Porcine-derived leuconostoc citreum with emission reduction and weight gain effects and application thereof

Abstract

The invention provides a pig source leuconostoc citreum (Leuconostoc citreum) with the effects of reducing emission and increasing weight and an application of the pig source leuconostoc citreum. The microorganism preservation number of the leuconostoc citreum S4 disclosed by the invention is CGMCC (China General Microbiological Culture Collection Center) No.31017. The strain provided by the invention is separated and screened from healthy pig manure, and has the characteristics of producing acid and exopolysaccharides. The invention discloses an optimized culture medium of a strain. The culture medium can improve the culture efficiency and polysaccharide yield of the strain. The invention further discloses a fermented feed containing the leuconostoc citreum S4, the fermented feed is added into a fattening pig feed, the daily gain of a pig herd can be remarkably increased, emission of fecal odor substances and escherichia coli in excrement can be remarkably reduced, and green and efficient development of pig breeding can be promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a porcine-derived Leuconostoc citrinum with the effects of reducing emissions and increasing weight, and an application thereof. Background Art

[0002] Entering a new era and a new development stage, the pig farming industry is pursuing green and efficient development. While ensuring the health of pigs and improving production performance, it should also reduce the discharge of breeding waste that has a negative impact on the ecological environment. The use of advanced feeding microbial technology is expected to optimize the structure of pig intestinal flora, improve feed conversion efficiency, improve pig growth performance, reduce the discharge of waste such as odorous substances and harmful microorganisms in pig manure, and promote high-quality development of the pig farming industry.

[0003] Leuconostoc citreum is one of the species in the genus Leuconostoc, a type of lactic acid bacteria with good probiotic properties. Studies have shown that Leuconostoc citreum can produce metabolites such as organic acids, polysaccharides and bacteriocins, which help improve the quality of human food. The existing published patent application "New Leuconostoc citreum and fermented foods and compositions using it as a starter" (CN 102625828 A) discloses that Leuconostoc citreum CJGN34 can be used as a starter to improve the overall taste quality of kimchi. The patent application "A strain of Leuconostoc citreum and its application" (CN 115895978A) discloses that Leuconostoc citreum SH12 was isolated from birch sap. The strain has a high yield of glucan and mannitol, and can be used to ferment sour soy milk, strawberry juice and bread, which can improve quality indicators. The patent application "A fermentation agent and fermentation method for fermented apple chips" (CN 118562645 A) shows that the use of a fermentation agent composed of Lactobacillus plantarum LNJ-002, ethanol-resistant Pediococcus and Leuconostoc citrinum can avoid contamination by other bacteria, and can produce a large amount of flavor substances, which can improve the taste of fermented apple chips. The patent application "A method for preparing a fermentation agent suitable for fruit and vegetable fermentation" (CN 117736897 A) discloses that the fermentation of vegetables and fruits with a fermentation agent composed of Lactobacillus plantarum, Lactococcus lactis, Weissella sinus and Leuconostoc citrinum has good fermentation flavor and taste. Patent publication number CN 117320736 A "Pharmaceutical composition containing lemon Leuconostoc strain as an effective ingredient for preventing or treating cholestatic liver disease", CN 117320737 A "Pharmaceutical composition containing lemon Leuconostoc strain as an effective ingredient for preventing or treating fibrosis" and CN 117337187 A "Pharmaceutical composition containing lemon Leuconostoc strain as an effective ingredient for preventing or treating intestinal damage" disclose that lemon Leuconostoc WiKim0104 can be used as a pharmaceutical composition or food composition, and has good health effects. The above patented technologies are mainly used in the preparation of human food, fermented fruits and vegetables, and health products, and do not involve porcine lemon Leuconostoc and its use as fermented feed and pig farming. Summary of the invention

[0004] The object of the present invention is to provide a pig-derived Leuconostoc citreum with the effect of reducing emissions and increasing weight and its application. The Leuconostoc citreum of the present invention is isolated and screened from healthy pig feces, and is named Leuconostoc citreum S4. After a series of experiments such as in vitro activity, acid production, exopolysaccharide production, and drug sensitivity, it is proved that it has good probiotic properties. The culture efficiency and the ability to produce exopolysaccharides of the strain are improved by optimizing the culture medium. The prepared fermented feed is added to the fattening pig feed, which can significantly increase the daily weight gain of the pig herd, reduce the content of fecal odor substances and Escherichia coli in feces, and help promote the green and efficient development of pig farming.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A Leuconostoc citreum S4, which was deposited on June 19, 2024 at the General Microbiology Center of the China Culture Collection Administration (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101), with the deposited name being Leuconostoc citreum and the deposited number being: CGMCC No.31017.

