CN1511945A - Lactobacillus strain and its isolation and breeding method and application - Google Patents
Lactobacillus strain and its isolation and breeding method and application Download PDFInfo
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- CN1511945A CN1511945A CNA021597545A CN02159754A CN1511945A CN 1511945 A CN1511945 A CN 1511945A CN A021597545 A CNA021597545 A CN A021597545A CN 02159754 A CN02159754 A CN 02159754A CN 1511945 A CN1511945 A CN 1511945A
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Abstract
本发明涉及由仔猪胃肠道中分离选育的格氏乳酸杆菌Lactobacillus gasseri,其保藏号为CGMCC No.0840;罗伊氏乳酸杆菌Lactobacillus reuteri,其保藏号为CGMCCNo.0841;嗜酸乳酸杆菌Lactobacillus acidophilus,其保藏号为CGMCC No.0842;和发酵乳酸杆菌Lactobacillus fermentum,其保藏号为CGMCC No.0843;该四种乳酸杆菌组合,用于制备有效防治仔猪断奶腹泻和促进生长的复合益生菌制剂,应用于仔猪断奶后日粮中。可提高仔猪的日增重,解决仔猪断奶后的腹泻问题。The invention relates to Lactobacillus gasseri isolated and selected from the gastrointestinal tract of piglets, the preservation number of which is CGMCC No.0840; Lactobacillus reuteri, whose preservation number is CGMCC No.0841; Lactobacillus acidophilus , whose preservation number is CGMCC No.0842; and fermenting lactobacillus Lactobacillus fermentum, whose preservation number is CGMCC No.0843; the combination of these four kinds of lactobacilli is used to prepare a compound probiotic preparation that can effectively prevent and treat piglet weaning diarrhea and promote growth, It is used in post-weaning diets of piglets. It can increase the daily gain of piglets and solve the problem of diarrhea in piglets after weaning.
Description
发明领域field of invention
本发明属于动物营养学和微生态学领域,特别涉及4种在仔猪消化道内具有互作效应的乳酸杆菌菌种及其分离选育方法和用途,含该乳酸杆菌菌种的复合制剂可应用于断奶仔猪的饲料添加剂,起到仔猪防病促生长效果。The invention belongs to the field of animal nutrition and microecology, and in particular relates to 4 kinds of lactobacillus strains with interactive effects in the digestive tract of piglets and their isolation and breeding methods and uses. The compound preparation containing the lactobacillus strains can be applied to Feed additive for weaned piglets, which can prevent diseases and promote growth of piglets.
背景技术Background technique
饲料添加剂是饲料的核心问题,药物饲料添加剂是随着上个世纪40年代初抗菌素问世后逐渐被广泛使用的。随着抗菌素品种的增多和生产水平的提高,抗菌药物的价格下降,在养殖业集约化程度日益提高的情况下,饲料中添加抗菌药物已成为提高养殖效益的一种有效手段。但是抗菌药物的耐药性和药物在动物性产品中的残留对人类具有潜在危害性。疯牛病、口蹄疫、盐酸克伦特罗中毒和“二恶英”事件等使消费者对食品安全,尤其是动物食品的安全更加重视。从上个世纪90年代以来,欧盟开始禁用和限用抗菌素作为畜禽生长促进剂的法规,2000年,欧盟做出了要在今后若干年内全面禁止抗菌素作为饲料添加剂使用的规定。我国由于饲养环境较差,对抗菌素的依赖更大,在日粮中大量使用抗菌素是限制我国畜牧业发展的重要因素和限制了我国动物产品的出口。断奶仔猪腹泻和生长受抑制是世界性的问题,由于抗菌素的不合理应用产生的耐药性问题,使治疗动物和人类疾病遇到很大困难,研究与开发抗菌素替代品是当前畜牧业和饲料工业的一个热点,益生素是公认的作为抗菌素替代品的一种制剂。但目前市场上的益生素产品作用效果不稳定是影响益生素在养殖业中广泛应用的一个重要原因,产品不稳定的原因主要有益生素产品的作用对象太广泛和产品质量不稳定变异大。Feed additives are the core issue of feed, and pharmaceutical feed additives have been widely used since the advent of antibiotics in the early 1940s. With the increase of antibiotic varieties and the improvement of production level, the price of antibacterial drugs has dropped. In the case of increasing intensification of the aquaculture industry, adding antibacterial drugs to feed has become an effective means to improve breeding efficiency. However, antimicrobial drug resistance and drug residues in animal products are potentially harmful to humans. Mad cow disease, foot-and-mouth disease, clenbuterol poisoning and "dioxin" incidents have made consumers pay more attention to food safety, especially animal food safety. Since the 1990s, the European Union has banned and restricted the use of antibiotics as livestock and poultry growth promoters. In 2000, the European Union made a regulation to completely ban the use of antibiotics as feed additives in the next few years. Due to the poor breeding environment in our country, we are more dependent on antibiotics. The large use of antibiotics in the diet is an important factor restricting the development of my country's animal husbandry and restricting the export of animal products in our country. Diarrhea and growth inhibition of weaned piglets are worldwide problems. Due to the problem of drug resistance caused by the irrational application of antibiotics, it is very difficult to treat animal and human diseases. Research and development of antibiotic substitutes is the current animal husbandry and feed industry. A hot topic in the industry, prebiotics are an agent recognized as an alternative to antibiotics. However, the instability of the effect of prebiotic products on the market is an important reason that affects the wide application of prebiotics in the aquaculture industry. The main reasons for the instability of the products are that the target of the prebiotic products is too wide and the quality of the products is unstable and variable.
