Homo / heterozygous lactic acid bacteria as well as breeding method and application thereof

Abstract

The invention discloses homozygous / heterozygous lactic acid bacteria as well as a breeding method and application thereof, and relates to the technical field of microorganisms. The homozygous lactic acid bacteria are named as pediococcus pentosaceus M46, the heterozygous lactic acid bacteria are named as lactobacillus fermentum 1761, and the homozygous lactic acid bacteria and the heterozygous lactic acid bacteria are preserved in the Guangdong Microbial Culture Collection Center on March 28, 2025, and the homozygous lactic acid bacteria and the heterozygous lactic acid bacteria are preserved in the Guangdong Microbial Culture Collection Center on March 28, 2025. The homozygous / heterozygous lactic acid bacteria are bred through heavy ion beam 12C6 + irradiation mutation, the lactic acid yield of the homozygous / heterozygous lactic acid bacteria is remarkably increased compared with that of an original strain, and an efficient strain resource is provided for rapid acidification of silage through the high-lactic-acid-yield characteristic.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically to a homo / hetero-fermentative lactic acid bacterium, a breeding method thereof, and an application thereof. Background Art

[0002] At present, as an effective feed storage method, silage technology creates an anaerobic environment to convert straw-like plant substances into active roughage, which can not only improve the value of straw itself but also increase the utilization rate of crops. As the dominant microorganisms in the silage process, lactic acid bacteria can utilize the sugars in plant cells for fermentation to produce organic acids such as lactic acid and acetic acid. These acidic substances can lower the pH of the silage and inhibit the reproduction of harmful microorganisms, further protecting the nutritional components in the straw. Therefore, the characteristics of lactic acid bacteria have an important impact on the quality of silage feed and its long-term preservation. After mutagenesis and breeding, lactic acid bacteria have stronger metabolic ability, faster reproduction speed, and higher acid production ability, which not only provides high-quality strain resources for silage feed but also extends the shelf life to solve the seasonal gap problem of silage feed, providing high-quality feed sources for ruminants such as cattle and sheep. Summary of the Invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a homo / hetero-fermentative lactic acid bacterium, a breeding method thereof, and an application thereof. By irradiating Pediococcus pentosaceus and Lactobacillus fermentum with heavy ions for mutagenesis, the technical problem of relatively low lactic acid production ability of Pediococcus pentosaceus and Lactobacillus fermentum is solved, providing high-quality strain resources for later application to silage fermentation.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A homo / hetero-fermentative lactic acid bacterium, wherein the homo-fermentative lactic acid bacterium is named Pediococcus pentosaceus ( Pediococcus pentosaceus ) M46, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on March 28, 2025, with the deposit number GDMCC NO: 66069, and the deposit address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou; the hetero-fermentative lactic acid bacterium is named Lactobacillus fermentum ( Limosilactobacillus fermentum ) 1761, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on March 28, 2025, with the deposit number GDMCC NO: 66068, and the deposit address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0005] Furthermore, the homo-fermentative lactic acid bacterium is obtained by heavy ion mutagenesis and breeding using Pediococcus pentosaceus ( Pediococcus pentosaceus ) as the starting strain; the hetero-fermentative lactic acid bacterium is obtained by heavy ion mutagenesis and breeding using Lactobacillus fermentum ( Lactobacillus fermentum) was obtained by heavy ion mutagenesis breeding of the starting bacteria.

[0006] Furthermore, the present invention also provides an application of the above-mentioned homo / heterofermentative lactic acid bacteria in the preparation of silage feed. Pediococcus pentosaceus ( Pediococcus pentosaceus ) M46 and Lactobacillus fermentum ( Limosilactobacillus fermentum ) 1761 are mixed and then inoculated into the silage raw materials, and fermented at room temperature in the dark for 28 - 32 days.

