TWI760923B - Novel lactobacillus and application thereof - Google Patents

Abstract

The present disclosure provides a novel Lactobacillus paracaseiwhich is deposited under Accession number BCRC 910990.

Description

Novel Lactobacillus and its Application

The present disclosure relates to a novel lactobacillus and its application, in particular, to a novel Lactobacillus paracasei strain, a method for preparing silage, and the prepared silage.

In addition to being eaten as fruit, citrus is also commonly used in processing applications such as juice production. However, after processing, a large amount of processing wastes such as peels and pomace are generated, and with the accumulation of these large amounts of processing wastes, environmental and economic cost problems will be caused. Therefore, in order to solve these problems, countries all over the world have also carried out research and development on the secondary utilization of agricultural waste in recent years in order to obtain additional economic value and achieve the concept of a circular economy with zero waste resources.

Nowadays, there have been studies on the use of agricultural wastes such as citrus as animal feed. However, one of the treatment methods of citrus pomace as feed is the silage method (Ensilage). The silage method is to compact and seal the fermented material, so that it can be fermented in an anaerobic environment to produce lactic acid as a silage microbial agent, so that the feed is weakly acidic, which can effectively inhibit the growth of spoilage bacteria and prevent secondary fermentation. , and effectively control organic acids and degrade lignocellulose. However, the general silage process is very long and requires proper microorganisms and proper environment to complete silage. If the fermentation of the silage is not ideal, the pH value of the silage may not drop normally during fermentation, which means that the fermentation is not in good condition.

Therefore, there is still an urgent need for a novel technology that shortens the silage time and can improve the quality of the silage.

The present disclosure provides a novel strain of Lactobacillus ( Lactobacillus ) as a highly active silage agent, more specifically, a strain named Lactobacillus paracasei GL1 ( Lactobacillus paracasei GL1), which is deposited in the Institute of Food Industry Development And the deposit number is BCRC 910990.

The present disclosure further provides a method for preparing silage, which includes: (a) inoculating a silage agent into a feed material to obtain a to-be-fermented product, wherein the silage agent comprises a Lactobacillus strain with deposit number BCRC 910990 ; and (b) the fermented material is fermented for a predetermined time in an anaerobic environment to obtain a silage.

The present disclosure further provides a silage material, which is prepared by the above-mentioned preparation method of the silage material.

In order to make the above-mentioned and other objects, features, and advantages of the present disclosure more obvious and easy to understand, the following specific embodiments are given in conjunction with the accompanying drawings, and the detailed descriptions are as follows:

The present disclosure can be understood through the related descriptions of various disclosure aspects, examples and tables disclosed in the following embodiments. Unless otherwise defined herein, terms (including technical and scientific terms) used in connection with this disclosure shall have the meanings that are understood by those of ordinary skill in the art to which this disclosure belongs. And it should be understood that, in the event of any potential ambiguity, the definitions of terms shall be accorded to those commonly used terms (as defined in the dictionary) unless the definitions provided herein indicate otherwise. It can be further understood that the terms used in this application are only used for the purpose of describing specific implementation aspects, rather than being used for limitation.

It must be noted that, unless clearly indicated to the contrary, the singular forms "a", "an" and "the" used in the specification or the scope of the patent application also include plural expressions. Accordingly, unless otherwise required by context, singular terms shall include pluralities and plural terms shall also include the singular.

In this article, "standard strain" refers to a type strain, which can be used as a strain reference for comparison to determine whether it is a strain with the same name.

The following describes the lactobacillus of the present disclosure and its application in detail.

In one aspect of the present disclosure, the present disclosure provides a novel Lactobacillus strain, more specifically, a Lactobacillus paracasei strain as a highly active silage agent. The above-mentioned lactobacillus can rapidly reduce the pH value of silage under anaerobic conditions, thereby obtaining high-quality livestock silage.

In a specific embodiment, the above-mentioned Lactobacillus strain can be Lactobacillus paracasei GL1, which was deposited in the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute of the Republic of China on April 20, 109, and the deposit number is BCRC 910990. In addition, in this specific embodiment, the above-mentioned ribosomal (Ribosomal) DNA gene of Lactobacillus paracasei GL1 can be the sequence of SEQ ID NO: 2.