发明内容Contents of the invention
本发明的目的在于:针对仔猪消化道的生理特点,分离选育出适合在仔猪胃发挥益生作用的格氏乳酸杆菌,在十二指肠发挥益生作用的罗伊氏乳酸杆菌,在空肠发挥益生作用的嗜酸乳酸杆菌和在结肠发挥益生作用的发酵乳酸杆菌;The purpose of the present invention is to isolate and select Lactobacillus gasseri suitable for exerting probiotic effects in piglet stomach, Lactobacillus reuteri exerting probiotic effect in duodenum, and developing probiotic effect in jejunum according to the physiological characteristics of piglet digestive tract. Lactobacillus acidophilus and Lactobacillus fermentum that exert probiotic effects in the colon;
通过优化上述四种乳酸杆菌的组合,可制备有效防治仔猪断奶腹泻和促进生长的复合益生菌制剂,应用于仔猪断奶后日粮中;By optimizing the combination of the above four kinds of lactobacilli, a compound probiotic preparation that can effectively prevent weaning diarrhea and promote growth of piglets can be prepared and applied to post-weaning diets of piglets;
本发明的另一目的在于,提供一种分离选育上述四种乳酸杆菌的分离选育方法;Another object of the present invention is to provide a method for isolating and breeding the above four kinds of Lactobacillus;
本发明的再一目的在于提供本发明的格氏乳酸杆菌的用途,即与在健康仔猪十二指肠中发挥益生作用的罗伊氏乳酸杆菌、在空肠中发挥益生作用的嗜酸乳酸杆菌和在结肠发挥益生作用的发酵乳酸杆菌组合,用于制备有效防治仔猪断奶腹泻和促进生长的复合益生菌制剂,应用于仔猪断奶后曰粮中。Another object of the present invention is to provide the use of Lactobacillus gasseri of the present invention, that is, Lactobacillus reuteri which exerts a probiotic effect in the duodenum of healthy piglets, and Lactobacillus acidophilus which exerts a probiotic effect in the jejunum and The combination of fermented Lactobacillus exerting probiotic effect in the colon is used to prepare a compound probiotic preparation that can effectively prevent weaning diarrhea and promote growth of piglets, and is used in post-weaning diets of piglets.
本发明的技术方案如下:Technical scheme of the present invention is as follows:
本发明提供的格氏乳酸杆菌,为由仔猪胃粘膜中分离选育的于2002年12月2日保藏于《中国微生物菌种保藏管理委员会普通微生物中心》的格氏乳酸杆菌(Lactobacillus gasseri),其保藏号为CGMCC No.0840;The Lactobacillus gasseri provided by the present invention is the Lactobacillus gasseri (Lactobacillus gasseri) that was isolated and selected from the gastric mucosa of piglets and preserved in the "General Microbiology Center of China Microbiological Culture Collection Management Committee" on December 2, 2002. Its preservation number is CGMCC No.0840;
本发明提供的格氏乳酸杆菌分离选育方法,其分离选育步骤如下:The separation and breeding method of Lactobacillus gasseri provided by the invention, its separation and breeding steps are as follows:
一、分离1. Separation
用Hungates管制备含分离培养基的无菌厌氧滚管:分离培养基组分为10克胰蛋白胨,5克酵母浸粉,10克葡萄糖,5克阿拉伯糖,5克蔗糖,15克乙酸钠,2克枸椽酸铵,6克磷酸二氢钾,0.30克无水硫酸镁,0.12克硫酸锰,0.03克硫酸亚铁,1克吐温-80,13克琼脂,1000毫升蒸馏水,用醋酸调节pH至5.4,再用浓盐酸调pH至4.5;并用Hungates管做成无菌厌氧滚管;Prepare sterile anaerobic roller tubes containing separation medium using Hungates tubes: separation medium components are 10 g tryptone, 5 g yeast extract powder, 10 g glucose, 5 g arabinose, 5 g sucrose, 15 g sodium acetate , 2 g of ammonium citrate, 6 g of potassium dihydrogen phosphate, 0.30 g of anhydrous magnesium sulfate, 0.12 g of manganese sulfate, 0.03 g of ferrous sulfate, 1 g of Tween-80, 13 g of agar, 1000 ml of distilled water, and acetic acid Adjust the pH to 5.4, and then adjust the pH to 4.5 with concentrated hydrochloric acid; and use Hungates tubes to make sterile anaerobic rolling tubes;
取健康断奶仔猪胃底部粘膜于无菌容器中,用无菌生理盐水冲洗除去粘膜上的食糜,将粘膜浸入装有15mlHEPES缓冲液的容器中,摇动5分钟,洗脱粘附在粘膜上的细菌;取其上清液注入装有上述无菌厌氧滚管中,轻轻滚动使上清液均匀分布在分离培养基表面;再放入5%-8%CO2培养箱,37℃培养24~72小时,出现白色针尖大小菌落;24-72小时之后,将上述白色针尖大小菌落分别无菌操作转移到厌氧无菌Rogosa肉汤里,37℃培养24-72小时,分别得到使肉汤变浑浊的菌株培养物;Take the mucous membrane at the bottom of the stomach of healthy weaned piglets in a sterile container, wash it with sterile saline to remove the chyme on the mucous membrane, immerse the mucous membrane in a container containing 15ml of HEPES buffer, shake it for 5 minutes, and wash off the adhering to the mucous membrane bacteria; take the supernatant and inject it into the above-mentioned sterile anaerobic roller tube, roll gently to make the supernatant evenly distributed on the surface of the separation medium; then put it into a 5%-8% CO 2 incubator, 37 After 24-72 hours of cultivation, white pinpoint-sized colonies appeared; after 24-72 hours, the above-mentioned white needle-point-sized colonies were aseptically transferred to anaerobic sterile Rogosa broth, cultured at 37°C for 24-72 hours, and obtained respectively. Cultures of strains whose broth becomes cloudy;
