1 Introduction
Silage is a storage-resistant and palatable feed made by chopping green fodder, filled in silage tower or silage, and fermented by microorganisms. It is one of the most important sources of roughage for ruminants. The quality of corn silage directly affects the production performance of ruminants and the economic benefits of producers, and the quality of silage has a great relationship with the lactic acid bacteria it contains. Lactic acid bacteria are a class of microorganisms that are of great industrial importance and are widely used in many industries such as light industry, food, medicine and feed industry. The importance of lactic acid bacteria can also be reflected in the research of more and more related fields, and the economic significance of lactic acid bacteria has been recognized by governments and enterprises in many countries. The European Commission and some commercial organizations have invested heavily in the long-term research and utilization of lactic acid bacteria. Lactic acid bacteria play an important role in the feed industry, especially in the field of silage preparation. Many countries, such as EU countries and the United States, have conducted extensive research on the application of lactic acid bacteria in silage preparation. The research includes the screening of lactic acid bacteria with excellent traits, mainly Enterococcus, Lactococcus lactis, Leuconostoc, Streptococcus and Lactobacillus (Cai Yimin et al., 1999); the effect of lactic acid bacteria on the fermentation quality of silage; silage supplemented with lactic acid bacteria The effect on feed intake of dairy cows and the inhibitory effect of lactic acid bacteria on secondary fermentation of silage (Weinberg et al., 1999). China's research and application of silage additive technology is very weak and urgently needs to be strengthened. Research reports on fermentation-promoting lactic acid bacteria additives in corn straw silage are rare, and most of the additives used in production practice are nutritional additives. Therefore, it is necessary to carry out research in this field in view of the specific situation in China.
The purpose of this study is to isolate lactic acid bacteria from natural fermented high quality silage samples, identify them, and screen out lactic acid bacteria with excellent traits, which lays a foundation for the preparation of excellent silage-specific starter.
2 Materials and methods
2.1 Materials
2.1.1 Sample Source
Samples were collected from silage of 12 farm and animal husbandry companies in Heilongjiang Province.
2.1.2 Standard strain
L. casei, codenamed ATCC15009; L. acidophilus, codenamed ATCC4356; Lact. lactis subsp. lactis, codenamed ATCC19435.
2.1.3 Equipment
Optical microscope (OLMPUS), METTLER TOLEDO Delta320 pH meter, electrothermal portable pressure steam sterilizer, constant temperature incubator, ultra clean bench.
2.1.4 Various media and reagents
The exercise test, gelatin liquefaction test, esculine hydrolysis test medium and MRS medium were prepared according to the method described by Ling Daiwen (1999); the test medium was in accordance with the bacterial classification group of the Institute of Microbiology, Chinese Academy of Sciences (1978). The method is introduced; the H2S production test, the nitrate reduction test and the various carbohydrate hydrolysis test media are all purchased from the Hangzhou Tianhe Microbial Reagent Co., Ltd. in Zhejiang Province; the microbiochemical reaction tubes; various biochemical reagents and dyeing The liquids were prepared according to the literature (Ling Daiwen, 1999; Du Lianxiang et al., 1992).
2.2 Method
2.2.1 Preparation of samples
Use the four-point method to evenly take about 200g of high-quality silage samples in the sealed bag, and put them in the ice box, and bring them back to the laboratory for storage in the refrigerator at 4 °C.
2.2.2 Isolation and purification of lactic acid bacteria
Weigh 10g of silage samples 1 to 12 in a sterile room, place them in a large test tube containing 15ml of sterile physiological saline for 2h, then dilute with physiological saline, select 3 dilutions, each dilution 0.1 ml of SL and M17 plates were separately applied, and cultured at 37 ° C for 24 to 72 hours, and then typical colonies were picked for Gram staining and microscopic examination. All Gram-positive strains were further isolated and purified. The purified strains were inoculated into MRS slant medium and stored in a refrigerator at 4 °C for use.
