WO2022207015A2 - 一种香肠乳杆菌sr2及其应用 - Google Patents

一种香肠乳杆菌sr2及其应用 Download PDF

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WO2022207015A2
WO2022207015A2 PCT/CN2022/107666 CN2022107666W WO2022207015A2 WO 2022207015 A2 WO2022207015 A2 WO 2022207015A2 CN 2022107666 W CN2022107666 W CN 2022107666W WO 2022207015 A2 WO2022207015 A2 WO 2022207015A2
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lactobacillus
content
sausage
group
rice straw
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French (fr)
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WO2022207015A3 (zh
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刘蓓一
丁成龙
田吉鹏
韦青
程云辉
张文洁
许能祥
李晟
顾洪如
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江苏省农业科学院
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Publication of WO2022207015A2 publication Critical patent/WO2022207015A2/zh
Publication of WO2022207015A3 publication Critical patent/WO2022207015A3/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
    • A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
    • A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
    • A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/141—Farciminis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the invention belongs to the technical field of lactobacillus, in particular to a kind of lactobacillus sausage SR2 and application thereof.
  • the cell wall components in rice straw account for more than 80%, and there are special structures (cellulose hierarchical structure and condensed structure) in the cell wall, which limit the digestion and utilization of straw in animals.
  • cellulose hierarchical structure and condensed structure due to the hard and rough texture of rice straw and low crude protein content, hemicellulose and cellulose are wrapped by lignin, which makes rice straw unpalatable and difficult to be utilized to the maximum extent. Fermentation can not only promote the conversion of crude fiber in rice straw into sugars, improve the nutritional value, but also improve its palatability.
  • WSC soluble carbohydrates
  • the present invention provides a kind of Lactobacillus sausages SR2, and the storage number of the Lactobacillus sausage SR2 is CCTCC NO: M 2022416.
  • the invention also provides the application of the Lactobacillus sausage SR2 as a plant feed additive.
  • the Lactobacillus sausage SR2 is used for plant feed silage.
  • the Lactobacillus sausifici SR2 is capable of high production of ferulic acid esterase.
  • the Lactobacillus sausage SR2 increases the lactic acid content in the plant feed, reduces the acetic acid content in the plant feed, and reduces the ammonia nitrogen content.
  • the Lactobacillus sausifici SR2 inhibits the production of aerobic bacteria and yeast in the silage.
  • the Lactobacillus sausifici SR2 significantly increases the content of dry matter and WSC, and significantly reduces the content of NDF, ADF and cellulose.
  • the plant feed includes rice straw.
  • the present invention also provides the application of the Lactobacillus sausificus SR2 in the preparation of bacteriostatic preparations.
  • the Lactobacillus saussureum SR2 inhibits Escherichia coli, Salmonella and Staphylococcus aureus.
  • the present invention also provides a bacteriostatic preparation, the active ingredient comprises the Lactobacillus sausage SR2 described in the above technical solution.
  • Figure 1 is the FA standard curve
  • Fig. 2 is the transparent circle screening of ferulic acid esterase-producing strains, wherein a is the dilution coating diagram of lactic acid bacteria, and b is the transparent circles produced by ferulic acid esterase-producing strains on a flat plate;
  • Figure 3 is a morphological identification diagram, in which a is the SR1 colony characteristic, b is the SR2 colony characteristic, c is the SR1 microscopic examination form (100 ⁇ ), and d is the SR2 microscopic examination form (100 ⁇ );
  • Fig. 4 is the growth curve diagram of SR1 strain
  • Figure 5 is a graph of the growth curve of the SR2 strain.
  • Lactobacillus farciminis SR2 was deposited in the China Center for Type Culture Collection (CCTCC) on April 14, 2022, the address is Wuhan University, Wuhan, China, and the deposit number is CCTCC No: M 2022416.
  • references herein to "one embodiment” or “an embodiment” refers to a particular feature, structure, or characteristic that may be included in at least one implementation of the present invention.
  • the appearances of "in one embodiment” in various places in this specification are not all referring to the same embodiment, nor are they separate or selectively mutually exclusive from other embodiments.
  • HPLC high performance liquid chromatography
  • (2) Preparation of fermentation broth transfer the lactic acid bacteria that produce FAE to the MRS liquid medium. After culturing overnight at 37°C, wash with 90% sterile saline for 2 to 3 times, and place the bacterial weight in deionized water. . The bacterial suspension was transferred according to 5% inoculum and fermented in fresh enzyme-producing liquid medium, cultured at 37°C for 48h, and the FAE activity of the primary screened lactic acid bacteria was determined for reference for further screening. The liquid to be tested was centrifuged at 8000-12000 rpm/min for 5 min to obtain the supernatant fermentation broth.
  • Injection volume 10 ⁇ L.
  • Fig. 2 is the dilution coating diagram of lactic acid bacteria
  • Fig. 2 b is the transparent circle produced by the ferulic acid esterase-producing strain on the plate.