将上述步骤得到的使肉汤变浑浊的菌株培养物分别进行革兰氏染色和接触酶试验,分离出呈革兰氏阳性和接触酶试验为阴性的乳酸杆菌;Carry out Gram staining and contact enzyme test respectively with the bacterial strain culture that makes broth become turbid that above-mentioned step obtains, isolate the lactobacillus that is Gram-positive and contact enzyme test is negative;
所述的厌氧无菌Rogosa肉汤组分包括:10克胰蛋白胨,5克酵母浸粉,10克葡萄糖,5克阿拉伯糖,5克蔗糖,15克乙酸钠,2克枸椽酸铵,6克磷酸二氢钾,0.30克无水硫酸镁,0.12克硫酸锰,0.03克硫酸亚铁,1克吐温-80,蒸馏水1000毫升,并用醋酸调节pH至5.4;Described anaerobic sterile Rogosa broth component comprises: 10 gram tryptones, 5 gram yeast extract powder, 10 gram glucose, 5 gram arabinose, 5 gram sucrose, 15 gram sodium acetate, 2 gram ammonium citrate, 6 grams of potassium dihydrogen phosphate, 0.30 grams of anhydrous magnesium sulfate, 0.12 grams of manganese sulfate, 0.03 grams of ferrous sulfate, 1 gram of Tween-80, 1000 ml of distilled water, and adjust the pH to 5.4 with acetic acid;
二、选育2. Breeding
将上述步骤得到的呈革兰氏阳性和接触酶试验为阴性的乳酸杆菌再进行以下选育:The gram-positive and contact enzyme test obtained by the above steps are negative lactobacillus and then carry out the following selection:
(一)耐酸选育:分别将上述乳酸杆菌24小时的培养物分别按1%接种量(乳酸杆菌与肉汤培养基的体积份配比为1∶100)接种在上述肉汤培养基中,选育出接种12-72小时后,在pH3.0的肉汤培养基中可生长的,即使肉汤变浑浊的耐酸乳酸杆菌;(1) Acid-resistant breeding: inoculate the 24-hour cultures of the above-mentioned lactobacillus in the above-mentioned broth culture medium by 1% inoculation amount (the volume ratio of the lactobacillus and the broth culture medium is 1:100), respectively, After 12-72 hours of inoculation, the acid-resistant Lactobacillus that can grow in the broth medium of pH 3.0, even if the broth becomes cloudy;
所述肉汤培养基组分为:10克胰蛋白胨;10克牛肉浸膏;5克酵母浸粉;2克磷酸氢二钟;2克枸椽酸二铵;20克葡萄糖;0.58克七水硫酸镁;0.25克四水硫酸锰;5克乙酸钠;1ml吐温-80;1000毫升蒸馏水;先冰醋酸调pH至5.2,再将pH用浓盐酸调至3.0;The broth medium components are: 10 gram tryptone; 10 gram beef extract; 5 gram yeast extract powder; 2 gram dibasic hydrogen phosphate; 2 gram diammonium citrate; 20 gram glucose; 0.58 gram heptahydrate Magnesium sulfate; 0.25 g of manganese sulfate tetrahydrate; 5 g of sodium acetate; 1 ml of Tween-80; 1000 ml of distilled water; first adjust the pH to 5.2 with glacial acetic acid, then adjust the pH to 3.0 with concentrated hydrochloric acid;
(二)耐高铜选育:将上述选育的耐酸乳酸杆菌分别与1克五水硫酸铜加热溶解后,混合装进Hungates管,趁热充1.5%CO21分钟,封口,高压灭菌,取出晾凉后,按1%接种量接种至肉汤培养基中,选育出接种后18-72小时能在该肉汤培养基中生长的同时耐酸和耐高铜的乳酸菌株;(2) High-copper-resistant breeding: heat and dissolve the above-mentioned acid-resistant lactobacillus with 1 gram of copper sulfate pentahydrate respectively, mix them into Hungates tubes, fill with 1.5% CO 2 for 1 minute while hot, seal, and autoclave , take it out and let it cool, inoculate it into a broth medium according to 1% inoculation amount, and select and breed lactic acid bacteria strains that can grow in the broth medium 18-72 hours after inoculation while being acid-resistant and high-copper resistant;
所述肉汤培养基组分为:10克胰蛋白胨;10克牛肉浸膏;5克酵母浸粉;2克磷酸氢二钾;2克枸椽酸二铵;20克葡萄糖;0.58克七水硫酸镁;0.25克四水硫酸锰;5克乙酸钠;1ml吐温-80;1000毫升蒸馏水;用冰醋酸调pH至5.4;The broth medium component is: 10 gram tryptone; 10 gram beef extract; 5 gram yeast extract powder; 2 gram dipotassium hydrogen phosphate; 2 gram diammonium citrate; 20 gram glucose; 0.58 gram heptahydrate Magnesium sulfate; 0.25 g of manganese sulfate tetrahydrate; 5 g of sodium acetate; 1 ml of Tween-80; 1000 ml of distilled water; adjust the pH to 5.4 with glacial acetic acid;
(三)抗大肠杆菌选育:(3) Breeding against Escherichia coli:
制作麦康凯培养基,溶解后高压灭菌,无菌操作倾倒平皿;用记号笔在平皿底部划线将平皿分成三个平行的区域,每个区域用接种棒划线接种大肠杆菌(K88、K99和987P)培养液(4.0×108cfu/ml)于培养基表面,然后在距平皿中线约3.5cm的地方挖一宽为0.5cm的沟,补底,沟与划线的培养物垂直;Make MacConkey culture medium, autoclave after dissolving, pour the plate aseptically; draw a line at the bottom of the plate with a marker pen to divide the plate into three parallel areas, and use an inoculation stick to inoculate Escherichia coli (K 88 , K 99 and 987P) culture solution (4.0×10 8 cfu/ml) on the surface of the medium, and then dig a 0.5cm wide groove at a distance of about 3.5cm from the center line of the plate, fill the bottom, and culture the groove and streak vertical;