[0007] The lemon lemon Leuconostoc (CGMCC No. 31017) is a Gram-positive rod, arranged singly, in pairs or in short chains; the colonies on the MRS agar medium are white, round, moist, opaque and have neat edges; the colonies on the agar medium containing sucrose are larger, slightly yellow, round, moist, translucent and easy to connect into pieces; it can grow at 20°C to 37°C, and its optimum growth temperature is 30°C; the bacterium is sensitive to multiple antibiotics such as erythromycin, bacitracin, gentamicin, tetracycline, lincomycin, amoxicillin and cefpirome; it can metabolize sucrose to produce products such as extracellular polysaccharides and lactic acid; it is cultured in a preferred fermentation medium for 24 hours, and the polysaccharide yield is 62.5g / L.

[0008] The invention discloses a preferred culture medium for improving Leuconostoc citreum S4 polysaccharide, which comprises: 20-150 g sucrose, 0-10 g glucose, 4-10 g soy peptone, 5-10 g beef extract, 5-10 g yeast extract powder, 5 g anhydrous sodium acetate, 2 g diammonium hydrogen citrate, 2 g K2HPO4·3H2O, 0.6 g MgSO4·7H2O, 0.2 g MnSO4·H2O, 1 mL Tween-80 and 1000 mL distilled water.

[0009] The preferred seed culture medium is 20 g sucrose, 5 g glucose, 8 g soy peptone, 8 g beef extract, 5 g yeast extract powder, 5 g anhydrous sodium acetate, 2 g diammonium hydrogen citrate, 2 g K2HPO4·3H2O, 0.6 g MgSO4·7H2O, 0.2 g MnSO4·H2O, 1 mL Tween-80, and 1000 mL distilled water;

[0010] The preferred fermentation medium is 120 g sucrose, 5 g soy peptone, 5 g beef extract, 6 g yeast extract powder, 5 g anhydrous sodium acetate, 2 g diammonium hydrogen citrate, 2 g K2HPO4·3H2O, 2 g MgSO 4· 7H2O 0.6 g, MnSO4·H2O 0.2 g, Tween-80 1 mL, distilled water 1000 mL.

[0011] Another object of the present invention is to provide the use of the above-mentioned Leuconostoc citrinum in fattening pig feed and breeding.

[0012] A feed additive or fermented feed additive containing Leuconostoc citrea as described above.

[0013] Use of the above-mentioned Leuconostoc citrinum or the above-mentioned feed additive or fermented feed additive or feed in improving animal production performance and / or reducing skatole.

[0014] Furthermore, the improving of animal production performance is to improve the daily weight gain of pigs. The feed is fermented feed, and the addition ratio is 0.4% to 4% of the daily feed weight.

[0015] The use of the lemongrass as described above in the preparation of a skatole-reducing preparation for animal breeding.

[0016] A feed containing Leuconostoc citrina S4 as described above.

[0017] The fermented feed containing the above-mentioned Leuconostoc citrinum S4 is preferably obtained by inoculating Leuconostoc citrinum S4 into a base material of the fermented feed for fermentation. The base material of the fermented feed is common feed raw materials such as corn, wheat bran, soybean meal, rice bran, or complete feed.

[0018] A method for preparing a feed fermented by Leuconostoc citrinum S4 as described above, characterized in that the method comprises the following steps:

[0019] ① Seed solution preparation: adopt the step-by-step expansion method, inoculate the lemon lemon S4 stored at -80℃ into the seed culture medium at a volume ratio of 1%, culture at 30℃ for 16h, repeat twice to obtain the seed solution;

[0020] ② Expanded culture: Inoculate the seed solution of Leuconostoc citrinum S4 in a volume ratio of 1% into the fermentation medium, and culture at 30°C for 24 hours to obtain the fermentation liquid;

[0021] ③ Further, the fermentation liquid, drinking water and base material are mixed in a weight ratio of 20-30:20-30:40-60, and the preferred ratio is 25:25:50. After mixing evenly, they are divided into fermentation barrels, compacted, sealed, and cultured at 30°C for 24 hours to obtain lemon S4 fermented feed.

[0022] A fermented feed, wherein the fermented feed contains the lemongrass S4 as described above.

[0023] As mentioned above, fermented feed is used to improve animal production performance and reduce fecal odor.

[0024] As mentioned above, fermented feed is used to increase the daily weight gain of fattening pigs and reduce skatole and ammonia nitrogen.