[0007] Further, the ratio of the viable cell numbers of Pediococcus pentosaceus ( Pediococcus pentosaceus ) M46 and Lactobacillus fermentum ( Limosilactobacillus fermentum ) 1761 is 1:1, and the total viable cell number inoculated into the silage raw materials is 1×10 6 CFU / g.

[0008] Compared with the prior art, the present invention has the following beneficial effects: (1) In the present invention, Pediococcus pentosaceus ( 12 C 6＋ ) M46 and Lactobacillus fermentum ( Pediococcus pentosaceus ) 1761 are selected by heavy ion beam Limosilactobacillus fermentum irradiation mutagenesis, and their lactic acid production is significantly improved compared with the original strains. Among them, the lactic acid content of Pediococcus pentosaceus M46 is increased from 7.9 g / L to 22.2 g / L, and the increase rate is as high as 181%; the lactic acid content of Lactobacillus fermentum 1761 is increased from 13.6 g / L to 15.8 g / L, and the increase rate is 16.18%. This high lactic acid production characteristic provides an efficient strain resource for the rapid acidification of silage feed, and can extend the shelf life of silage feed, making the silage feed not restricted by seasons.

[0009] (2) By selecting and breeding the strains through the selection method provided by the present invention, after continuous 9 generations of subculture, the lactic acid production does not show significant fluctuations, indicating that the acid production performance of the selected strains is stable and heritable. This characteristic ensures the long-term reliability of the strains in industrial production, avoids the need for frequent rejuvenation, and can reduce the application cost.

[0010] (3) When the present invention applies Pediococcus pentosaceus ( Pediococcus pentosaceus ) M46 and Lactobacillus fermentum ( Limosilactobacillus fermentum ) 1761 in a 1:1 mass ratio to oat straw silage, the lactic acid content reaches 16.4 g / kg, which is 11.11 g / kg higher than that of natural fermentation (CK treatment). The synergistic effect of homofermentative and heterofermentative lactic acid bacteria not only accelerates the pH drop, but also improves the flavor and palatability of the feed, provides a better feed source for ruminants, and solves the problem of seasonal feed shortage at the same time. Description of the Drawings

[0011] Figure 1 It is the operation step diagram of the present invention; Figure 2 They are the growth curve and acid reduction curve of the original Pediococcus pentosaceus; Figure 3 They are the growth curve and acid reduction curve of the original Lactobacillus fermentum; Figure 4 They are the lactic acid content of the secondary screening of Pediococcus pentosaceus; Figure 5 They are the lactic acid content of the secondary screening of Lactobacillus fermentum; Figure 6 They are the growth curve and acid reduction curve of Pediococcus pentosaceus M46; Figure 7 They are the growth curve and acid reduction curve of Lactobacillus fermentum 17 - 25 - 61; Figure 8 They are the effects of different strains on the lactic acid content of oat straw silage. Specific embodiments

[0012] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The embodiments of the present invention include but are not limited to the following examples.

[0013] Example 1 The culture media used in this example are as follows: (1) MRS screening medium: Take 10 g of peptone, 20 g of glucose, 5 g of sodium acetate, 0.05 g of manganese sulfate, 5 g of beef extract powder, 1 g of Tween 80, 2 g of ammonium citrate, 4 g of yeast extract powder, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 20 g of agar powder, and 1000 mL of distilled water. Sterilize it at 115 °C for 30 min using an autoclave, and adjust the pH to 6.8.

[0014] (2) Seed medium: Take 10 g of peptone, 20 g of glucose, 5 g of sodium acetate, 0.05 g of manganese sulfate, 5 g of beef extract powder, 1 g of Tween 80, 2 g of ammonium citrate, 4 g of yeast extract powder, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, and 1000 mL of distilled water. Sterilize it at 115 °C for 30 min using an autoclave, and adjust the pH to 6.8.

[0015] (3) Fermentation medium: Take 10 g of peptone, 20 g of glucose, 5 g of sodium acetate, 0.05 g of manganese sulfate, 5 g of beef extract powder, 1 g of Tween 80, 2 g of ammonium citrate, 4 g of yeast extract powder, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 15 g of calcium carbonate, and 1000 mL of distilled water. Sterilize it at 115 °C for 30 min using an autoclave, and adjust the pH to 6.8.