  • the fermentation broths of strains SR1 and SR2 were subjected to high performance liquid chromatography according to step 1.3, and it was found that both strains SR1 and SR2 had ferulic acid esterase (FAE) activity, and the enzyme of strain SR2 had ferulic acid esterase (FAE) activity.
  • the activity was higher than that of SR1 at 10.36 mU/mL, and the results are shown in Table 3.
  • the activity of ferulic acid esterase produced by strain SR2 is high, which is 10.36mU/mL, which is higher than that of strain SR1, and is higher than that of existing Lactobacillus plantarum (enzyme activity 8.34mU/mL), lactic acid tablets
  • the enzyme activity of ferulic acid esterase produced by coccus (enzyme activity 5.12mU/mL) and Lactobacillus brevis (enzyme activity 7.56mU/mL).
  • strain SR1 forms a milky white, round, regular edge, raised center and shiny colony on the plate
  • strain SR2 is A milky-white colony with a slightly raised center, but with irregular edges and a flat, moist surface.
  • the staining results of the smears after 48 hours of culture are shown in c and d in Figure 3.
  • the results show that SR1 and SR2 are both G+ Brevibacterium in microscopic examinations.
  • strain SR1 grows slowly at 0-2h and is in the stagnant phase; 2-16h strain SR1 enters the logarithmic growth phase, and the growth rate is the fastest; after 16h, it enters the flat phase, and the growth gradually stabilizes; , the final pH of strain SR1 is 3.94; strain SR2 uses sugar to produce lactic acid during the growth process, which makes the acidity of the culture medium continuously increase, resulting in damage to the bacteria and inhibition of growth.
  • strain SR2 accelerated its reproduction after 2h, entered the logarithmic growth phase, and at the same time, the acid-producing capacity also increased correspondingly, and the pH value decreased rapidly; after 18h, it gradually became stable, and the pH value decreased slowly, and 24h
  • the final pH of the post-strain SR2 was 3.83.
  • the strain SR1 can grow well in the MRS medium at pH 4.0-7.0, 25-37°C, and 3% salt concentration; the acid resistance of the strain SR2 is higher than that of SR1, and can be grown at pH 3.0-7.0, 25-37°C , 3% salt concentration of MRS medium growth well.
  • SR1 is rhamnose lactobacillus Bacillus and SR2 are Lactobacillus sausage.
  • the 16S rDNA sequence identification was completed by Nanjing Qingke Biological Company.
  • the identification results of 16SrDNA method are as follows: the genomic DNA of the test sample is used as the template, and the template is amplified by PCR with primers to obtain the PCR product, and 5 ⁇ L of the product is taken for verification by gel electrophoresis, and the target fragment at about 1.5kbp is obtained as expected. .
  • the qualified PCR products were sent to Nanjing Qingke Company for sequencing.
  • the sequence lengths of SR1 and SR2 were 1408bp and 1478bp, respectively.
  • the cryopreserved SR2 strain was inoculated into the MRS medium, cultured at 37°C for 24 hours, and the activated SR2 strain was obtained by subculture for 2 times.
  • the activated SR2 strain was re-transferred to the MRS liquid culture at 3% of the inoculum.
  • culture medium cultured for 24 hours, and the bacterial liquid was centrifuged to obtain the supernatant.
  • Bacteria Escherichia coli, Staphylococcus aureus, Salmonella
  • nutrient agar medium was used as the medium for indicator bacteria, and the bacteria were activated. Pour 10 mL of autoclaved water agar into the petri dish for bottoming.
  • Lactobacillus sausage SR2 of the present invention can inhibit Escherichia coli, Salmonella and Staphylococcus aureus.
  • Lactobacillus sausage SR2 was higher than that of Pediococcus lactis (4.32mU/mL) and Lactobacillus brevis (7.56mU/mL). Lactobacillus sausifici SR2 is more tolerant to acidity.
  • the microbial starter suitable for silage should have a consistent fermentation pathway, which can not only use sugar to increase acid production, but also need to have a certain acid resistance, so that the pH value of silage can be reduced to 4.0 as soon as possible, thereby hindering the activities of harmful microorganisms , improve feed quality.
  • Silage for test In October 2021, it was harvested from the earless japonica rice straw harvested at the experimental base of the Institute of Grain Crops, Jiangsu Academy of Agricultural Sciences.
  • Tested strains Lactobacillus rhamnosus SR1 and Lactobacillus sausage SR2.
  • inoculum Activated Lactobacillus rhamnosus SR1 and Lactobacillus saussureum SR2, transferred to MRS medium at 5% inoculum, cultured at 37°C until the number of viable bacteria reached 10 8 cfu/mL or more, and centrifuged at 10,000 rpm. The cells were obtained by centrifugation for 2 min to remove the supernatant, and the cells were diluted to 10 8 cfu/mL with sterile normal saline.
  • Treatment group settings 3 kinds of additives, Lactobacillus rhamnosus SR1(R), Lactobacillus chorus SR2(F), Lactobacillus rhamnosus SR1, and Lactobacillus chorizus SR2(RF), were set up in the experiment, and CK control group without additive was set at the same time. , a total of 4 treatments, the addition amount of each group is shown in Table 7 below.