In another aspect of the present disclosure, the present disclosure provides a method for preparing silage. The silage is obtained by inoculating a silage agent into a feed material and fermenting it for a predetermined time in an anaerobic environment. Wherein the feed raw material can be the peel residue of citrus plants.

The "Citrus" system described herein is used to collectively refer to plants belonging to the genus Citrus in plant taxonomy, called Citrus. Specifically, as examples of citrus, for example, tangerines, tangerines, sweet oranges, grapefruits, lemons, limes, limes, kumquats, etc. are mentioned, but the varieties of citrus are not limited to these, that is, the technical field Anyone with ordinary knowledge in the genus can buy any plant belonging to the genus Citrus for use, or collect citrus or cultivated plants of the genus Citrus for use in nature. According to an embodiment of the present disclosure, the above-mentioned citrus plant can use lemon.

The preparation method of the silage of the present disclosure may include the following steps, but is not limited thereto.

First, as shown in FIG. 1, in step S110, the citrus peel residue is ground and placed in a PE plastic bag, and the citrus peel residue is fully mixed with the silage agent containing Lactobacillus paracasei GL1 to form a fermented thing. However, the storage of the to-be-fermented product is not limited to PE plastic bags, that is, as long as it is a space that can stably store the to-be-fermented product in an anaerobic environment, such as a silage tank.

In step S120, the plastic bag storing the to-be-fermented product is degassed, so that the air in the plastic bag is completely exhausted to create an oxygen-free environment.

Next, in step S130, the plastic bag is sealed with a sealing machine, so that the to-be-fermented material can be stably stored in an anaerobic environment and maintained at a temperature of 25 to 30°C.

Finally, in step S140, after a predetermined time, the fermentation is terminated, and the obtained fermentation end product can be used as silage.

In addition, before step S110 is performed, the silage agent containing Lactobacillus paracasei GL1 of BCRC 910990 of the present disclosure can be inoculated into the MRS medium to activate the silage agent.

In one embodiment, in the method for preparing silage of the present disclosure, the inoculation concentration of the silage inoculum is 1×10 ⁹ CFU/ml to 3×10 ⁹ CFU/ml, for example, 1×10 ⁹ CFU/ml, 1.5× 10 ⁹ CFU/ml, 2 x 10 ⁹ CFU/ml, 2.5 x 10 ⁹ CFU/ml, 3 x 10 ⁹ CFU/ml.

In one embodiment, the predetermined time period used in the method for preparing silage of the present disclosure may be 18 days to 45 days, such as 20 days, 21 days, 24 days, 26 days, 28 days, 30 days, 32 days, 34 days, 36 days, 38 days, 40 days, 42 days, 44 days.

In one embodiment, the pH value of the silage prepared by the method for preparing silage of the present disclosure is 3.0 to 5.0, such as 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9.

According to the preparation method of the present disclosure, agricultural wastes such as skin residue can be effectively converted into silage, and the shortcomings of liquid ammonia ammoniation and urea ammoniation are solved simultaneously. In addition, since the silage inoculant of the present disclosure does not add chemical substances that are costly and easy to pollute the environment, it can avoid environmental damage during the process of manufacturing silage, and can effectively preserve various nutrients in the slag.

Furthermore, according to the preparation method of the present disclosure, since the silage agent containing the novel lactobacillus of the present disclosure is combined and fermented under specific silage conditions, the silage time can be shortened to about one and a half months compared with the conventional silage technology. Therefore, the efficiency of the silage process can be effectively improved.

The following examples are provided to assist those skilled in the art in implementing the present disclosure. Even so, these examples should not be viewed as limitations of the present disclosure, as modifications to the embodiments discussed herein may be made by persons of ordinary skill in the art to which this disclosure pertains without departing from the spirit or scope of this disclosure and change, and still fall within the scope of this disclosure.

Example

Example 1

Isolation and culture of strains

First, the isolation step of the strain was performed using MRS agar (DifcoTM Lactobacilli MRS Broth; Sparks, MD, USA).