[0025] The feed containing the above-mentioned Leuconostoc citrinum S4 is characterized in that the addition ratio of the Leuconostoc citrinum S4 fermented feed in the daily feed is 0.4% to 4%, and the preferred ratio is 2%.

[0026] The beneficial effects of the present invention are:

[0027] The porcine Leuconostoc citreum S4 provided by the present invention has the characteristics of easy cultivation, fast growth, acid production, and extracellular polysaccharide production. The fermented feed can effectively increase the daily weight gain of fattening pigs and reduce the content of fecal odor substances and Escherichia coli in feces. When 2% of fermented feed containing Leuconostoc citreum S4 is added to the fattening pig feed, the average daily weight gain of the pig herd is significantly increased by 6.29%; the feed-to-weight ratio is significantly reduced by 4.83%; the skatole content in the fattening pig feces is reduced by 20.69%, the ammonia nitrogen content is reduced by 15.08%; and the number of Escherichia coli in the feces is reduced by 58.36%. Adding an appropriate amount of Leuconostoc citreum S4 to pig feed is helpful to promote the green and efficient development of pig farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention provides the bacterial morphology of Leuconostoc citrinum S4.

[0029] Figure 2 The colony morphology of Leuconostoc citrinum S4 is shown in Figure 2-A (MRS agar medium) and 2-B (MRS agar medium containing sucrose) after culturing at 30°C for 24 hours.

[0030] Figure 3 OD of Leuconostoc citrinum S4 in MRS liquid medium 600nm Change curve.

[0031] Figure 4 This is the pH change curve of Leuconostoc citrinum S4 in MRS liquid culture medium.

[0032] Figure 5 These are the results of screening carbon sources in the culture conditions of Leuconostoc citrinum S4.

[0033] Figure 6 These are the results of screening of nitrogen sources in the culture conditions of Leuconostoc citrinum S4.

[0034] Figure 7 The results of extracellular polysaccharide production of different strains in fermentation medium. DETAILED DESCRIPTION

[0035] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

[0036] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in this method are analytical grade or above; the MRS medium used in this method is a conventional medium for the culture and counting of lactic acid bacteria, and its formula is 20 g of glucose, 10 g of peptone, 10 g of beef extract, 5 g of yeast extract powder, 5 g of anhydrous sodium acetate, 2 g of diammonium hydrogen citrate, 2 g of K2HPO4·3H2O, 0.58 g of MgSO4·7H2O, 0.19 g of MnSO4·H2O, 1 mL of Tween-80, 1000 mL of distilled water, pH 6.5±0.2; MRS agar medium is prepared by adding 1.5% agar to MRS liquid medium.

[0037] Example 1 Isolation, screening and identification of porcine Leuconostoc citrinum S4

[0038] Fresh fecal samples were collected from a large-scale pig farm in Beijing. Under sterile conditions, an appropriate amount of samples were placed in MRS liquid culture medium for 24 hours, shaken evenly and diluted tenfold, spread on MRS agar plates, and cultured anaerobically at 37°C. After 48 hours, milky white colonies suspected to be lactic acid bacteria were picked and cultured anaerobically to obtain 7 purified strains. The isolated and purified strains were numbered S1-S7 and stored at -80°C.

[0039] The S1-S7 strains were streaked on an MRS agar plate, and a single colony was picked and inoculated into 25 mL of MRS liquid medium, cultured at 37°C for 12 h, and the cells were collected. The bacterial DNA was extracted using a DNA extraction kit (Beijing Solebold Technology Co., Ltd.), and the extracted bacterial DNA was used as a template. The universal amplification primers 27F and 1492R of bacterial 16SrDNA (Shanghai Sangon Biotechnology Co., Ltd.) were used for PCR amplification. The strains were compared by sequencing the amplified products, and the comparison results were Enterococcus faecalis, Lactobacillus fermentans, Streptococcus nonlactis, Leuconostoc citrinum, Lactobacillus reuteri and Leuconostoc mesenteroides subsp. The strains were activated twice, inoculated in an MRS liquid medium containing 2% sucrose at a volume ratio of 1%, and compared with the characteristics of viable bacteria count, acid production, and exopolysaccharide production after culture at 37°C for 24 h. The results are shown in Table 1.