  • Silage preparation cut the rice straw to 2-3cm long, spray the activated SR1 and SR2 strains on the cut rice straw with a spraying can at the spraying rate of 5 ⁇ 10 5 cfu/g rice straw, Among them, the CK group was sprayed with the same amount of water.
  • Gloves Disposable clinker gloves are mixed evenly, put into silage bags, and sealed by vacuum sealing machine. Each bag weighs 300g. After 60 days of fermentation at room temperature, the bags were opened and samples were taken for quality inspection. In addition, the raw materials of rice straw were stored in a -20°C refrigerator for subsequent testing.
  • Sample Determination Determination of sample DM (dry matter), NDF (neutral detergent fiber), ADF (acid detergent fiber), CP (crude protein), WSC (soluble carbohydrate), in vitro digestibility (IVDMD), pH value, organic Acid (lactic acid, acetic acid, propionic acid, butyric acid), ammonia nitrogen content.
  • the lactic acid content in group F increased by 114.53%, 97.57%, and 10.36%, respectively;
  • the addition of acetic acid (AA) had a greater effect, significantly reducing the acetic acid content in the F group and the RF group, and increasing the lactic acid/acetic acid value.
  • the LA/AA (lactic acid/acetic acid) value reflects the homogenous fermentation of lactic acid bacteria in the silage. Except for the R group, the LA/AA value of the F group and the RF group increased by 114.53% and 94.39% compared with the CK group, which was significantly higher than that of the CK group. Among them, the LA/AA value of the F group was the highest.
  • the content of AN (ammonia nitrogen) reflects the decomposition degree of protein in the silage, and the level of the content affects the feeding value of the feed.
  • the ammonia nitrogen is produced by the degradation of crude protein in the original silage by spoilage microorganisms (such as Clostridium). high, indicating that the fermentation effect is worse.
  • the AN content of group F and group RF decreased by 60.63% and 49.84% respectively compared with group CK. It indicated that the addition of Lactobacillus sausage SR2 could effectively reduce the degradation of crude protein in rice straw by spoilage bacteria, thereby improving the quality of silage rice straw.