[0016] In this example, the shallow treatment and biological irradiation terminal (TR4) of the Heavy Ion Research Facility in Lanzhou (HIRFL, 31111.02) of the Institute of Modern Physics, Chinese Academy of Sciences was used to irradiate and mutate to screen for high-yield lactic acid strains, such as Figure 1 as shown. The specific method is as follows: 1. Study on the characteristics of the original homo / hetero-lactic acid strains: Two original strain cultures, Pediococcus pentosaceus Pediococcus pentosaceus and Lactobacillus fermentum Lactobacillus fermentum (two lactic acid bacteria strains previously screened from silage in the laboratory) were taken out from -80 °C, thawed at room temperature and activated, and then inoculated into 100 mL of seed medium at an inoculation amount of 1% respectively. They were statically cultured at 37 °C, with the seed medium as the blank control. Every 2 h, 2 mL of samples were taken to measure OD 600 and pH until 24 h. With time as the abscissa and OD 600 and pH as the ordinate, growth curves and acid reduction curves were plotted. Lactic acid bacteria can be divided into homo-lactic acid bacteria and hetero-lactic acid bacteria according to the fermentation type. Among them, homo-lactic acid bacteria such as Pediococcus pentosaceus have a high lactic acid production capacity. During the fermentation process, they can rapidly reduce the pH value of silage and limit the activities of harmful microorganisms, thereby improving the fermentation quality of silage; hetero-lactic acid bacteria such as Lactobacillus fermentum can produce other metabolites in addition to lactic acid during the fermentation process, such as acetic acid, which can effectively inhibit fungi, thereby improving aerobic stability.

[0017] The growth curve and acid reduction curve of Pediococcus pentosaceus are as Figure 2 shown; the growth curve and acid reduction curve of Lactobacillus fermentum are as Figure 3 shown. From Figure 2 , 3 it can be seen that Pediococcus pentosaceus was in the growth lag phase from 0 h to 4 h and in the logarithmic growth phase from 4 h to 20 h. Its pH value dropped from 6.44 to 4.21, and the acidity decreased significantly; Lactobacillus fermentum was in the growth lag phase from 0 h to 2 h and in the logarithmic growth phase from 2 h to 10 h. Its pH value dropped from 6.38 to 4.35, and the acidity also decreased significantly. Subsequently, Lactobacillus fermentum and Pediococcus pentosaceus entered the stationary phase from 10 h to 16 h and from 20 h to 24 h, and OD 600 slowly decreased during the subsequent culture, entering the decline phase.

[0018] 2. Take 2 mL of the starting bacterial liquid in the logarithmic growth phase of fermentation, place it in an irradiation dish with a diameter of 35 mm, and seal the bacterial liquid in the irradiation dish with a sealing film. Then place it on the rotating rack at the terminal of the heavy ion irradiation device, and use the heavy ion beam provided by the shallow treatment and biological irradiation terminal (TR4) of the Heavy Ion Research Facility in Lanzhou (HIRFL, 31111.02) of the Institute of Modern Physics, Chinese Academy of Sciences 12 C 6＋Irradiation is carried out, and heavy ion beams are set 12 C 6＋ Irradiation parameters for irradiating lactic acid bacteria, including an irradiation dose rate of 40 Gy / min, and gradient irradiation is carried out on the original strain, with irradiation doses of 0 Gy, 25 Gy, 50 Gy, 75 Gy, 100 Gy, 125 Gy, 150 Gy, 175 Gy, 200 Gy, and 225 Gy.