2.2.3 Identification test of lactic acid bacteria
According to the literature (Ling Daiwen, 1999; Bacterial Classification Group, Institute of Microbiology, Chinese Academy of Sciences, 1978; Du Lianxiang, 1992; Yudeng Gyulle, 2001a, 2001b; He Yinfeng, 2002), all Gram-positive, catalase test negative The strain was initially identified as a lactic acid bacterium, and then an identification test of the genus Lactobacillus was carried out. The determination of the genus Lactobacillus requires a nitrate reduction test, a gelatin liquefaction test, a sputum test, an H2S production test, and a pH 4.5 growth test. Identification of lactic acid bacteria requires temperature test, salt tolerance test, glucose gas production test, etc. to distinguish between Enterococcus, Streptococcus, Lactococcus, Pediococcus and Leuconostoc.
2.2.4 Identification test of lactic acid bacteria
According to the literature (Ling Daiwen, 1999; Bacterial Classification Group, Institute of Microbiology, Chinese Academy of Sciences, 1978; Du Lianxiang et al., 1992), the identification of lactic acid bacteria is mainly based on the fermentation acid production test of carbohydrates by test strains, that is, by monosaccharides, Disaccharides, trisaccharides, polysaccharides, and the utilization of carbohydrates such as glycosides and sugar alcohols are distinguished.
2.2.5 Excellent lactic acid bacteria screening test for silage starter
The 17 strains of lactic acid bacteria isolated and identified were respectively introduced into MRS liquid medium at a dose of 3%, and cultured at 37 ° C with constant temperature. The pH value of the fermentation broth of different strains was measured every 2h, and the acid production rate curve was the change of the pH value of the fermentation broth corresponding to different fermentation time (h). The strains suitable for use as silage are screened according to the acid production capacity and acid production rate of different strains.
3 Results and discussion
3.1 Identification results of Lactobacillus species
The genus identification results of the tested strains are shown in Table 1. Twenty-one strains were identified as Lactobacillus, and they were all able to grow at 15 ° C and 45 ° C. Among them, Ssd3 and Ssd1-1 can use glucose, lactose, mannitol, maltose, cellobiose, galactose, fructose, mannose, melezitose, salicin to produce acid, and can not use melibiose, raffin, arab Sugar and sorbitol produce acid, can not hydrolyze escin, and have similar properties to Lactobacillus corniculans, but they can use xylose to produce acid (weak acidity), which is different from the model strain. Ssd2-1, Ssd2-3, Sld1-2, and Sld2-1 can be used to produce acid by using glucose, fructose, arabinose, melibiose, and sucrose, and can not use cellobiose, mannose, sorbitol, Salicin produces acid, which is similar to that of Lactobacillus brevis, but Ssd2-1 and Sld1-2 cannot use maltose to produce acid, which is different from the model strain. Slm1-3 can use glucose, mannitol, cellobiose, galactose, fructose, mannose, melezitose, sorbitol to produce acid, can not use melibiose, cottonseed to produce acid, can hydrolyze escin, and cheese Lactobacillus has similar fermentation properties, but it can utilize xylose and arabinose to produce acid (less acidic), which is different from the model strain. Stj1-1, Stj2-1, Shw3-3, Shw3-6 heteroferment fermentation, can ferment glucose, fructose, maltose, arabinose, melibiose to produce acid, can not use cellobiose, mannose, melezitose, sorbitol Acid production, similar to the nature of Lactobacillus. Shj1-3, Shj1-4, and Sks3-5 can all utilize glucose, lactose, mannitol, maltose, cellobiose, galactose, fructose, xylose, arabinose, mannose, melibiose, and sorbitol to produce acid. It can hydrolyze escin, which is similar to Lactobacillus pentosus, but none of the three strains can produce acid by using raffinose, which is different from the model strain. Sld3, Sfq2-2, Sfq3-1, Sfq3-3, Shw4-1, Shj1-1, and Shj1-5 are still unable to determine which species they belong to. In the isolated strains, Ssd3 and Ssd1-1 can utilize xylose to produce acid, and Slm1-3 can utilize xylose and arabinose to produce acid, which is different from the model strain. This may be due to the long-term growth of the isolated strain in silage. Adapted to the growing environment of silage, xylose and arabinose are widely present in many plants, so that xylose or arabinose can be used to produce acid.