  • the number of microorganisms is also an important indicator for evaluating the quality of feed fermentation.
  • the number of lactic acid bacteria in the F group and RF group was significantly higher than that in the CK group (P ⁇ 0.001); the number of aerobic bacteria in the F group and RF group was significantly lower than that in the CK group (P ⁇ 0.001).
  • the yeast count in group F was significantly lower than that in other treatment groups (P ⁇ 0.001), and there was little difference between group R, group RF and group CK.
  • the DM (dry matter) content of the F group and RF group was significantly higher than that of the CK group (P ⁇ 0.001), and the DM content of the R group was no different from the CK group; the CP content of the F group was the highest , significantly higher than the CK and F groups.
  • the level of fiber directly affects the feeding and digestive performance of animals.
  • the cellulose content of the F group was the lowest, which was significantly lower than that of the CK and R groups.
  • the IVDMD content of the F group was the highest.
  • the IVDMD increased by 2.25%.

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Abstract

本发明公开了一种香肠乳杆菌SR2及其应用，本发明提供的香肠乳杆菌SR2在酸性pH＝3.0条件下表现出较好的耐受性，在高温45℃及高盐(10％NaCl)环境中有较强的适应性，具有抑制大肠杆菌、金黄色葡萄球菌、沙门氏菌等有害菌的生长，且具有高产阿魏酸酯酶的能力，能显著降低稻秸青贮饲料pH，显著增加乳酸含量，显著减少氨态氮含量，显著增加干物质和WSC含量，显著降低NDF、ADF和纤维素含量，提高稻秸青贮饲料的发酵品质，可广泛应用于稻秸发酵饲料制备领域。

Description

一种香肠乳杆菌SR2及其应用
本申请要求于2022年05月09日提交中国专利局、申请号为CN202210500994.8、发明名称为“一种香肠乳杆菌及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于乳杆菌技术领域，具体涉及一种香肠乳杆菌SR2及其应用。
背景技术
我国是草食家畜生产大国，优质青贮饲料的均衡供给已经成为制约肉羊产业良性发展的瓶颈。因此，充分利用和开发农区潜在的庞大非常规饲料资源成为当务之急。秸秆综合治理已成为头等大事，秸秆禁烧、禁抛与综合利用成为生态文明建设和资源高效利用的重中之重。在当前我国畜牧业发展面临粗饲料资源紧缺的情况下，合理有效地开发利用秸秆成为畜牧业发展的方向。
稻秸中细胞壁成分占80％以上，细胞壁中存在特殊结构(纤维素层次结构和凝聚态结构)，限制了秸秆在动物体内的消化利用。同时，由于稻秸质地坚硬粗糙、粗蛋白质含量低，半纤维素和纤维素被木质素包裹，使稻秸适口性差、难以被最大限度的利用。发酵不仅能够促进稻秸中粗纤维转化为糖类物质，提高营养价值，还能改善其适口性。但由于稻秸茎叶上自然附着乳酸菌少，可溶性碳水化合物(WSC)含量很低，单独青贮很难制成优质的青贮饲料。实际生产中由于受管理条件和天气影响，青贮饲料二次发酵霉变在所难免，霉变的青贮饲料不仅造成饲料营养的损失，而且可引起家畜产奶量下降、下痢、流产和乳房炎等问题，影响家畜的生产性能。所以筛选出优良的乳酸菌菌剂至关重要。
发明内容
本部分的目的在于概述本发明实施例的一些方面以及简要介绍一些较佳实施例。在本部分以及本申请的说明书摘要和发明名称中可能会做些简化或省略，以避免使本部分、说明书摘要和发明名称的目的模糊，而这种简化或省略不能用于限制本发明的范围。
本发明提供了一种香肠乳杆菌SR2，所述香肠乳杆菌SR2保藏编号为CCTCC NO：M 2022416。
本发明还提供所述的香肠乳杆菌SR2作为植物饲料添加剂的应用。
优选的，所述香肠乳杆菌SR2用于植物饲料青贮。