分别用上述选育出的同时耐酸和耐高铜的乳酸菌18-24小时的培养物将沟填满,置普通培养箱37℃培养18-24小时后,观察出现大肠杆菌菌落的位置,大肠杆菌的菌落为红色,测定离小沟最近的大肠杆菌菌落与小沟边缘的距离;选育出大肠杆菌菌落距离小沟边缘最远的同时耐酸、耐高铜和抗大肠杆菌的乳酸杆菌;Fill the ditch with the 18-24 hour cultures of acid-resistant and high-copper-resistant lactic acid bacteria bred above, and place them in an ordinary incubator at 37°C for 18-24 hours to observe the position where E. coli colonies appear. E. coli The colony is red, and the distance between the E. coli colony closest to the small ditch and the edge of the small ditch is measured; the E. coli colony is the farthest from the edge of the small ditch, and the acid-resistant, copper-resistant and E. coli-resistant lactic acid bacteria are selected at the same time;
(四)乳酸浓度为82mg/100ml的乳酸杆菌的选育:(4) the lactic acid concentration is the breeding of the lactobacillus of 82mg/100ml:
将上述同时耐酸、耐高铜和抗大肠杆菌的乳酸杆菌培养物按1%接种量接种到肉汤培养基中,培养20小时,离心,取上清液,用超纯水稀释10倍后,在Technicon RA100生化仪上用酶法测定上清液中的乳酸浓度;选育得到一株乳酸浓度为82mg/100ml的乳酸杆菌;经中国科学院微生物研究所鉴定,该乳酸杆菌为格氏乳酸杆菌(Lactobacillus gasseri);Inoculate the above-mentioned lactobacillus culture of acid resistance, high copper resistance and Escherichia coli at the same time into the broth medium according to the inoculum size of 1%, cultivate for 20 hours, centrifuge, take the supernatant, and dilute it 10 times with ultrapure water, On the Technicon RA100 biochemical instrument, the lactic acid concentration in the supernatant was determined by enzymatic method; a strain of lactic acid bacteria with a lactic acid concentration of 82mg/100ml was obtained through breeding; identified by the Institute of Microbiology, Chinese Academy of Sciences, the lactobacillus was Lactobacillus gasseri ( Lactobacillus gasseri);
所述肉汤培养基组分为:10克胰蛋白胨;10克牛肉浸膏;5克酵母浸粉;2克磷酸氢二钾;2克枸椽酸二铵;20克葡萄糖;0.58克七水硫酸镁;0.25克四水硫酸锰;5克乙酸钠;1ml吐温-80;1000毫升蒸馏水;用冰醋酸调pH至5.4,混合后装进Hungates管,每管装适量肉汤,趁热充1.5%CO21分钟,封口,高压灭菌;The broth medium component is: 10 gram tryptone; 10 gram beef extract; 5 gram yeast extract powder; 2 gram dipotassium hydrogen phosphate; 2 gram diammonium citrate; 20 gram glucose; 0.58 gram heptahydrate Magnesium sulfate; 0.25 g of manganese sulfate tetrahydrate; 5 g of sodium acetate; 1 ml of Tween-80; 1000 ml of distilled water; adjust the pH to 5.4 with glacial acetic acid, mix and put into Hungates tubes. 1.5% CO 2 for 1 minute, seal and autoclave;
中国科学院微生物研究所对该乳酸杆菌为格氏乳酸杆菌(Lactobacillus gasseri)的鉴定结果如下:
本发明的格氏乳酸杆菌(Lactobacillus gasseri)的生物学特性为:The biological characteristic of Lactobacillus gasseri (Lactobacillus gasseri) of the present invention is:
(一)耐酸存活率:在pH2.0处理6小时之后的存活率为133%;(1) acid-resistant survival rate: the survival rate after 6 hours of pH2.0 treatment was 133%;
其耐酸测试步骤如下:The acid resistance test steps are as follows:
制备肉汤培养基,其组分为:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,溶解后先用冰醋酸调pH至5.6,再用浓盐酸将pH调至2.0;Prepare broth medium, its component is: 10 gram tryptones, 10 gram beef extracts, 5 gram yeast extract powder, 2 gram dipotassium hydrogen phosphate, 2 gram diammonium citrate, 20 gram glucose, 0.58 gram seven Magnesium sulfate water, 0.25 g manganese sulfate tetrahydrate, 5 g sodium acetate, 1 ml Tween-80, 1000 ml distilled water, after dissolving, first adjust the pH to 5.6 with glacial acetic acid, then adjust the pH to 2.0 with concentrated hydrochloric acid;
将制备的肉汤培养基置于Hungates管中,每个管放置20mlMRS肉汤培养基,趁热充1.5%CO21分钟,加塞封口,高压灭菌,凉下来后每管中加1ml的格氏乳酸杆菌培养物,在开始时和第6小时分别取样,进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,每个梯度做3个平行样,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,取平皿中的菌落数50-150的平皿计数,以平均值表示结果;Place the prepared broth medium in Hungates tubes, place 20ml of MRS broth medium in each tube, fill with 1.5% CO 2 for 1 minute while hot, seal with a stopper, and sterilize under high pressure. After cooling down, add 1ml of each tube For the culture of Lactobacillus subtilis, samples were taken at the beginning and at the 6th hour. After gradient dilution, 0.3ml dilution was taken from each gradient and evenly spread on MRS agar (pH5.2), and 3 parallel samples were made for each gradient. , the plate is placed at 37°C, 8.5% CO in a carbon dioxide incubator with a concentration of 24-48 hours, and the number of colonies in the plate is counted for 50-150, and the result is expressed as an average value;
耐酸存活率的计算公式为:The formula for calculating the acid-resistant survival rate is:
S酸=n6/n0 S acid = n 6 /n 0
S酸为经过pH2.0处理后的乳酸杆菌存活率;S acid is the survival rate of lactobacillus after pH2.0 treatment;
n0为pH2.0处理前涂布的每ml活菌数;n 0 is the number of live bacteria per ml coated before pH2.0 treatment;
n6为pH2.0处理6小时的每ml活菌数,得出在pH2.0处理6小时之后的存活率为133%;n 6 is the number of viable bacteria per ml treated at pH 2.0 for 6 hours, and the survival rate after 6 hours at pH 2.0 is 133%;