[0019] 3. Preliminary screening of lactic acid bacteria: Strains with high lactic acid production ability are screened according to colony morphological characteristics and growth conditions. The specific method is as follows: 100 μL of the bacterial liquid treated by heavy ion beam irradiation mutagenesis is taken and added to 900 μL of physiological saline for gradient dilution. The dilution gradients are 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 . At each dilution gradient, 0.1 mL of the bacterial liquid is taken and added to the MRS screening medium containing 1% calcium carbonate, and then spread evenly. Anaerobic culture is carried out at 37°C for 48 h. The colony morphology is observed, and the HC value (the ratio of the diameter of the calcium dissolution circle to the colony diameter) is calculated. Single colonies with large calcium dissolution circles are picked out, and the strains with an HC value increased by 20% or more compared to the original strain are used as the preliminary screening strains.

[0020] In this example, 25 strains of Pediococcus pentosaceus and 12 strains of Lactobacillus fermentum are screened out. The HC values are all increased by 20% or more compared to the original strain, and they are named according to the irradiation dose of the strain. The preliminary screening results are shown in Table 1. The 25 strains of Pediococcus pentosaceus and 12 strains of Lactobacillus fermentum screened out are used for further re-screening.

[0021] Table 1 Preliminary screening results

[0022] 4. Re-screening of lactic acid bacteria: The 25 strains of Pediococcus pentosaceus and 12 strains of Lactobacillus fermentum screened in Table 1 are respectively inoculated into the seed medium for culture. When the culture reaches the latter half of the logarithmic phase at 37°C, they are respectively inoculated into the fermentation medium at an inoculation amount of 10%. After static culture at 37°C for 24 h, the fermentation broth is obtained. Finally, a lactic acid standard curve is made according to the method of DB15 / T 1458-2018, and the lactic acid content in the fermentation broth is detected.

[0023] Three parallel repeated experiments are carried out on the lactic acid content of the 25 strains of Pediococcus pentosaceus and 12 strains of Lactobacillus fermentum. The experimental data are as Figure 4 、 5 shown, whereFigure 4 For the rescreening of lactic acid content of Pediococcus pentosaceus; Figure 5 For the rescreening of lactic acid content of Lactobacillus fermentum.

[0024] From Figure 4 , 5 It can be seen that among the 25 mutagenized strains of Pediococcus pentosaceus and 12 strains of Lactobacillus fermentum with positive mutations, the lactic acid content has increased to varying degrees compared with the original strains. The Pediococcus pentosaceus and Lactobacillus fermentum with the highest lactic acid content are strain M46 and strain 17-25-61 respectively, and their lactic acid contents are 22.2 g / L and 15.8 g / L respectively. Compared with the lactic acid contents of 7.9 g / L and 13.6 g / L of the original strains of Pediococcus pentosaceus and Lactobacillus fermentum, the improvement rates of Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61 after mutagenesis are 181% and 16.18% respectively.

[0025] 5. Study on the genetic stability of lactic acid strains Genetic stability analysis was carried out on the two mutagenized strains of Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61. Method: The two strains of Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61 were subcultured continuously for 9 generations, and the lactic acid fermentation experiments were carried out every other generation. Specific method: The two mutagenized lactic acid bacteria were inoculated into the seed medium at an inoculation amount of 1% respectively, and the 1st generation seed liquid was obtained after static culture at 37°C for 12 h. Then, the 1st generation seed liquid was inoculated into the fermentation broth at an inoculation amount of 10% respectively. The 1st generation seed liquid of the two lactic acid bacteria was inoculated into the seed medium at an inoculation amount of 1% respectively, and the 2nd generation seed liquid was obtained after static culture at 37°C for 12 h. Then, the 2nd generation seed liquid was inoculated into the fermentation broth at an inoculation amount of 10% respectively. And so on, the two strains of Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61 were subcultured continuously for 9 generations, and the lactic acid content was measured according to DB15 / T1458-2018 every other generation.

[0026] The lactic acid contents of the 1st, 3rd, 5th, 7th, and 9th generations of the two strains of Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61 were measured, and 3 parallel repeated experiments were carried out for each. The lactic acid contents of Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61 strains are shown in Table 2.