优选的，所述香肠乳杆菌SR2能够高产阿魏酸酯酶。
优选的，所述香肠乳杆菌SR2提高植物饲料中乳酸含量，降低植物饲料中乙酸含量，降低氨态氮含量。
优选的，所述香肠乳杆菌SR2抑制青贮中好氧细菌和酵母菌产生。
优选的，所述香肠乳杆菌SR2显著增加干物质、WSC含量，显著降低NDF、ADF、纤维素含量。
优选的，所述植物饲料包括稻秸。
本发明还提供所述的香肠乳杆菌SR2在制备抑菌制剂中的应用。
优选的，所述香肠乳杆菌SR2抑制大肠杆菌、沙门氏菌和金黄色葡萄球菌。
本发明还提供了一种抑菌制剂，活性成分包括上述技术方案所述的香肠乳杆菌SR2。
本发明的有益效果：本发明提供的香肠乳杆菌SR2在酸性(pH＝3.0)条件下表现出较好的耐受性，在高温45℃及高盐(10％NaCl)环境中有较强的适应性，能够抑制大肠杆菌、金黄色葡萄球菌和沙门氏菌等有害菌的生长，且具有高产阿魏酸酯酶的能力，能显著降低稻秸青贮饲料pH，显著增加乳酸含量，显著减少氨态氮含量，显著增加干物质、WSC含量，显著降低NDF、ADF、纤维素含量，提高稻秸青贮饲料的发酵品质，可广泛应用于稻秸发酵饲料制备领域。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。。
图1为FA标准曲线；
图2为产阿魏酸酯酶菌株的透明圈筛选，其中a为乳酸菌的稀释涂布图，b为产阿魏酸酯酶菌株在平板上产生的透明圈；
图3为形态学鉴定图，其中a为SR1菌落特征，b为SR2菌落特征，c为SR1镜检形态(100×)，d为SR2镜检形态(100×)；
图4为SR1菌株的生长曲线图；
图5为SR2菌株的生长曲线图。
生物保藏说明
香肠乳杆菌(Lactobacillus farciminis)SR2，于2022年04月14日保藏于中国典型培养物保藏中心(CCTCC)，地址为中国武汉市武汉大学，保藏编号为CCTCC No：M 2022416。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是本发明还可以采用其他不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似推广，因此本发明不受下面公开的具体实施例的限制。
其次，此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。
实施例1
香肠乳杆菌的分离和鉴定：
1.分离方法：
1.1样品处理：
选择健壮的湖羊，使用瘤胃导管从胃部预留孔中采集胃液，置于无菌保温瓶中，并迅速带回实验室，在超净工作台上，采用无菌纱布过滤，进而梯度稀释。
1.2产阿魏酸酯酶菌株的初筛：选择三个合适梯度，各取100μL稀释液按三点法加到MRS培养基平板上，均匀涂布后，置于37℃培养箱中倒置厌氧培养48h。挑选形态各异的菌落继续在MRS培养基上反复划线，直至得到纯化的单菌落，用无菌牙签挑取单菌落点接在筛选培养基上，30℃培养12～72h，观察是否在平板上出现明显的透明圈。若出现透明圈，可初步认为该菌株具有产FAE能力。此外，将初筛菌株继续进行革兰氏染色和H 2O 2酶试验，观察该菌株镜检结果是否为紫色单细胞以及H 2O 2酶反应是否为阴性，若符合以上特征，可判定 其为乳酸菌。
1.3产阿魏酸酯酶菌株的复筛：
(1)FA标准曲线的制定：取6个100mL容量瓶，从1～5编号，参照表1加入试剂，每组做3个平行。
表1 FA标准曲线制定
Figure PCTCN2022107666-appb-000001
按上述步骤混匀后，经0.22μm滤膜，进行高效液相色谱(HPLC)测定，测得数值绘制阿魏酸标准曲线，见图1。
由图1可知FA的函数方程为y＝58.225x-392.6，FA浓度(50～200μg/mL)与峰面积的相关系数为R 2＝0.9980，具有良好的线性关系，可用于后续菌株FAE酶活力的测定。
(2)发酵液的准备：将初筛产FAE的乳酸菌移接到MRS液体培养基中，37℃培养过夜后，90％无菌生理盐水洗涤2～3次，菌体重置于去离子水。按5％接种量移接菌悬液于新鲜的产酶液体培养基中发酵，37℃培养48h，测定初筛乳酸菌的FAE活力，为进一步筛选作参考。待测液8000～12000rpm/min离心5min，取得上清发酵液。
(3)酶活测定：取2支试管，参照表2，加入试剂，每组做3个平行。
表2 FAE的活力测定表
Figure PCTCN2022107666-appb-000002
上述步骤完成后，沸水浴10min，8000～12000rpm/min离心5min，取上清过滤膜，进行HPLC测定。根据标准曲线计算阿魏酸的浓度，以每1min内分解 FAE，产生1μmol FA需要的酶量定义成1个酶活单位(U)，计算公式：
酶活(mU/mL)＝反应液中阿魏酸的含量(μg)/阿魏酸摩尔质量×反应时间(min)
(4)HPLC色谱条件：
C18色谱柱：Synergi Hydro-RP80(250×4.6mm 4um)
流动相：A-甲醇；B-1％冰乙酸(28:72)
流速：0.6mL/min
柱温：40℃
检测波长：UV320nm
进样量：10μL。
产阿魏酸酯酶菌株的筛选结果：