[0040] Table 1 Lactic acid bacteria screening results

[0041]

[0042] As shown in Table 1, the activity and extracellular polysaccharide production of the S4 strain were significantly higher than those of the other strains. The S4 strain was sent to the Institute of Microbiology, Chinese Academy of Sciences for cell morphology, physiological and biochemical characteristics, 16SrRNA gene, rpoA gene, etc., and was identified as Leuconostoc citreum. Figure 1 As shown, the cells of Leuconostoc citrinum S4 are Gram-positive rods, arranged singly, in pairs or in short chains.

[0043] like Figure 2 As shown, the colonies of Leuconostoc citrinum S4 on MRS agar medium are small, white, round, moist, opaque, and have neat edges; the colonies on MRS agar medium containing 2% sucrose are large, slightly yellow, round, moist, translucent, and easy to connect into pieces, indicating that sucrose can promote the growth of Leuconostoc citrinum S4 and produce extracellular polysaccharides.

[0044] The physicochemical characteristics of Leuconostoc citrinum S4 are shown in Table 2. The amplification primers used for the determination of the 16S rRNA gene sequence were 27F (SEQ ID NO.1: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO.2: 5'-TACGACTTAACCCCAATCGC-3'); the amplification primers used for the determination of the rpo A gene sequence were 21F (SEQ ID NO.3: 5'-ATGATYGA RTTTGAAAAACC-3') and 22R (SEQ ID NO.4: 5'-ACYTTVATCATNTC WGVYTC-3'), and the sequencing results of the amplified products are shown below.

[0045] Table 2 Physiological and biochemical characteristics of Leuconostoc citreum S4

[0046]

[0047]

[0048] Note: “+” indicates positive; “-” indicates negative.

[0049] 16S rRNA gene sequence (SEQ ID NO.5):

[0050] GCGAGAGGTGCTTGCACCTTTCAAGCGAGTGGCGAACGGGTGAGTAA

[0051] CACGTGGATAACCTGCCTCAAGGCTGGGGAATACATTTGGAAACAGAT

[0052] GCTAATACCGAATAAAACTTAGTATCGCATGATATAAAGTTAAAAGGC

[0053] GCTATGGCGTCACCTAGAGATGGATCCGCGGTGCATTAGTTAGTTGGT

[0054] GGGGTAAAGGCTTACCAAGACAATGATGCATAGCCGAGTTGAGAGAC

[0055] TGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAG

[0056] GCTGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAA

[0057] CGCCGCGTGTGTGATGAAGGCTTTCGGGTCGTAAAGCACTGTTGTATG

[0058] GGAAGAAATGCTAAAATAGGGAATGATTTTAGTTTGACGGTACCATAC

[0059] CAGAAAGGGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTAT

[0060] GTCCCGAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGACGG

[0061] TTGATTAAGTCTGATGTGAAAGCCCGGAGCTCAACTCCGGAATGGCAT

[0062] TGGAAACTGGTTAACTTGAGTGTTGTAGAGGTAAGTGGAACTCCATGT

[0063] GTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGG

[0064] CGGCTTACTGGACAACAACTGACGTTGAGGCTCGAAAGTGTGGGTAGC

[0065] AAACAGGATTAGATACCCTGGTAGTCCACACCGTAAACGATGAATACT

[0066] AGGTGTTAGGAGGTTTCCGCCTCTTAGTGCCGAAGCTAACGCATTAAG

[0067] TATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTG

[0068] ACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAA

[0069] CGCGAAGAACCTTACCAGGTCTTGACATCCTTTGAAGCTTTTAGAGAT

[0070] AGAAGTGTTCTCTTCGGAGACAAAGTGACAGGTGGTGCATGGTCGTCG

[0071] TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC

[0072] CCTTATTGTTAGTTGCCAGCATTCAGTTGGGCACTCTAGCGAGACTGCC

[0073] GGTGACAAACCGGAGGAAGGCGGGGACGACGTCAGATCATCATGCCC

[0074] CTTATGACCTGGGCTACACACGTGCTACAATGGCGTATACAACGAGTT

[0075] GCCAACCTGCGAAGGTGAGCTAATCTCTTAAAGTACGTCTCAGTTCGG

[0076] ACTGCAGTCTGCAACTCGACTGCACGAAGTCGGAATCGCTAGTAATCG

[0077] CGGATCAGCACGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTAATGCCCAAAGCCGGTGGCCTA。

[0078] rpoA gene sequence (SEQ ID NO.6):

[0079] .

[0080] The strain S4 was patented and deposited at the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) on June 19, 2024, with the deposit number CGMCC No.31017, the deposit name S4, and the classification name: Leuconostoc citreum.