The above separation steps are as follows: take 100 μl of aquaculture water samples and add 900 μl of secondary water to form a mixed solution, take 0.1 mL of the mixed solution and apply it to the MRS agar medium with an L stick respectively. Next, anaerobic culture was performed at 30°C for 24 hours. Colonies on the medium were picked and plated on MRS agar medium for strain purification. After culturing under the same conditions for 24 hours, single colonies were picked out and screened to a single bacterial phase by electron microscopy using the Gram staining method. After adding 20% glycerol to the bacterial solution, it was frozen and stored in a -80°C refrigerator for preservation, and the sample strains were named GB6, CS3, SC1, SC2, SC3, GL1, GL1-1 and GL2-1 respectively.

Bacteria identification

The sample strains GB6, GL1, GL1-1 and GL2-1 were entrusted to Mingxin Biotechnology Co., Ltd. to carry out nucleic acid identification, and the 16S rDNA sequence of the sample strain GB6 (sequence identification number: 1) and the 16S rDNA sequence of the sample strain GL1 ( SEQ ID NO: 2), the 16S rDNA sequence of the sample strain GL1-1 (SEQ ID NO: 3), and the 16S rDNA sequence of the sample strain GL2 (SEQ ID NO: 4).

After aligning the 16S rDNA sequence of the sample strain GL1 with the gene database on the NCBI website, it was found that the 16S rDNA sequence of the sample strain GL1 was similar to, for example, the 16S ribosomal partial sequence of the Lactobacillus paracasei strain 4G330 [Genbank accession number] MK026811.1] with 100% similarity.

According to the above results, the sample strain GL1 of the present disclosure is Lactobacillus paracasei through preliminary identification, in order to confirm whether Lactobacillus paracasei GL1 is a novel Lactobacillus paracasei isolate, Lactobacillus paracasei GL1 is further subjected to the following microbial characteristic analysis .

Microbial properties

Investigations were carried out using the API ZYM strip (bioMérieux, product name API ZYM). Each test well contains different substrates, so during the incubation period of the sample, the color change caused by the metabolic reaction in the reagent strip is used to determine the enzyme activity reaction. The specific steps are as follows.

Pick sample strains and standard strains (Lactobacillus paracasei paracasei 206 and Lactobacillus paracasei paracasei -1) from the medium and mix them with about 2 ml of sterile water to make a bacterial suspension with a concentration of 5-6 McFarland. Next, add about 5 ml of sterile water into the culture box, keep the culture box at a certain humidity, and move the reagent strip into the culture box. Add 65 μl (about 2 drops) of the sample suspension to each test well of the reagent strip dropwise, cover the incubation box, and incubate for 4 hours at 35-37 °C. After culturing, one drop of ZYM A and one drop of ZYM B reagent was added to each test well, and the results were read after standing for 5 minutes. The results are shown in Table 1 below.

[Table 1] Numbering Enzyme name used for analysis Lactobacillus paracasei ssp . paracasei 206 _ Lactobacillus paracasei ssp . paracasei - 1 Lactobacillus paracasei GL1 1 control group - - - 2 Alkaline phosphatase - + - 3 Esterase (C4) - + - 4 Esterase lipase - + + 5 Lipase - - - 6 Leucine arylamidase + + + 7 Valine arylamidase + + + 8 Cystine arylamidase - - - 9 Trypsin - - - 10 α-Chymotrypsin (α-Chymotrypsin) - - - 11 Acid phosphatase - + - 12 Naphthol-AS-BI-phosphohydrolase + + + 13 α-Galactosidase (α-Galactosidase) - - - 14 β-Galactosidase + - - 15 β-Glucuronidase (β-Glucuronidase) - - - 16 α-Glucosidase (α-Glucosidase) + - + 17 β-Glucosidase (β-Glucosidase) - - - 18 N-Acetyl-β-glucosaminidase + - - 19 α-Mannosidase (α-Mannosidase) - - - 20 α-Fucosidase (α-Fucosidase) - - -

Based on the above results, it can be seen that Lactobacillus paracasei GL1 of the present disclosure possesses esterase lipase, leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase and α- - The functional activity of glucosidase is inconsistent with the functional activities of standard strains (Lactobacillus paracasei 206 and Lactobacillus paracasei -1), indicating that the Lactobacillus paracasei GL1 of the present disclosure is a novel strain.