[0027] Table 2 Determination of genetic stability of Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61 strains

[0028] It can be seen from Table 2 that after 9 generations of subculture of Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61, the difference in lactic acid content between generations is not significant, indicating that both lactic acid bacteria have the acid-producing performance with stable inheritance.

[0029] The obtained homofermentative lactic acid bacterium Pediococcus pentosaceus M46 was named Pediococcus pentosaceus M46, and it was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 28, 2025, with the deposit number GDMCC NO: 66069, and the deposit address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou. The obtained heterofermentative lactic acid bacterium 17-25-61 was named Limosilactobacillus fermentum 1761, and it was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 28, 2025, with the deposit number GDMCC NO: 66068, and the deposit address is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0030] Example 2 In this example, two strains of homo / heterofermentative lactic acid bacteria that had been screened were used for silage fermentation of oat straw. The specific method is as follows: 1. Study on the characteristics of homo / heterofermentative lactic acid strains after mutagenesis and breeding The seed solutions of Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61 after breeding were inoculated into 100 mL of seed medium at an inoculation amount of 1% respectively, and cultured statically at 37°C. Using the seed medium as a blank control, 2 mL of samples were taken every 2 h to measure OD 600 and pH until 24 h. With time as the abscissa and OD 600 and pH as the ordinate, growth curves and acid reduction curves were plotted.

[0031] The growth curve and acid reduction curve of Pediococcus pentosaceus M46 are as Figure 6 shown; the growth curve and acid reduction curve of Lactobacillus fermentum 17-25-61 are as Figure 7 shown.

[0032] As Figure 6 、 7 can be seen, both Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61 after mutagenesis and breeding had a growth lag phase from 0 h to 2 h. Subsequently, the cells of Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61 increased geometrically from 4 h to 18 h and from 4 h to 10 h respectively, and the strains were in the logarithmic growth phase during this stage. In addition, the pH of Pediococcus pentosaceus M46 decreased from 6.86 to 4.28, and the pH of Lactobacillus fermentum 17-25-61 decreased from 6.71 to 4.12. The acidity of both Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61 decreased significantly. Subsequently, Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61 entered the stationary phase from 18 h to 22 h and from 10 h to 18 h respectively, and OD 600 slowly decreased during the subsequent culture, entering the decline phase.

[0033] 2. Production of oat straw: Harvest the oat straw planted in Wuchuan County, cut the green oat straw to about 2 cm, mix evenly and reserve. Two mutagenized and selected lactic acid bacteria agents were used in the experiment. Among them, Pediococcus pentosaceus M46 (GDMCC NO.66069), at 37 °C, was inoculated into the seed medium and statically cultured for 24 h, and the viable bacteria count > 1.76×10 15 CFU / g), Lactobacillus fermentum 17 - 25 - 61 (GDMCC NO.66068), at 37 °C, was inoculated into the seed medium and statically cultured for 24 h, and the viable bacteria count > 1.12×10 8 CFU / g).

[0034] 3. Oat straw silage method: Set up 3 treatment groups: ① Control treatment (CK), without adding any bacteria agent; ② Pediococcus pentosaceus (T), the addition amount is 1×10 6 CFU / g; ③ Lactobacillus fermentum (B), the addition amount is 1×10 6 CFU / g; ④ Pediococcus pentosaceus M46:Lactobacillus fermentum 1761 = 1:1 (TB), the total addition amount is 1×10 6 CFU / g. Dissolve the lactic acid bacteria agent in distilled water with 3% molasses by fresh weight, and evenly spray it onto the oat straw sample. The control group is added with an equal amount of sterile water containing 3% molasses by fresh weight. All treatments are repeated 3 times, each repetition has 3 respiration bags, and 500 g of oat straw is weighed for each respiration bag and put into a polyethylene valve bag for sealing and exhausting. After storing at room temperature in the dark for 30 d, open the package and take samples, and determine the lactic acid content according to DB15 / T 1458 - 2018.