[0081] Example 2 Biological characteristics of Leuconostoc citrinum S4

[0082] 2.1 Growth curve

[0083] Absorbance OD 600nm As an indicator, the growth characteristics of Leuconostoc citrinum S4 were determined. Figure 3 OD of Leuconostoc citrinum S4 in MRS liquid medium600nm Change curve. Figure 3 It can be seen that the OD of the bacterial solution of Leuconostoc citrinum S4 was 600nm The value increases rapidly; during 10 to 16 hours, the bacterial solution OD 600nm The change gradually decreased, indicating that it had reached the end of logarithmic growth; after 16 hours, OD 600nm The change was not obvious, indicating that the bacteria had entered a stable growth period.

[0084] 2.2 pH curve

[0085] The acid production characteristics of Leuconostoc citrinum S4 were determined using pH as an indicator. Figure 4 pH change curve of Leuconostoc citrinum S4 in MRS liquid medium. Figure 4 It can be seen that the pH of Leuconostoc citrinum S4 decreased rapidly during 2 to 14 hours, and the pH change was not obvious after 16 hours.

[0086] 2.3 Growth temperature

[0087] After activating Leuconostoc citrinum S4, it was inoculated into MRS liquid culture medium at a volume ratio of 1%, and placed in static culture at 20°C, 25°C, 30°C, and 37°C, and the number of viable bacteria was measured at 12h and 24h. As shown in Table 3, the number of viable bacteria was the highest when cultured at 30°C, indicating that 30°C is the appropriate culture temperature for it.

[0088] Table 3 The number of viable bacteria of Leuconostoc citrinum S4 at different culture temperatures (CFU / mL)

[0089]

[0090] 2.4 Extracellular polysaccharide determination

[0091] The phenol-sulfuric acid method was used to determine the content of extracellular polysaccharides in the fermentation broth of Leuconostoc citrinum S4. After Leuconostoc citrinum S4 was activated, it was inoculated into MRS liquid medium containing 2% sucrose at a volume ratio of 1%, cultured at 30°C, and samples were taken at 6h, 12h, 24h and 48h to determine the polysaccharide content in the fermentation broth. As shown in Table 4, the content of extracellular polysaccharides in the fermentation broth reached a peak when Leuconostoc citrinum S4 was cultured for 24h, and decreased slightly at 48h.

[0092] Table 4 Extracellular polysaccharide content in the fermentation broth of Leuconostoc citrinum S4

[0093]

[0094] 2.5 Drug sensitivity test

[0095] The antibiotic sensitivity test was performed using the agar diffusion method. The drug sensitivity test of 8 categories and 20 antibiotics (OXOID drug sensitivity paper) was performed on Leuconostoc citrinum S4, and the results are shown in Table 5. As shown in Table 5, Leuconostoc citrinum S4 is resistant to sulfamethoxazole, streptomycin, ciprofloxacin and polymyxin E, and is sensitive to other antibiotics.

[0096] Table 5 Results of drug sensitivity test of Leuconostoc citrinum S4

[0097]

[0098] Note: S stands for sensitive; I stands for moderately sensitive; R stands for resistant.

[0099] Example 3 Preparation of Leuconostoc citrinum S4 fermented feed

[0100] 3.1 Improvement of the culture medium of Leuconostoc citrinum S4

[0101] According to the biological characteristics of Leuconostoc citrinum S4, the carbon and nitrogen sources were optimized and screened based on MRS medium, with the number of viable bacteria and the production of extracellular polysaccharides as indicators. Specific operation methods: (1) Using the single factor experimental method, 4 different carbon sources (glucose, maltose, sucrose, D-fructose) and 4 different nitrogen sources (peptone, yeast extract powder, beef extract and soy peptone) were selected to replace the carbon source and nitrogen source in MRS medium to screen out the key carbon and nitrogen sources that affect the number of viable bacteria and the production of extracellular polysaccharides; (2) The orthogonal experimental method was used to optimize the combination of key factors. Results are shown in Figure 5 , Figure 6 .