3.2 Identification results of species
Four strains of cocci were isolated in this study, and the results are shown in Table 2.
Three strains were identified as Enterococcus and one strain was Pediococcus. Among them, Slm1-5, Ssm3-1 and Shw3-7 can grow at 10 ° C and 45 ° C, and resist 6.5% NaCl, which is determined to be Enterococcus. Slm1-5, Ssm3-1 can not use mannitol, xylose, arabinose, melibiose, raffinose, melezitose, sorbitol, sorbose to produce acid, similar to the properties of strong enterococci; Shw3-7 can be utilized Lactose, mannitol, melezitose, sorbitol produce acid, can not use xylose, melibiose, cottonseed to produce acid, similar to the nature of Enterococcus faecalis. The cells of Slm3-4 are arranged in a quadruple arrangement. In the fermentation test of various carbohydrates, only monosaccharides (glucose, fructose, mannose) can be used to produce acid, sorbitol and starch cannot be used, and gas can not be produced from glucose. It is determined to be a Pediococcus, but it is not certain which species it belongs to.
Since the types and the number of bacteria of lactic acid bacteria are very small in naturally fermented silage, and lactobacilli mainly in fermented mature silage, there are very few lactic acid bacteria (Lin, 1992). Therefore, in this study, only 5 kinds of lactobacilli and 2 kinds of lactic acid bacteria were isolated from silage of 12 different sources.
3.3 Determination of acid production rate
As shown in Fig. 1 and Fig. 2, the Ssm3-1 and Shw3-7 strains in the cocci were better in acid production, and the Ssm3-1 strain had the strongest acid-producing ability, and the pH value reached 3.6 in about 10 hours. The Ssd3 and Sld2-1 strains in the bacillus have good acid-producing properties, and can reach a very low acidity in about 14h. Especially, the Ssd3 strain has a very strong acid-producing ability, and the pH value can be reduced to 3.22 at around 14h. The acid production rate is one of the important characteristics of good lactic acid bacteria activity, and there are large differences among different strains. Rapid and drastic reduction in pH during silage preparation significantly inhibits the growth of microorganisms in silage corn materials that have an adverse effect on silage quality, such as butyric acid bacteria, filamentous fungi, yeasts and molds. Improve the quality of silage. Therefore, cocci Ssm3-1 and Shw3-7, bacilli Ssd3 and Sld2-1 can be used to make an excellent corn silage starter.
4 Conclusion
4.1 In this study, 25 strains of lactic acid bacteria were isolated from silage of 12 different sources, and 17 of them were identified: 2 strains of Lactobacillus brevis, 4 strains of Lactobacillus brevis, 4 strains of Lactobacillus brevis, and Lactobacillus pentosus Three strains, one strain of Lactobacillus casei, one strain of Enterococcus faecalis, and two strains of Enterococcus faecalis.
4.2 From the identified lactic acid bacteria, the cocci Ssm3-1 and Shw3-7 strains, Bacillus Ssd3 and Sld2-1 strains with good acid production performance were screened out. In MRS liquid medium, the acid production rate of cocci Ssm3-1 and bacillus Ssd3 was very fast, the pH value of cocci Ssm3-1, 10h reached 3.6; the pH value of Bacillus Ssd3, 14h reached 3.22. Can be used to make silage starter. (Liu Fei Yang Lijie Hou Juncai Guo Dong Huoguicheng)
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