The above-mentioned Lactobacillus paracasei GL1 has been deposited in the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute of the Republic of China on April 20, 109, and the deposit number is BCRC 910990.

activation and preservation

The medium for activation and preservation is not particularly limited, and the medium generally used for culturing lactic acid bacteria can be appropriately modified as necessary. That is, for example, the carbon source may use galactose, glucose, fructose, mannose, sorbose, mannitol, salicin, cellobiose, maltose, sucrose, cocoon honey, starch hydrolyzate, waste Sugars such as molasses. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, and nitrates can be used. Moreover, as an inorganic salt, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate, etc. can be used. In addition, organic ingredients such as protein gluten, wine dregs, whey, soybean meal, defatted soybean meal, meat extract, and yeast extract can also be used. In addition, as the prepared medium, for example, MRS medium or its correction medium can be used.

The sample strain can be used directly from the culture obtained after culturing, or the obtained culture solution can be diluted or concentrated for use, or the bacteria recovered from the culture can be used. In addition, various additional operations such as freeze-drying can also be performed after the culture without impairing the effect of the present disclosure. The additional operation is suitable for those with higher survival rate of viable bacteria.

Specifically, sample strains stored at -20°C can be streaked on MRS solid medium and cultured at 37°C for 16 hours, single colonies are picked and placed in MRS liquid medium, cultured at 37°C for 16 hours, and fresh bacteria Transfer 300 μl of the liquid to a 1.5 ml microcentrifuge tube, add 700 μl of glycerol, mix well and store at -20°C.

In one embodiment, the formula of the MRS liquid medium is 10 g/L of No. 3 protein P, 10 g/L of beef extract, 5 g/L of yeast extract, 20 g/L of glucose, 1 g/L of L polysorbate 80, 2 g/L ammonium citrate, 5 g/L sodium acetate, 0.1 g/L magnesium sulfate, 0.05 g/L manganese sulfate, 2 g/L dipotassium hydrogen phosphate, Then add 2g/L of soluble starch, 2 to 4g/L of corn steep liquor, 0.2 to 0.5g/L of yeast powder, 0.05 to 0.15g/L of CaCO ₃ , 0.05 to 0.15g/L of Tween 80 (Tween80 ), sterilize at 121°C for 25 to 40 minutes, pH 6.8-7.0.

In another embodiment, the MRS solid culture medium is added with 15 g/L agar in the above formula, stirred until completely dissolved, and sterilized at 121° C. for 15 to 20 minutes.

Example 2

Bacteria count and acid production ability test

The isolated sample strains GB6, CS3, SC1, SC2, SC3, GL1, GL2 were cultivated at 30°C using the aforementioned MRS liquid culture, and the number of bacteria and the pH value of the culture solution were determined every hour, and Continuous monitoring for 24 hours.

Based on the results, Lactobacillus paracasei GL1 was able to reach a stable growth phase (the number of bacteria 10 ⁹ cfu/ml) after 12 hours of culture in MRS liquid medium, and as shown in Figure 2, after 24 hours of culture, paracasei The pH value of the culture solution of Lactobacillus paracasei GL1 decreased from 6.0 to pH 3.9, indicating that the Lactobacillus paracasei GL1 of the present disclosure has excellent acid-producing ability.

Example 3

Preparation of silage inoculants

Inoculate 10 ul of the sample strain into the MRS liquid medium for 12 hours, and use a spectrophotometer (Biochrom, model LibraS22) to measure the OD value until the colony concentration reaches 10 ⁹ cfu/ml, then the sample strain can be taken out as silage. Bacterial use.

Preparation of bark silage

The lemon peel residue after being squeezed was used as feed material, and the bacterial concentration of 1 × 10 ⁹ CFU/ml was inoculated with every 5 kg of feed material, and the sample strains GB6, CS3, SC1, SC2, SC3, GL1 and GL2, fully mix the lemon peel residue in the PE plastic bag with the sample strain to produce the fermented product. In addition, the lemon peel residue without inoculated strain is used as the blank group (Blank), and the air in the plastic bag is completely exhausted. After forming an anaerobic environment, use a sealing machine to seal and store at 25 to 28 ° C for 5 weeks, and measure the pH value and lactic acid content of the silage every week, and visually evaluate the appearance quality of the silage.