本试验将湖羊瘤胃液处理后稀释涂布于MRS培养基中，经分离纯化获得156株乳酸菌，将156株乳酸菌分别点接在以阿魏酸乙酯为唯一碳源的筛选平板上，培养12h后发现，仅有2株编号为SR1和SR2的菌株产生较大的透明圈，可初步判断菌株SR1、SR2能利用碳源阿魏酸乙酯，可能具有FAE活力，其中SR1、SR2的透明圈直径分别为8mm、12mm，与菌株SR1相比，菌株SR2的直径更大，表明该菌株降解阿魏酸乙酯性能更好。具体透明圈结果如图2所示，其中图2的a为乳酸菌的稀释涂布图；图2的b为产阿魏酸酯酶菌株在平板上产生的透明圈。此外，依据透明圈的初筛结果，将菌株SR1、SR2的发酵液按照步骤1.3，通过高效液相色谱法发现菌株SR1、SR2都具有阿魏酸酯酶(FAE)活性，且菌株SR2的酶活力高于SR1，为10.36mU/mL，结果见表3。根据表3可以看出，菌株SR2产阿魏酸酯酶的活性高，为10.36mU/mL，高于菌株SR1，并且高于现有的植物乳杆菌(酶活8.34mU/mL)、乳酸片球菌(酶活5.12mU/mL)和短乳杆菌(酶活7.56mU/mL)产阿魏酸酯酶的酶活。
表3菌株的透明圈大小及其酶活测定
Figure PCTCN2022107666-appb-000003
2.高产阿魏酸酯酶菌株的鉴定：
2.1形态学鉴定
取一环产FAE乳酸菌在MRS培养基上划线，37℃下倒置，具体的，将菌种SR1、SR2分别划线接种于含琼脂的MRS培养基上，37℃厌氧培养48h，观察菌落的形状、颜色、大小及边缘等特征，同时，取单菌落，使用革兰氏染色试剂盒(北京索莱宝科技有限公司)进行染色，镜检菌体形态。结果如图3所述，具体的，菌落特征见图3中a和b，结果显示，菌株SR1在平板上形成乳白色、圆形、边缘规则、中心凸起且表面有光泽的菌落，菌株SR2为中心稍凸起的乳白色菌落，但其边缘不整齐，表面湿润呈扁平状。培养48h后涂片染色结果见图3中c和d，结果显示，SR1、SR2镜检均为G+短杆菌。
2.2生长曲线测定：
取一环乳酸菌接种至MRS液体培养基中，37℃培养，培养48h；再取菌悬液分别接种(按5％接种量)至液体培养基中，37℃培养，培养0～24h，每间隔两小时取一管测定其OD 600nm值和pH值，绘制菌株生长曲线，结果如图4～5所示。
根据图4～5可以看出，随着菌株SR1的生长，培养液pH值逐渐下降，可能是由于菌株SR1在生长过程中产生了乳酸等代谢物质。培养基中乳酸的积累量与菌体SR1的培养时间有关，培养时间越久，产生的乳酸越多，从而导致培养液的pH不断下降，干扰了菌株的生长代谢。从OD值结果来看，0～2h菌株SR1生长缓慢，处在停滞期；2～16h菌株SR1步入生长对数期，生长速率最快；16h后步入平缓期，生长逐渐稳定；24h后，菌株SR1的最终pH为3.94；菌株SR2在生长过程中利用糖产生乳酸，使得培养液酸度不断提高，导致菌体受损，生长受抑制。从OD值与pH来看，菌株SR2在2h后繁殖速度加快，步入对数生长期，同时产酸能力也相应提高，pH值快速下降；18h后逐渐趋于稳定，pH值缓慢降低，24h后菌株SR2的最终pH为3.83。
2.3生长特性鉴定：
2.3.1耐酸试验：
取一环乳酸菌接种至MRS液体培养基中，37℃培养，培养48h；再取菌悬液分别接种(按5％接种量)至不同pH(3.5、4、4.5、5、5.5、6、6.5)的MRS液体培养基中，37℃培养，培养48h，目测菌株生长情况，结果如表4。
2.3.2耐温试验：
取一环乳酸菌接种至MRS液体培养基中，37℃培养，培养48h；再取菌 悬液分别接种(按5％接种量)至MRS液体培养基中，分别在不同温度(5℃、25℃、30℃、37℃、45℃)培养，其中，5℃与15℃下培养120h，25℃与35℃下培养48h，45℃下培养96h，结果如表4。
2.3.3菌株耐盐试验：
取一环乳酸菌接种至MRS液体培养基中，37℃培养，培养48h；再取菌悬液分别接种(按5％接种量)至含3％、6.5％、10％、15％盐浓度的MRS液体培养基中，37℃培养，培养48h，目测菌株生长情况，结果如表4。
表4菌株在不同条件下的生长情况
Figure PCTCN2022107666-appb-000004
注：-，不生长；+，微弱生长；++，一般生长；+++，良好生长。
由表4可知，在pH＝2.0条件下，2株菌均不能生长；在pH＝3.0时，SR1微弱生长，SR2一般生长；在pH值4.0～7.0时，2菌株都能良好的生长。2株菌在5℃条件下，不能生长；在25～37℃条件下，生长良好；而在45℃时，生长微弱。2株菌在含3％～10％NaCl的培养液中生长，而盐浓度达到15％时，2株菌都不生长。表明，菌株SR1能够在pH4.0～7.0、25～37℃、3％盐浓度的MRS 培养液中良好生长；菌株SR2的耐酸能力比SR1高，能够在pH3.0～7.0、25～37℃、3％盐浓度的MRS培养液中很好生长。
2.3.4生化鉴定：
分别挑取乳酸菌的单菌落进行生化检测，使用购买于杭州天和的细菌生化鉴定管进行测定，按反应管试剂盒中说明书方法操作，之后培养2～3d不等，记录菌株反应情况，并结合《乳酸细菌分类鉴定及实验方法》推测菌株大致所属类型，结果如表5。
表5菌株的生化反应情况
Figure PCTCN2022107666-appb-000005
注：+，阳性；-，阴性。
由表5可知，SR1、SR2与乳糖、蔗糖、麦芽糖、葡萄糖等的生化管反应均显示黄色，为阳性，硝酸盐还原试验、接触酶试验均为阴性，SR1发生葡萄糖产气，SR2发生精氨酸产氨现象；SR1与阿拉伯糖、核糖、甘露醇、山梨醇的生化管反应为阴性，而SR2为阴性。以上结果与胡博、章德法等人对鼠李糖乳杆菌及香肠乳杆菌的理化试验鉴定结果基本一致，同时结合凌代文的《乳酸细菌分类鉴定及实验方法》，可初步判断SR1为鼠李糖乳杆菌、SR2为香肠乳杆菌。