(二)耐热存活率:本发明的格氏乳酸杆菌的耐热存活率为72%;(2) heat-resistant survival rate: the heat-resistant survival rate of Lactobacillus gasseri of the present invention is 72%;
其耐热存活测试方法如下:Its heat-resistant survival test method is as follows:
制备肉汤培养基,其组分为:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,溶解后用冰醋酸调pH为6.7;Prepare broth medium, its component is: 10 gram tryptones, 10 gram beef extracts, 5 gram yeast extract powder, 2 gram dipotassium hydrogen phosphate, 2 gram diammonium citrate, 20 gram glucose, 0.58 gram seven Magnesium sulfate water, 0.25g manganese sulfate tetrahydrate, 5g sodium acetate, 1ml Tween-80, 1000ml distilled water, adjust the pH to 6.7 with glacial acetic acid after dissolving;
将制备的肉汤培养基置于Hungates管中,每个管放置20mlMRS肉汤,趁热充1.5%CO21分钟,加塞封口,高压灭菌,凉下来后每管中加1ml格氏乳酸杆菌培养物,放置在37℃培养箱中16小时后,取样进行活菌浓度计数,同时将Hungates管放置在75℃水浴中加热15分钟,取样进行活菌浓度计数。取样进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,每个梯度做3个平行样,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,取平皿中的菌落数50-150的平皿计数,以平均值表示结果;Put the prepared broth medium in Hungates tubes, place 20ml of MRS broth in each tube, fill with 1.5% CO 2 for 1 minute while hot, seal with a stopper, autoclave, add 1ml of Lactobacillus gasseri to each tube after cooling down After the culture was placed in a 37°C incubator for 16 hours, samples were taken to count the concentration of viable bacteria. At the same time, the Hungates tube was placed in a water bath at 75°C and heated for 15 minutes, and samples were taken to count the concentration of viable bacteria. After sampling for gradient dilution, take 0.3ml dilution for each gradient and spread evenly on MRS agar (pH5.2), make 3 parallel samples for each gradient, place the plate at 37°C, and culture in carbon dioxide with a concentration of 8.5% CO 2 In the box, cultivate for 24-48 hours, get the plate count of 50-150 bacterial colonies in the plate, and express the result with the average value;
耐热存活率的计算公式为:The formula for calculating the heat-resistant survival rate is:
S热=n1/n0 S heat = n 1 /n 0
S热为经过75℃加热处理后的乳酸杆菌存活率;S heat is the survival rate of lactobacillus after heat treatment at 75°C;
n0为加热处理前每ml的活菌数;n 0 is the number of viable bacteria per ml before heat treatment;
n1为加热处理15分钟后每ml的活菌数,得出耐热存活率为72%;n 1 is the number of viable bacteria per ml after heat treatment for 15 minutes, and the heat-resistant survival rate is 72%;
(三)耐贮藏存活率为38%;(3) The storage-resistant survival rate is 38%;
耐贮藏存活率测试方法如下:The storage survival rate test method is as follows:
制备肉汤培养基,其组分为:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,溶解后用冰醋酸调,pH为6.7;Prepare broth medium, its component is: 10 gram tryptones, 10 gram beef extracts, 5 gram yeast extract powder, 2 gram dipotassium hydrogen phosphate, 2 gram diammonium citrate, 20 gram glucose, 0.58 gram seven Magnesium sulfate water, 0.25g manganese sulfate tetrahydrate, 5g sodium acetate, 1ml Tween-80, 1000ml distilled water, adjust with glacial acetic acid after dissolving, pH is 6.7;
将制备的肉汤培养基置于Hungates滚管中,每个管放置20mlMRS肉汤,趁热充1.5%CO21分钟,加塞封口,高压灭菌,凉下来后每管中加1ml乳酸杆菌培养物,放置在37℃培养箱中16小时后,取样进行活菌浓度计数培养。室温下放置30天后,取样进行活菌浓度计数培养。取出1ml进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,每个梯度做3个平行样,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,取平皿中的菌落数50-150个的平皿计数,以平均值表示结果;Put the prepared broth medium in Hungates roller tubes, place 20ml of MRS broth in each tube, fill with 1.5% CO 2 for 1 minute while hot, seal with a stopper, autoclave, add 1ml of Lactobacillus to each tube after cooling down After being placed in a 37°C incubator for 16 hours, samples were taken to count and culture the viable bacteria concentration. After standing at room temperature for 30 days, samples were taken to count and culture the viable bacteria concentration. After taking out 1ml for gradient dilution, take 0.3ml dilution for each gradient and spread evenly on MRS agar (pH5.2), make 3 parallel samples for each gradient, and place the plate at 37 ° C, 8.5% CO 2 concentration of carbon dioxide In the incubator, cultivate for 24-48 hours, take the plate count of 50-150 colonies in the plate, and express the result with the average value;
耐贮藏存活率的计算公式为:The formula for calculating the storage-resistant survival rate is:
S贮=n1/n0 S storage =n 1 /n 0