[0035] The effects of homo / heterofermentative lactic acid bacteria on the lactic acid production ability of oat straw silage are as Figure 8 shown.

[0036] As Figure 8 shown, different treatment groups have obvious change rules for the lactic acid content of oat straw silage. Compared with natural fermentation (CK treatment), Pediococcus pentosaceus (T), and Lactobacillus fermentum (B), the addition of Pediococcus pentosaceus M46 and Lactobacillus fermentum 17 - 25 - 61 in a mass ratio of 1:1 (TB) can significantly increase the lactic acid content, and the lactic acid content reaches 16.4 g / kg, an increase of 11.11 g / kg compared with natural fermentation (CK treatment). The level of lactic acid content is also an index to distinguish the storage time. Using the mixed bacteria provided in this example to ferment oat straw silage, its lactic acid content is significantly higher than that of the prior art (natural fermentation). Therefore, the strain resources provided in this example can also extend the shelf life of silage to a certain extent and make it not restricted by seasons.

[0037] In summary, the present invention uses heavy ion beams12 C 6＋ Pediococcus pentosaceus M46 and Lactobacillus fermentum 17-25-61 were screened through steps such as original strain characteristic research, primary screening of lactic acid bacteria, secondary screening of lactic acid bacteria, research on lactic acid production ability and genetic stability of lactic acid bacteria strains. Finally, two strains of lactic acid bacteria with high yield and stable performance were selected. The characteristics of homo / hetero-fermentative lactic acid bacteria strains after mutagenesis and screening were studied. The two selected lactic acid bacteria strains were applied to oat straw for silage fermentation. Through the production of oat straw and the method of oat straw silage, the change of lactic acid content was observed. It provides high-quality strain resources for extending the shelf life of mixed-strain fermented silage, improving the flavor and palatability of feed, etc., and lays a foundation for industrial production.

[0038] The above embodiments are only one of the preferred embodiments of the present invention and should not be used to limit the protection scope of the present invention. Any meaningless changes or polishing made on the main design concept and spirit of the present invention, as long as the technical problems solved are still consistent with the present invention, should be included in the protection scope of the present invention.

Claims

1. A homo / hetero-fermentative lactic acid bacterium, characterized in that, The homofermentative lactic acid bacterium is named Pediococcus pentosaceus ( Pediococcus pentosaceus ), M46, and was deposited at the Guangdong Provincial Microbial Culture Collection Center on March 28, 2025, with the deposit number GDMCC NO: 66069, and the deposit address being: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou; the heterofermentative lactic acid bacterium is named Lactobacillus fermentum ( Limosilactobacillus fermentum ), 1761, and was deposited at the Guangdong Provincial Microbial Culture Collection Center on March 28, 2025, with the deposit number GDMCC NO: 66068, and the deposit address being: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

2. The homo / hetero-fermentative lactic acid bacterium according to claim 1, wherein The homofermentative lactic acid bacteria are obtained by heavy ion mutagenesis and selection using Pediococcus pentosaceus ( Pediococcus pentosaceus ); the heterofermentative lactic acid bacteria are obtained by heavy ion mutagenesis and selection using Lactobacillus fermentum ( Lactobacillus fermentum ).

3. The application of the homofermentative or heterofermentative lactic acid bacteria according to claim 2 in the preparation of silage feed, characterized in that, Pediococcus pentosaceus ( Pediococcus pentosaceus ), M46 and Lactobacillus fermentum ( Limosilactobacillus fermentum ) 1761 were mixed and inoculated into the silage raw materials, and fermented at room temperature in the dark for 28 - 32 days.

4. Use of the homofermentative or heterofermentative lactic acid bacteria according to claim 3, characterized in that, Pediococcus pentosaceus ( Pediococcus pentosaceus ), and Lactobacillus fermentum ( Limosilactobacillus fermentum ) with a mixed viable cell count ratio of 1:

1. The total viable cell count after mixing and inoculating into the silage raw material is 1×10 6 CFU / g.

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