[0102] Depend on Figure 5 , Figure 6It can be seen that the carbon sources with higher viable counts of Leuconostoc citrinum S4 are glucose and sucrose, and the nitrogen sources are yeast extract powder and soy peptone. The carbon sources with higher extracellular polysaccharide production are sucrose and the nitrogen sources are yeast extract powder. The combination of glucose, sucrose, yeast extract powder and soy peptone was further optimized, and the final medium composition was screened out as follows: sucrose 20-150g, glucose 0-10g, soy peptone 4-10g, beef extract 5-10g, yeast extract powder 5-10g, anhydrous sodium acetate 5g, diammonium hydrogen citrate 2g, K2HPO4·3H2O 2g, MgSO4·7H2O 0.6g, MnSO4·H2O 0.2g, Tween-80 1mL, distilled water 1000mL. The study found that as the sucrose content in the culture medium increased, the production of extracellular polysaccharides increased significantly, the fermentation broth became viscous, the fluidity decreased, and the lemon S4 bacteria were difficult to separate, affecting the preparation of its bacterial agent. Therefore, in order to obtain the optimal viable count and the optimal polysaccharide yield, combined with economy and ease of operation, further, its preferred seed culture medium is 20g sucrose, 5g glucose, 8g soy peptone, 8g beef extract, 5g yeast extract, 5g anhydrous sodium acetate, 2g diammonium hydrogen citrate, K2HPO4·3H2O2g, 0.6g MgSO4·7H2O, 0.2g MnSO4·H2O, 1mL Tween-80, and 1000mL distilled water; further, its preferred fermentation medium is 120g sucrose, 5g soy peptone, 5g beef extract, 6g yeast extract, 5g anhydrous sodium acetate, 2g diammonium hydrogen citrate, K2HPO4·3H2O 2g, 0.6g MgSO4·7H2O, 0.2g MnSO4·H2O, 1mL, distilled water 1000mL; mix all the components of the culture medium evenly, heat to dissolve, adjust the pH to 6.5, package, and sterilize at 121℃ for 15min.

[0103] The activated Leuconostoc citrinum S4 was inoculated into the above medium and MRS liquid medium at a volume ratio of 1% at the same time, cultured at 30°C, and samples were taken at 12h and 24h for viable count determination. The viable count reached 3.2×10 9 CFU / mL, the effect was better than that of MRS medium. The results are shown in Table 6.

[0104] Table 6 The number of viable bacteria of Leuconostoc citrinum S4 in different culture media (CFU / mL)

[0105]

[0106] The activated lemon Leuconostoc S4 was inoculated into MRS medium, seed medium and fermentation medium at a volume ratio of 1% at the same time, cultured at 30°C, and samples were taken at 12h and 24h, respectively, and the exopolysaccharide yield in the fermentation broth was determined by the phenol-sulfuric acid method. As shown in Table 7, the average exopolysaccharide yield of lemon Leuconostoc S4 reached 62.5g / L after being cultured in the optimized fermentation medium for 24h, which is 110 times the exopolysaccharide yield using conventional MRS medium.

[0107] Table 7 Polysaccharide yields of Leuconostoc citrinum S4 in different culture media (g / L)

[0108]

[0109] The strains isolated and screened in the same batch were selected and inoculated into the fermentation medium in the same way. After culturing for 24 hours under the same conditions, samples were taken to determine the extracellular polysaccharide yield in the fermentation broth. The results are shown in Figure 7 .Depend on Figure 7 It can be seen that the average extracellular polysaccharide production of fermented Lactobacillus mucilaginosus S2, Lactobacillus mucilaginosus S5 and S7, and Leuconostoc mesenteroides subspecies S6 were 0.86, 0.55, 0.78 and 34.72 g / L, respectively, which were significantly lower than the extracellular polysaccharide production of Leuconostoc citrinum S4 (62.5 g / L), indicating that the optimized fermentation medium has a significant effect on the sugar production of Leuconostoc citrinum S4.

[0110] 3.2 The preparation method of the fermented feed of Leuconostoc citrinum S4 comprises the following process steps:

[0111] ① Seed solution preparation: adopt the step-by-step expansion method, inoculate the lemon lemon S4 stored at -80℃ into the seed culture medium at a volume ratio of 1%, culture at 30℃ for 16h, repeat twice to obtain the seed solution;

[0112] ② Expanded culture: Inoculate the seed liquid of Leuconostoc citrinum S4 in a volume ratio of 1% into the fermentation medium, and culture at 30°C for 24 hours to obtain the fermentation liquid;

[0113] ③ Further, the fermentation liquid, drinking water and base material are mixed in a weight ratio of 20-30:20-30:40-60, and the preferred ratio is 25:25:50. After mixing evenly, the mixture is divided into fermentation barrels, compacted, sealed, and cultured at 30°C for 24 hours to obtain a fermented feed of Leuconostoc citrinum S4. The number of viable bacteria in the fermented feed reaches 1.0×10 9 CFU / g, pH reached below 4.4.