S贮为经过30天贮藏后的乳酸杆菌存活率;S storage is the lactobacillus survival rate after 30 days of storage;
n0为贮藏前每ml的活菌数;n 0 is the number of viable bacteria per ml before storage;
n1为贮藏30天后每ml的活菌数,得出耐贮藏存活率为38%;n 1 is the number of viable bacteria per ml after storage for 30 days, and the storage-resistant survival rate is 38%;
(四)本发明的格氏乳酸杆菌在与大肠杆菌混合培养24小时后可使大肠杆菌(K88、K99和987P)下降105个数量级:(4) Lactobacillus gasseri of the present invention can reduce Escherichia coli (K 88 , K 99 and 987P) by 105 orders of magnitude after being mixed with Escherichia coli for 24 hours:
其测试方法如下:Its test method is as follows:
(五)取5ml格氏乳酸杆菌培养物(108cfu/ml)与15ml无菌普通营养肉汤混合,制成含格氏乳酸杆菌培养物的营养肉汤,接种10%大肠杆菌培养物,大肠杆菌的起始浓度为:107cfu/ml,起始培养液中格氏乳酸杆菌的数量为大肠杆菌的10倍,用0.1N的NaOH调培养液的pH为7.0,置37℃,2.5%CO2培养箱培养24个小时后,取出培养液,培养液经梯度稀释后,取0.3ml稀释液均匀涂布MRS琼脂培养基上,每个梯度的培养液作3个平行样,MRS琼脂培养基置5%CO237℃培养箱培养18-48小时,取菌落数在30-150个的平皿计数,以平均值表示格氏乳酸杆菌的数量,计算每毫升培养液中格氏乳酸杆菌的数量;同样地,在培养24小时后取出培养液稀100倍后,取0.3ml稀释液置于麦康凯琼脂上,均匀涂布,做3个平行样,将麦康凯琼脂平板置37℃,普通培养箱培养18-48个小时,计算每个平板的大肠杆菌菌落数,以平均值表示,其结果为本发明的格氏乳酸杆菌在与大肠杆菌混合培养24小时后可使大肠杆菌(K88、K99和987P)下降105个数量级:(5) Get 5ml of Lactobacillus gasseri culture (10 8 cfu/ml) and mix with 15ml aseptic ordinary nutrient broth to make a nutrient broth containing Lactobacillus gasseri culture, inoculate 10% Escherichia coli culture, The initial concentration of Escherichia coli is: 10 7 cfu/ml, the number of Lactobacillus gasseri in the initial culture solution is 10 times that of Escherichia coli, the pH of the culture solution is adjusted to 7.0 with 0.1N NaOH, set at 37°C, 2.5 After culturing in the % CO2 incubator for 24 hours, take out the culture solution. After the culture solution is serially diluted, take 0.3ml of the diluted solution and spread it evenly on the MRS agar medium. Make 3 parallel samples of the culture solution of each gradient. MRS agar Place the culture medium in a 5% CO 2 37°C incubator for 18-48 hours, count the plates with 30-150 colonies, and use the average value to express the number of Lactobacillus gasseri, and calculate the amount of Lactobacillus gasseri per ml of culture solution Similarly, after 24 hours of cultivation, take out the culture solution and dilute it 100 times, take 0.3ml of the diluted solution and place it on MacConkey agar, spread it evenly, make 3 parallel samples, place the MacConkey agar plate at 37°C, Common incubator is cultivated 18-48 hour, calculates the coliform colony number of each plate, expresses with mean value, and its result is that Lactobacillus gasseri of the present invention can make Escherichia coli (K 88 , K 99 and 987P) drop by 10 5 orders of magnitude:
(六)本发明的格氏乳酸杆菌在培养后20小时达到的最高生长浓度为1010cfu/ml;(6) The highest growth concentration reached by Lactobacillus gasseri of the present invention in 20 hours after cultivation is 10 10 cfu/ml;
其测试方法如下:Its test method is as follows:
制备肉汤培养基,其组分为:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,在500ml的锥形瓶里装300ml的MRS肉汤,高压灭菌并晾凉;Prepare broth medium, its component is: 10 gram tryptones, 10 gram beef extracts, 5 gram yeast extract powder, 2 gram dipotassium hydrogen phosphate, 2 gram diammonium citrate, 20 gram glucose, 0.58 gram seven Magnesium sulfate water, 0.25 g of manganese sulfate tetrahydrate, 5 g of sodium acetate, 1 ml of Tween-80, 1000 ml of distilled water, 300 ml of MRS broth in a 500 ml Erlenmeyer flask, autoclaved and allowed to cool;
晾凉后的肉汤培养基中接种5%的格氏乳酸杆菌培养物,分别在培养后24小时内每个小时、28、32、36、40、44、48小时取培养液进行梯度稀释后,取0.3ml各梯度稀释液注入装有MRS琼脂(pH6.5)的厌氧Hungates管中,用手轻轻滚匀,培养24-48小时计算滚管中的菌落数,以每管中菌落数在50-150个的管的梯度作为计算用,每个梯度做3个平行,以平均值表示结果,其结果为:本发明的格氏乳酸杆菌在培养后20小时达到最高生长浓度为1010cfu/ml。Inoculate 5% Lactobacillus gasseri culture into the cooled broth medium, and take the culture solution every hour, 28, 32, 36, 40, 44, and 48 hours within 24 hours after cultivation for gradient dilution , take 0.3ml of each gradient dilution and inject it into the anaerobic Hungates tube equipped with MRS agar (pH6.5), roll it evenly by hand, and cultivate for 24-48 hours to calculate the number of colonies in the rolling tube, and take the number of colonies in each tube The gradient in the tube of 50-150 is used as calculation, and each gradient is done 3 parallels, expresses the result with the mean value, and the result is: Lactobacillus gasseri of the present invention reaches the highest growth concentration in 20 hours after cultivation and is 10 10 cfu/ml.