Determination of pH

The silage samples of each group were sampled every week, and the pH value of each group of silage was measured by an acid-base analyzer.

Determination of lactic acid content

The silage samples of each group were sampled every week, and the silage samples were placed in a 1.5ml microcentrifuge tube and stored at 4 °C, and the lactic acid standard was used as the reference point for comparison. After the 96-well plate, a YSI analyzer (YSI Incorporated, model 2900D) was used to quantify the lactic acid content of the silage samples.

result

As shown in the results in Figure 3, in terms of pH value, the silage of the group using the sample strain GL1 was significantly lower than that of the other sample strain groups at the 4th and 5th week, and the pH value could be reduced to below about 3.5. In addition, as shown in the results of Fig. 4, in terms of lactic acid content, the silage of the sample strain GL1 group was superior to the other sample strain groups at the 2nd week, and had up to 2 g at the 4th and 5th week. /L or more lactic acid content. In terms of the appearance of the silage, the silage of the sample strain GL1 group was stored for 5 weeks without mold, rot, deterioration, etc., indicating that the quality of the silage was good. Based on the above results, the sample strain GL1 has excellent lactic acid production activity when lemon is used as a feed material, and then the pH value of the silage can be rapidly reduced, and its pH value can be maintained for at least 1 month.

Example 4

Respectively inoculate a concentration of 1×10 ⁹ CFU/ml per 5 kg of feed raw materials. The inoculated sample strains are strains GL1, GL2 and standard strain (BCRC17484), and the lemon peel residue is fully mixed with the sample strain and the standard strain. Produce to be fermented, in addition, the lemon peel residue that is not inoculated with bacteria is used as blank group, and then the silage process is carried out and stored for 5 weeks in the same manner as in Example 3, and the silage is measured weekly in the same manner as in Example 3. pH value and lactic acid content, and visually evaluate the appearance quality of the silage.

result

As shown in Fig. 5, in terms of pH, the silage of the sample strain GL1 group was significantly lower than the standard sample strain 17484 group from the 2nd week, and maintained until the 5th week. In addition, as shown in the results of Fig. 6, in terms of lactic acid content, the silage of the group using the sample strain GL1 had higher lactic acid content than that of the standard sample strain 17484 group from the second week. In terms of the appearance of the silage, the silage of the sample strain GL1 group was stored for 5 weeks without mold, rot, deterioration, etc., indicating that the quality of the silage was good. Based on the above results, it was shown that the sample strain GL1 had better silage capacity than the standard sample strain 17484.

Example 5

Respectively inoculate a concentration of 1×10 ⁹ CFU/ml per 5 kg of feed raw materials, inoculate sample strains GL1, GL2 and commercially available silage strains ( ^XinfawangTM -grass silage), mix lemon peel residue with Commercially available silage strains and standard strains are fully mixed to produce a fermented product. In addition, the lemon peel residue that is not inoculated with the strain is used as a blank group, and then the silage process is carried out in the same manner as in Example 3 and stored for 5 weeks. Example 3 The pH value and lactic acid content of the silage were measured weekly in the same manner, and the appearance quality of the silage was visually evaluated.

result

As shown in the results in Figure 7, in terms of pH, the silage of the group using the sample strain GL1 showed a pH similar to that of the commercially available silage strain after 5 weeks of silage process. In addition, as shown in the results of Fig. 8, in terms of lactic acid content, the silage of the group using the sample strain GL1 had a higher lactic acid content than the commercially available silage strain at both the 3rd week and the 4th week. In terms of the appearance of the silage, the silage using the sample strain GL1 group and the commercially available silage strain had no mold, rot, deterioration after 5 weeks of storage, indicating that the quality of the silage was good. Based on the above results, it was shown that the sample strain GL1 was able to achieve silage capacity similar to that of the commercial silage strain.

Example 6

Storage test 1

The silage of different weeks of Example 4 was sampled, and cultivated at 30 °C for 12 hours using MRS culture, and the OD value was determined after serial dilution to calculate the number of bacteria, and to evaluate whether the bacteria phase type is not. For the inoculated sample strains or standard strains, the results are shown in Table 2 below.

[Table 2]

According to the results in the above table, both GL1 and GL2 can maintain their cell numbers during the silage period, while the cell numbers of the standard strains are less than 1. In addition, after the bacterial phase type evaluation, it was confirmed that it was the sample strain and standard strain of the initial inoculation.