[0114] Example 4 Application of Leuconostoc citrinum S4 in fattening pig feed and breeding

[0115] 4.1 Experimental materials and methods

[0116] A total of 90 healthy long × large fattening pigs with an average body weight of (52.02 ± 1.12) kg were randomly divided into three groups (control group, experimental group 1 and experimental group 2). The control group was fed a diet without fermented feed, and the experimental group 1 was fed a diet containing 2% S4 fermented feed (the final effective viable count in the complete feed was 2.1 × 10 7 CFU / g), the diet of the experimental group 2 contained 2% S6 fermented feed (the final effective viable count in the complete feed was 2.0×10 7 CFU / g) were repeated 3 times in each group, with 10 pigs in each repeat. The pigs were fed and drank freely, and the indoor temperature was 24-30℃. The feeding and management were carried out according to the routine. The experimental period was 70 days.

[0117] The fermented feed of Leuconostoc citrinum S4 used was the fermented feed prepared in Example 3, and the base material used was wheat bran. At the same time, the strain of Leuconostoc mesenteroides subspecies S6 isolated from the same batch was selected and the fermented feed was prepared using the same method.

[0118] At the beginning and end of the experiment, the fasting weight was measured, the daily feed consumption was recorded, and the average daily weight gain, daily feed intake and feed-to-weight ratio were calculated; after the end of the experiment, 2 fresh fecal samples were randomly collected from each replicate, and a total of 18 fecal samples were collected for the determination of the number of lactic acid bacteria and Escherichia coli live bacteria, skatole and ammonia nitrogen content in the feces. Lactic acid bacteria in feces were cultured with MRS medium and counted anaerobically at 37°C for 48 hours; Escherichia coli were cultured with eosin-methylene blue medium and counted aerobically at 37°C for 24 hours; skatole content was determined by high performance liquid chromatography; ammonia nitrogen content was detected by potassium chloride solution extraction-spectrophotometry. The experimental diet was prepared in accordance with "Nutritional Requirements of Pigs" GB / T 39235-2020. The composition and nutritional level of the control and experimental diets are shown in Table 8.

[0119] Table 8 Composition and nutritional level of fattening pig diets (feeding basis) %

[0120]

[0121] Note: 1) The compound premix provides Cu 10mg, Fe 80mg, Zn 60mg, Mn 30mg, Se0.3mg, I 0.6mg, VA 5000IU, VD3 1800IU, VE 16mg, VK 1.8mg, VB1 1.0mg, VB2 4mg, VB62.0mg, VB 12 0.02mg; Biotin 0.1mg; Folic acid 0.8mg; Niacin 16mg; Pantothenic acid 9mg; L-Lysine 3.0g; L-Threonine 1.4g; DL-Methionine 0.5; L-Tryptophan 0.3g; Calcium 5g; Total phosphorus 1.8g.

[0122] 2) Dry matter, crude protein, calcium and total phosphorus are measured values.

[0123] 4.2 Test results

[0124] The results of average daily weight gain, daily feed intake and feed-to-weight ratio of each group are shown in Table 9. The results of the number of lactic acid bacteria and Escherichia coli in fattening pig feces are shown in Table 10. The results of skatole content and ammonia nitrogen content in fattening pig feces are shown in Table 11.

[0125] Table 9 Production performance of fattening pigs

[0126]

[0127] Note: Different lowercase letters in the same row indicate significant differences (P<0.05). The data in the table are mean ± standard deviation; the same as in the following table.

[0128] As shown in Table 9, compared with the control group, the average daily weight gain of the pigs in the experimental group 1 increased by 6.29% (P<0.05), and the feed-to-weight ratio decreased by 4.83% (P<0.05); the average daily weight gain of the pigs in the experimental group 2 increased by 2.51% (P>0.05), and the feed-to-weight ratio decreased by 1.37% (P>0.05); compared with the experimental group 2, the average daily weight gain of the pigs in the experimental group 1 increased by 4.23% (P<0.05), and the feed-to-weight ratio decreased by 3.62% (P>0.05). The results show that adding fermented feed containing Leuconostoc citrifolia S4 to the diet of fattening pigs can better improve the production performance of the pigs.

[0129] Table 10 Number of lactic acid bacteria and Escherichia coli in fattening pig feces

[0130]

[0131] As shown in Table 10, compared with the control group, after adding the fermented feed of Leuconostoc citrinum S4 to the diet, the number of lactic acid bacteria in the feces of fattening pigs increased by 31.88% and the number of Escherichia coli decreased by 58.36%.

[0132] (P<0.05), and the ratio of lactic acid bacteria to Escherichia coli increased significantly (P<0.05).