本发明还从健康断奶仔猪十二指肠粘膜中分离选育出一种罗伊氏乳酸杆菌(Lactobacillus reuteri),于2002年12月2日保藏于《中国微生物菌种保藏管理委员会普通微生物中心》其保藏号为CGMCC No.0841;The present invention also isolates and breeds a kind of Lactobacillus reuteri (Lactobacillus reuteri) from the duodenal mucosa of healthy weaned piglets. Its preservation number is CGMCC No.0841;
该罗伊氏乳酸杆菌的分离选育步骤如下:The isolation and breeding steps of the Lactobacillus reuteri are as follows:
一、分离1. Separation
用Hungates管制备含分离培养基的无菌厌氧滚管:分离培养基组分为10克胰蛋白胨,5克酵母浸粉,10克葡萄糖,5克阿拉伯糖,5克蔗糖,15克醋酸钠,2克枸椽酸铵,6克磷酸二氢钾,0.30克无水硫酸镁,0.12克硫酸锰,0.03克硫酸亚铁,1克吐温-80,13克琼脂,1000毫升蒸馏水,用醋酸调节pH至5.0;高压蒸汽灭菌,并用Hungates管做成无菌厌氧滚管;Prepare sterile anaerobic roller tubes containing separation medium using Hungates tubes: separation medium components are 10 g tryptone, 5 g yeast extract powder, 10 g glucose, 5 g arabinose, 5 g sucrose, 15 g sodium acetate , 2 g of ammonium citrate, 6 g of potassium dihydrogen phosphate, 0.30 g of anhydrous magnesium sulfate, 0.12 g of manganese sulfate, 0.03 g of ferrous sulfate, 1 g of Tween-80, 13 g of agar, 1000 ml of distilled water, and acetic acid Adjust the pH to 5.0; high-pressure steam sterilization, and use Hungates tubes to make sterile anaerobic roller tubes;
取健康断奶仔猪十二指肠的粘膜于无菌容器中,先用无菌生理盐水冲洗粘膜上的食糜,将粘膜浸入装有15mlHEPES缓冲液的容器中,摇动5分钟,洗脱粘附在粘膜上的细菌;取其上清液注入装有上述含分离培养基的Hungates无菌厌氧滚管中,轻轻滚动使上清液均匀分布在分离培养基表面;再放入5%-8%CO2培养箱,37℃培养24~72小时,出现白色针尖大小菌落;24-72小时之后,在生物无菌操作下,将上述白色针尖大小菌落分别转移到厌氧无菌Rogosa肉汤里,37℃培养24-72小时,分别得到使肉汤变浑浊的菌株培养物;Take the mucous membrane of the duodenum of healthy weaned piglets in a sterile container, wash the chyme on the mucous membrane with sterile physiological saline, immerse the mucous membrane in a container containing 15ml of HEPES buffer, shake it for 5 minutes, and wash off the adhering Bacteria on the mucous membrane; take its supernatant and inject it into the Hungates sterile anaerobic roller tube containing the above-mentioned separation medium, roll gently to make the supernatant evenly distributed on the surface of the separation medium; then put 5%-8 % CO2 incubator, culture at 37°C for 24-72 hours, white pinpoint-sized colonies appear; 24-72 hours later, under biological aseptic operation, transfer the above-mentioned white pinpoint-sized colonies to anaerobic sterile Rogosa broth , cultivated at 37°C for 24-72 hours to obtain strain cultures that make the broth turbid;
所述的厌氧无菌Rogosa肉汤组分:10克胰蛋白胨,5克酵母浸粉,10克葡萄糖,5克阿拉伯糖,5克蔗糖,15克醋酸钠,2克枸椽酸铵,6克磷酸二氢钾,0.30无水硫酸镁克,0.12克硫酸锰,0.03克硫酸亚铁,1克吐温-80,蒸馏水1000毫升,并用醋酸调节pH至5.4;Described anaerobic sterile Rogosa broth component: 10 gram tryptones, 5 gram yeast extract powder, 10 gram glucose, 5 gram arabinose, 5 gram sucrose, 15 gram sodium acetate, 2 gram ammonium citrate, 6 gram of potassium dihydrogen phosphate, 0.30 gram of anhydrous magnesium sulfate, 0.12 gram of manganese sulfate, 0.03 gram of ferrous sulfate, 1 gram of Tween-80, 1000 milliliters of distilled water, and adjust the pH to 5.4 with acetic acid;
将上述步骤得到的使肉汤变浑浊的菌株培养物分别进行革兰氏染色和接触酶试验,分离出呈革兰氏阳性和接触酶试验为阴性的乳酸杆菌;Carry out Gram staining and contact enzyme test respectively with the bacterial strain culture that makes broth become turbid that above-mentioned step obtains, isolate the lactobacillus that is Gram-positive and contact enzyme test is negative;