2.3.5 16SrDNA法鉴定：
16SrDNA序列鉴定由南京擎科生物公司完成。
16SrDNA法鉴定结果如下：将供试样品的基因组DNA作模板，通过引物对模板进行PCR扩增，得到PCR产物，取产物5μL进行凝胶电泳验证，获得与预期相符大约在1.5kbp处的目的片段。将合格PCR产物送至南京擎科公司测序，SR1、SR2的序列长度分别为1408bp、1478bp，通过DNAStar SeqMan软件对SR1、SR2的原始序列进行校正、拼接，获得有效序列，提交至NCBI，并采用BLAST软件比对分析，结果显示，SR1与Lactobacillus rhamnosus的同源性为98.78％，SR2与Lactobacillus farciminis的相似度为99.73％，符合预期结果。综合菌株SR1、SR2的形态学、理化特征及16SrDNA信息结果，将从湖羊瘤胃液中分离出的具有阿魏酸酯酶活性的菌株SR1、SR2确定为鼠李糖乳杆菌、香肠乳杆菌。
2.3.6抑菌试验：
将冷冻保存的SR2菌株接种于MRS培养基中，在温度37℃下培养24h，如此传代培养2次得到活化的SR2菌株，活化后的SR2菌株按3％的接种量重新转接至MRS液体培养基中，培养24小时，菌液离心得上清液。细菌(大肠杆菌、金黄色葡萄球菌、沙门氏菌)为指示菌，以营养琼脂培养基作为指示细菌的培养基，并进行菌种活化，用生理盐水分别将指示菌液稀释到OD 600吸光度为0.1。向培养皿中倾注10mL高压灭菌过的水琼脂进行铺底，凝固后将灭菌的牛津杯置于琼脂纸上，吸取1mL指示菌菌液加入到100mL恒温至50±5℃的营养肉汤培养基中，摇匀，倒入到放有牛津杯的凝固的琼脂培养基中，每个培养皿倒20mL，冷却凝固。用镊子取出牛津杯，吸取200μL的SR2菌液上清液注入到孔内，以MRS培养液为空白对照，放入4℃冰箱内放置2h，再将培养皿放入37℃培养箱培养，18h观察SR2菌株对指示细菌的抑菌效果，结果如下表6。
表6 SR2抑菌实验结果
病原菌 大肠杆菌 沙门氏菌 金黄色葡萄球菌
抑菌圈(mm) 20.3±0.7 16.5±0.6 17.4±0.5
根据表6可以看出，本发明香肠乳杆菌SR2能够抑制大肠杆菌、沙门氏菌和金黄色葡萄球菌。
综上，与其他细菌相比，香肠乳酸菌SR2的产阿魏酸酯酶的活力要高于乳酸片球菌(4.32mU/mL)、短乳杆菌(7.56mU/mL)。香肠乳杆菌SR2对酸度的 耐受性更强。适合用于青贮的微生物发酵剂应持有一致的发酵途径，既可以利用糖提高产酸量，又需要具备一定的耐酸性，使青贮饲料的pH值尽快地降至4.0，从而阻碍有害微生物活动，改善饲料品质。菌株SR2在酸性(pH＝3.0)条件下均表现出较好的耐受性，且在高温(45℃)及高盐度(10％NaCl)环境中有较强的适应性，具有抑菌效果，且具有产阿魏酸酯酶的能力。
实施例2
香肠乳杆菌SR2在稻秸饲料化利用中的应用效果：
材料与方法：
试验材料：
供试青贮料：于2021年10月，取自江苏省农业科学院粮食作物研究所试验基地收获的去穗粳稻秸秆。
供试菌株：鼠李糖乳杆菌SR1和香肠乳杆菌SR2。
菌剂制取：将鼠李糖乳杆菌SR1和香肠乳杆菌SR2活化后按5％接种量转移至MRS培养基，37℃培养至活菌数达10 8cfu/mL以上，于离心机中10000rpm离心2min去上清获得菌体，用无菌生理盐水将菌体稀释至10 8cfu/mL。
试验设计：
处理组设置：试验共设置鼠李糖乳杆菌SR1(R)、香肠乳杆菌SR2(F)、鼠李糖乳杆菌SR1和香肠乳杆菌SR2(RF)3种添加剂，同时设置无添加剂CK对照组，共4个处理，各组添加量情况如下表7所示。
表7试验设计
Figure PCTCN2022107666-appb-000006
青贮调制：将稻秸切至2～3cm长，把活化好的SR1与和SR2菌株按5×10 5cfu/g稻秸的喷施量，用喷壶雾状喷撒于切断的稻秸中，其中，CK组喷洒等量的水。手套一次性熟料手套混匀，装入青贮袋，使用真空封口机抽气封口，每袋重300g。室温发酵60d后开袋取样进行品质检测。另取稻秸原料放置-20℃ 冰箱保存，已备后续检测。
样品测定：测定样品DM(干物质)、NDF(中性洗涤纤维)、ADF(酸性洗涤纤维)、CP(粗蛋白)、WSC(可溶性碳水化合物)、体外消化率(IVDMD)、pH值、有机酸(乳酸、乙酸、丙酸、丁酸)、氨态氮含量。
实验结果：
不同菌剂处理稻秸青贮饲料的发酵品质分析结果如表8：
表8稻秸青贮60d后的发酵品质分析
Figure PCTCN2022107666-appb-000007
Figure PCTCN2022107666-appb-000008
注：同行标注不同小写字母表示不同菌株处理发酵后差异显著(P<0.05)。PA，丙酸；AA,丁酸。“---”表示未检测到。
由表8可知，稻秸青贮发酵60d后，与CK组相比，所有处理组的pH值均显著低于对照组(P<0.001)，其中，F组处理的稻秸pH值最低(P<0.001)。
