viable bacterial agent obtained in Production Example 5 was added, pressed into a plastic container with a lid, and aged at 30°C for 22 days to prepare silage, and then aged at 30°C for 22 days. After that, it was further aged at 37°C for 10 days. In addition, Lactobacillus obtained in Production Example 4 in the same proportion
A plot to which the viable bacterial agent of C. casei C-1631 was added alone was used as an independent plot, and a plot to which no viable bacterial agent was added was defined as a control plot.

The results are shown in Table 1.

In addition, pH is 10% (v/v%) of shredded silage.
The filtrate of the extract (5°C, 24 hours) was used as a sample. The organic acid was determined by gas chromatographic analysis of the above sample.

30℃ 22 days Single section 4.4 3.1
0 further 37℃, 10 days Single section 4.4
2.9 0.1 As is clear from Table 1, 30
When aged for 22 days at ℃, the viable bacteria added group was superior in both the P)l value and the amount of lactic acid. Furthermore, among the groups to which viable bacteria were added, the combined group in which Streptococcus lactis C-6513 was further added was superior to the group in which Lactobacillus casei C-1631 was added alone. Furthermore, when aged at 37°C for 10 days, considerable deterioration was observed in the control group, whereas in the single group.

It was hardly observed in the combined use area. Among the single plot and combination plot, a small amount of new butyric acid production was observed in the single plot, while there was almost no deterioration in the combination plot.
It can be seen that high quality silage can be prepared even during periods of high temperature. In addition, in a sensory test conducted by four panel members, under all aging conditions, both the single and combined varieties were superior in terms of flavor and color. Furthermore, among the single and combination plots, the combination plot was the best. In this way, silage of excellent quality could be prepared under both aging conditions.

Example 2 Cutted orchard grass was dried in the sun for half a day and then shredded, and 0.75 g each of the viable bacteria obtained in Production Example 4 and Production Example 5 was added to 1.5 kg of it as a combined plot. Then, press it into a thick polyethylene bag and stuff it 25
Silage was prepared by aging for 23 days at
It was aged for 14 days at ℃. As a single group, 0.75 g of the probiotic agent obtained in Production Example 4 was added, and the mixture was aged in the same manner as the combined group. In addition, a control group was one in which no probiotics were added at all.

The results are shown in Table 2.

Note that the sample preparation and analysis methods were the same as in Example 1.

25℃ 23 days Single section 5.2 Further 37℃, 1
4th Single Ward 5.0 As is clear from Table 2, 2
Under the two ripening conditions, the P)I value was lower in the bacteria-added area,
Also, in terms of flavor and color, the group with the addition of viable bacteria was superior. Also.

Lactobacillus casei C-1 in the live bacteria addition area
The pH value was 0 in the combined area where Streptococcus lactis C-6513 was added than in the area where 631 was added alone.
．． 1 was lower, and the flavor and color tone were also slightly better.

Example 3 Harvested alfalfa was dried in the sun for half a day and then shredded, and 57.5 g of the viable bacterial agent obtained in Production Example 4 was added to 115 kg of the harvested alfalfa, and then 200
Silage was prepared by pressing and stuffing into a Q-capacity sealed drum and aging for 28 days. In addition, as a combined area, 57.5 g of the viable bacterial agent obtained in Production Example 5 was added to the area where the viable bacterial agent was added alone.
and prepared in the same manner.

The moisture content of the stuffed alfalfa was 74.9%, and the average temperature during the ripening period was about 20°C. In addition, a control group was one in which no probiotics were added at all.

The results are shown in Table 3.

Note that the sample preparation and analysis methods were the same as in Example 1. In addition, grass silage was evaluated using a sensory test of 40 points, pH of 60 points, and organic acid of 100 points (according to Frieg's scoring method), for a total of 200 points.As is clear from Table 3, viable bacteria It was possible to prepare silage of superior quality in the area where the additive was added. In addition, Lactobacillus casei C-163
The combination plot in which Streptococcus lactis C-6513 was further added was superior to the single plot in which Streptococcus lactis C-6513 was added.

Agent Patent Attorney 1) Parent Male

Claims (1)

[Scope of Claims] 1. A lactic acid bacteria starter for silage preparation containing Lactobacillus casei C-1631 having the following mycological properties as an effective bacterium. (A) Mycological properties of Lactobacillus casei C-1631. (1) Gram positive (2) Formation of spores: - (3) Bacillus (4) Production of CO_2 from glucose: - (5) Production of catalase: - (6) Reducibility of nitrate: - (7) Liquefaction of gelatin :- (8) H_2S 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: + Aesculin: + Salicin: + 2, Lactobacillus casei C-1631 and Streftococcus lactis C-6 having the following mycological properties
A lactic acid bacteria starter for silage preparation containing 513 as an effective bacteria. (A) Mycological properties of Lactobacillus casei C-1631. (1) Gram positive (2) Formation of spores: - (3) Bacillus (4) Production of CO_2 from glucose: - (5) Production of catalase: - (6) Reducibility of nitrate: - (7) Liquefaction of gelatin :- (8) H_2S 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: + Aesculin: + Salicin: + (B) Mycological properties of Streftococcus lactis C-6513. (1) Gram positive (2) Spore formation: - (3) Cocci (4) CO_2 production from glucose: - (5) Catalase production: - (6) Growth in 0.1% methylene blue milk: + ( 7) Growth in 6.5% NaCl: - (8) Growth in 40% bile: + (9) Growth in pH 9.6: - (10) Survival in treatment at 60°C for 30 minutes: - (11) Hemolytic properties of horse blood: - (12) Liquefaction of gelatin: - (13) Hydrolysis of starch: - (14) Decomposition of sodium hippurate: - (15) Hydrolysis of esculin: - (16) Arginine NH_2 production from: + (17) Growth at 10°C: + (18) Growth at 45°C: - (19) Fermentability of sugars Arabinose: + Xylose: + Rhamnose: - Glucose: + Mannose: + Fructose: + Galactose: + Sucrose: + Maltose: + Cellobiose: + Lactose: + Trehalose: + Melibiose: - Raffinose: - Mannitol: + Sorbitol: - Salicin: +