Storage test 2

The silage of different weeks of Example 5 was sampled, and cultivated at 30 °C for 12 hours using MRS culture, and the OD value was determined after serial dilution to calculate the number of bacteria, and to evaluate whether the bacteria phase type is not. For inoculated sample strains or standard strains, the results are shown in Table 3 below.

[table 3]

According to the results in the above table, GL1, GL2 and commercially available silage bacteria can maintain the number of bacteria during the silage period (for measurement methods, please refer to the aforementioned determination of lactic acid content). In addition, through the evaluation of bacterial phase type, it was confirmed that it was the sample strain of the initial inoculation and the commercial silage bacteria.

To sum up, according to the above results, Lactobacillus paracasei GL1 of the present disclosure can grow well in silage during silage, and can effectively produce lactic acid and rapidly reduce the pH value of silage, and can maintain at least 1 months.

Furthermore, the Lactobacillus paracasei GL1 of the present disclosure can achieve a similar effect to the commercially available silage bacteria, and even has a lactic acid production amount superior to that of the commercially available silage bacteria, so compared with the conventional silage process time, it can be more shortened. its process time.

S110~S140: steps.

Figure 1 shows a flow chart of a method for preparing silage according to the present disclosure. Figure 2 shows a graph of pH changes of each sample strain in MRS medium for 24 hours. Figure 3 shows a graph of pH changes for each sample strain under silage treatment for each week. Figure 4 shows a graph of the lactic acid content of each sample strain under silage treatment for each week. Figure 5 shows a graph of the pH change over the weeks under silage treatment for each sample strain and the standard strain (BCRC 17484). Figure 6 shows a graph of the lactic acid content of each sample strain and the standard strain (BCRC 17484) under silage treatment for each week. Figure 7 shows a graph of the pH changes of each sample strain and commercial silage bacteria under silage treatment for each week. Figure 8 shows a graph of the lactic acid content of each sample strain and commercially available silage bacteria for each week under silage treatment.

Domestic storage information [please note in the order of storage institution, date and number] 1. Lactobacillus paracasei GL1 Bioresource Conservation and Research Center of the Food Industry Development Research Institute of the Republic of China 4/20/109 BCRC 910990

Claims (15)

A novel Lactobacillus paracasei strain with accession number BCRC910990. The lactobacillus according to claim 1, wherein the ribosomal DNA gene of the lactobacillus is the sequence of SEQ ID NO: 2. The lactobacillus as claimed in claim 1, wherein after the lactobacillus is cultivated for 24 hours, the pH value of the culture solution drops from 6.0 to 3.6-3.9. The lactobacillus according to claim 1, wherein the lactobacillus has esterase lipase, leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase and α-glucosidase activity. A preparation method of silage, comprising: (a) inoculating a silage to a feedstuff to obtain a fermented product, wherein the silage comprises the Lactobacillus strain with deposit number BCRC 910990; and (b) fermenting the to-be-fermented material for a predetermined time in an anaerobic environment to obtain a silage. The method for preparing silage according to claim 5, wherein the feed material is a citrus plant. The method for preparing silage according to claim 6, wherein the citrus plant is tangerine, tangerine, sweet orange, grapefruit, lemon, lime, lime or kumquat. The method for preparing silage according to claim 5, wherein before step (a) is performed, the silage inoculum is inoculated into the MRS medium, and the strain is activated. The method for preparing silage according to claim 5, wherein the inoculation concentration of the silage inoculation is 1×10 ⁹ CFU/ml to 3×10 ⁹ CFU/ml. The method for preparing silage according to claim 5, wherein the predetermined time is 18 to 45 days. The method for preparing silage according to claim 5, wherein in step (b), the fermented material is fermented at a temperature of 20 to 30°C. A silage material produced by the method for preparing silage material according to any one of claims 5 to 11. The silage of claim 12, wherein the pH of the silage is between 3.0 and 5.0. The silage according to claim 12, wherein the number of bacterial cells in the silage is 1.0×10 ⁷ CFU/ml or more. The silage according to claim 12, wherein the lactic acid content of the silage is 1.5 g/L or more.

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