[0133] Table 11 Contents of skatole and ammonia nitrogen in fattening pig feces mg / kg

[0134]

[0135] As shown in Table 11, compared with the control group, the skatole content in the feces of the pigs in the experimental groups 1 and 2 decreased by 20.69% (P<0.05) and 6.28% (P>0.05), respectively, and the ammonia nitrogen content decreased by 15.08% (P<0.05) and 7.65% (P>0.05), respectively; compared with the experimental group 2, the skatole content in the feces of the pigs in the experimental group 1 decreased by 15.38% (P<0.05), and the ammonia nitrogen content decreased by 8.04% (P<0.05). The results show that adding fermented feed in the diet can reduce the emission of skatole and ammonia nitrogen in the feces, and the emission reduction effect of feeding the fermented feed of Leuconostoc citrinum S4 is more significant.

[0136] In summary, adding an appropriate amount of Leuconostoc citrinum S4 fermented feed to the fattening pig diet significantly increased the daily weight gain of the pig herd, significantly reduced the feed-to-weight ratio, significantly reduced the number of E. coli in pig manure, significantly increased the ratio of lactic acid bacteria to E. coli, and significantly reduced the content of skatole and ammonia nitrogen. These indicators are conducive to reducing emissions and increasing efficiency in fattening pig farming, and are conducive to promoting the green and efficient development of pig farming, and have good development and application prospects.

Claims

1. A Leuconostoc citreum S4, characterized in that: Its deposit number is CGMCC No.31017.

2. A feed additive or fermented feed additive or feed containing the lemongrass S4 according to claim 1.

3. Use of the lemongrass S4 according to claim 1 or the feed additive or fermented feed additive or feed according to claim 2 in improving animal production performance and / or reducing skatole.

4. The use as claimed in claim 3, wherein the improving animal production performance is improving the daily weight gain of pigs.

5. The use according to claim 3, wherein the feed is fermented feed, and the addition ratio thereof is 0.4% to 4% of the daily feed weight.

6. Use of the lemongrass S4 according to claim 1 in preparing a skatole-reducing preparation for animal breeding.

7. A fermented feed containing the lemon Leuconostoc S4 according to claim 1, characterized in that: The method is obtained by inoculating lemon Leuconostoc S4 into a base material of fermented feed for fermentation. The base material of the fermented feed is one or more of corn, bran, soybean meal, rice bran and complete compound feed.

8. The fermented feed according to claim 7, characterized in that During fermentation, the fermentation liquid, drinking water and base material are mixed in a weight ratio of 20-30:20-30:40-60. The fermentation liquid is prepared by inoculating lemongrass S4 into a fermentation medium. The formula of the fermentation medium is 20-150 g of sucrose, 0-10 g of glucose, 4-10 g of soy peptone, 5-10 g of beef extract, 5-10 g of yeast extract powder, 5 g of anhydrous sodium acetate, 2 g of diammonium hydrogen citrate, 2 g of K2HPO4·3H2O, 0.6 g of MgSO4·7H2O, 0.2 g of MnSO4·H2O, 1 mL of Tween-80 and 1000 mL of distilled water.

9. A culture medium for increasing the polysaccharide of Leuconostoc citrinum, characterized in that: The formula is: 20-150g sucrose, 0-10g glucose, 4-10g soy peptone, 5-10g beef extract, 5-10g yeast extract powder, 5g anhydrous sodium acetate, 2g diammonium hydrogen citrate, 2g K2HPO4·3H2O, 0.6g MgSO4·7H2O, 0.2g MnSO4·H2O, 1mL Tween-80, and 1000mL distilled water.

10. The culture medium according to claim 9, characterized in that The medium formula for the seed culture of Leuconostoc citrinum is 20 g sucrose, 5 g glucose, 8 g soy peptone, 8 g beef extract, 5 g yeast extract powder, 5 g anhydrous sodium acetate, 2 g diammonium hydrogen citrate, 2 g K2HPO4·3H2O, 0.6 g MgSO4·7H2O, 0.2 g MnSO4·H2O, 1 mL Tween-80, and 1000 mL distilled water; The culture medium formula for the fermentation culture of Leuconostoc citrinum is 120 g sucrose, 5 g soy peptone, 5 g beef extract, 6 g yeast extract powder, 5 g anhydrous sodium acetate, 2 g diammonium hydrogen citrate, 2 g K2HPO4·3H2O, 2 g MgSO 4· 7H2O 0.6g, MnSO4·H2O 0.2g, Tween-80 1mL, distilled water 1000mL.

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