有机酸是影响饲料发酵品质的重要因素。与CK组相比，F组、RF组的LA(乳酸)含量显著高于CK组(P<0.001)，其中，F组的LA含量最高，显著高于RF组。添加香肠乳杆菌SR2(F组)处理后促进了乳酸的生成，F组与CK、R、RF三个处理组相比，乳酸含量分别增加了114.53％、97.57％、10.36％；香肠乳杆菌的添加对乙酸(AA)的影响较大，明显降低了F组和RF组的乙酸含量，提高了乳酸/乙酸值。
LA/AA(乳酸/乙酸)值反映了青贮中乳酸菌的同质发酵情况。除R组外，F组和RF组的LA/AA值比CK组增加了114.53％、94.39％，显著高于CK组，其中，F组的LA/AA值最高。
AN(氨态氮)含量反映了青贮料中蛋白质的分解程度，含量高低影响饲料的饲用价值，氨态氮以腐败微生物(如梭菌)降解青贮原样中的粗蛋白而产生，其含量越高，表明发酵效果越差。F组、RF组的AN含量分别比CK组减少了60.63％、49.84％。说明添加香肠乳杆菌SR2能有效减少腐败菌对稻秸原料中粗蛋白的降解，进而改善青贮稻秸的品质。
微生物数目也是评定饲料发酵品质的一项重要指标。F组和RF组的乳酸菌数显著高于CK组(P<0.001)；F组和RF组的好氧细菌数都显著低于CK组(P<0.001)，其中，F组的好氧细菌数最少；F组的酵母菌数显著低于其他处理组(P<0.001)，R组、RF组与CK组之间差异不大。
不同菌剂处理稻秸饲料青贮的营养品质分析结果如表9：
表9稻秸青贮60d后的营养品质分析
Figure PCTCN2022107666-appb-000009
Figure PCTCN2022107666-appb-000010
注：同行标注不同小写字母表示不同菌株处理发酵后差异显著(P<0.05)。
由表9可知，除R组外，F组和RF组的DM(干物质)含量显著高于CK组(P<0.001),R组的DM含量与CK组无差异；F组的CP含量最高，显著高于CK和F组。
WSC含量反映饲料在发酵期间对糖的消耗。F组、RF组的WSC含量分别比CK组增加了42.67％、27.7％，且都显著高于CK组(P＝0.003)。
纤维高低直接影响动物的采食和消化性能。与CK组相比，F组的NDF含量显著低于CK组(P＝0.006)；F组的ADF含量最低，显著低于CK组与R组。F组的纤维素含量最低，显著低于CK组、R组；虽然各组间IVDMD含量差异不显著，但F组的IVDMD含量最高，与CK组相比，IVDMD提高2.25％。有研究发现，添加产阿魏酸乳酸菌苜蓿青贮试验中发现，接种产阿魏酸酯酶乳酸菌没有提高青贮苜蓿的发酵品质及纤维降解能力。而我们的研究发现添加产阿魏酸酯酶的香肠乳杆菌SR2(F组)稻秸饲料中NDF、ADF、纤维素含量降低，碳水化合物含量增加。
SR2序列：
Figure PCTCN2022107666-appb-000011
Figure PCTCN2022107666-appb-000012
SR1序列：
Figure PCTCN2022107666-appb-000013
Figure PCTCN2022107666-appb-000014
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (11)

  1. 一种香肠乳杆菌(Lactobacillus farciminis)SR2，其特征在于：所述香肠乳杆菌SR2保藏编号为CCTCC NO：M 2022416。
  2. 权利要求1所述的香肠乳杆菌SR2作为植物饲料添加剂的应用。
  3. 根据权利要求2所述的应用，其特征在于：所述香肠乳杆菌SR2用于植物饲料青贮。
  4. 根据权利要求2或3所述的应用，其特征在于：所述香肠乳杆菌SR2能够高产阿魏酸酯酶。
  5. 根据权利要求3所述的应用，其特征在于：所述香肠乳杆菌SR2提高植物饲料中乳酸含量，降低植物饲料中乙酸含量，降低氨态氮含量。
  6. 根据权利要求3所述的应用，其特征在于：所述香肠乳杆菌SR2抑制青贮中好氧细菌和酵母菌产生。
  7. 根据权利要求3所述的应用，其特征在于：所述香肠乳杆菌SR2显著增加干物质、可溶性碳水化合物含量，显著降低中性洗涤纤维、酸性洗涤纤维、纤维素含量。
  8. 根据权利要求2或3所述的应用，其特征在于：所述植物饲料包括稻秸。
  9. 权利要求1所述的香肠乳杆菌SR2在制备抑菌制剂中的应用。
  10. 根据权利要求9所述的应用，其特征在于：所述香肠乳杆菌SR2抑制大肠杆菌、沙门氏菌和金黄色葡萄球菌。
  11. 一种抑菌制剂，其特征在于，所述抑菌制剂的活性成分包括权利要求1所述的香肠乳杆菌SR2。
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CN116574630A (zh) * 2023-01-31 2023-08-11 江苏省农业科学院 具有抑菌和抗氧化活性的乳杆菌、适用于秸秆青贮的复合微生物菌剂及其应用

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CN116574630A (zh) * 2023-01-31 2023-08-11 江苏省农业科学院 具有抑菌和抗氧化活性的乳杆菌、适用于秸秆青贮的复合微生物菌剂及其应用
CN116574630B (zh) * 2023-01-31 2024-02-20 江苏省农业科学院 具有抑菌和抗氧化活性的乳杆菌、适用于秸秆青贮的复合微生物菌剂及其应用

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