二、选育2. Breeding
将上述步骤得到的呈革兰氏阳性和接触酶试验为阴性的乳酸杆菌再进行以下选育:The gram-positive and contact enzyme test obtained by the above steps are negative lactobacillus and then carry out the following selection:
(一)耐酸选育:(1) Breeding for acid resistance:
分别将上述乳酸杆菌24小时培养物按1%接种在肉汤培养基中,在试验开始时和第8小时分别用无菌注射器取出1ml进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,计算活菌数,计算存活率。Inoculate the 24-hour culture of the above-mentioned Lactobacillus in the broth medium at 1% respectively, take out 1ml with a sterile syringe at the beginning of the test and the 8th hour for gradient dilution, and take 0.3ml dilution for each gradient in the MRS Spread evenly on the agar (pH5.2), place the plate in a carbon dioxide incubator with 8.5% CO2 concentration at 37° C., and cultivate for 24-48 hours to calculate the number of viable bacteria and calculate the survival rate.
所述肉汤培养基组分为:10克胰蛋白胨;10克牛肉浸膏;5克酵母浸粉;2克磷酸氢二钾;2克枸椽酸二铵;20克葡萄糖;0.58克七水硫酸镁;0.25克四水硫酸锰;5克乙酸钠;1ml吐温-80;1000毫升蒸馏水;先用冰醋酸调pH至5.2,再将pH用浓盐酸调至2.5;The broth medium component is: 10 gram tryptone; 10 gram beef extract; 5 gram yeast extract powder; 2 gram dipotassium hydrogen phosphate; 2 gram diammonium citrate; 20 gram glucose; 0.58 gram heptahydrate Magnesium sulfate; 0.25 g of manganese sulfate tetrahydrate; 5 g of sodium acetate; 1 ml of Tween-80; 1000 ml of distilled water; first adjust the pH to 5.2 with glacial acetic acid, then adjust the pH to 2.5 with concentrated hydrochloric acid;
存活率的计算公式为:The formula for calculating the survival rate is:
S酸=n8/n0 S acid = n 8 /n 0
S酸为经过pH2.5处理后的乳酸杆菌存活率;S acid is the lactobacillus survival rate after pH2.5 treatment;
n0为pH2.5处理前涂布的每ml活菌数;n 0 is the number of viable bacteria per ml coated before pH2.5 treatment;
n8为pH2.5处理8小时的每ml活菌数。n 8 is the number of viable bacteria per ml treated with pH 2.5 for 8 hours.
选育出在pH2.5处理8小时后存活率大于18%的乳酸杆菌;Breeding Lactobacillus with a survival rate greater than 18% after pH 2.5 treatment for 8 hours;
(二)耐胆盐选育(2) Breeding for tolerance to bile salts
制备斜面厌氧琼脂培养基,其组分为:10克胰蛋白胨;10克牛肉浸膏;5克酵母浸粉;2克磷酸氢二钾;2克枸椽酸二铵;20克葡萄糖;0.58克七水硫酸镁;0.25克四水硫酸锰;5克乙酸钠;1ml吐温-80;1000毫升蒸馏水;3克猪胆盐;1000毫升蒸馏水,用冰醋酸调pH至5.2,将上述培养基溶解完后,分装在Hungates管,每管装15ml左右,充1.5%二氧化碳40秒,加盖封口后,高压灭菌,取出后摆成斜面培养基;Prepare slant anaerobic agar medium, its component is: 10 gram tryptones; 10 gram beef extracts; 5 gram yeast extract powder; 2 gram dipotassium hydrogen phosphate; 2 gram diammonium citrate; 20 gram glucose; Magnesium sulfate heptahydrate; 0.25 grams of manganese sulfate tetrahydrate; 5 grams of sodium acetate; 1ml Tween-80; 1000 milliliters of distilled water; 3 grams of pig bile salt; After dissolving, divide into Hungates tubes, each tube contains about 15ml, fill with 1.5% carbon dioxide for 40 seconds, cover and seal, autoclave, take it out and place it as a culture medium on a slant;
分别将上述耐酸性强的乳酸杆菌培养物,用无菌生理盐水稀释1000倍后,取0.2ml稀释液接种到斜面厌氧琼脂培养基中,轻轻摇荡使其均匀分散在培养基表面,置37℃培养箱培养18-48小时;选育出能长出菌落的乳酸杆菌;Dilute the above-mentioned acid-resistant Lactobacillus cultures 1000 times with sterile saline, inoculate 0.2ml of the diluted solution into an anaerobic agar medium on a slant, shake it gently to disperse it evenly on the surface of the medium, and place Cultivate in a 37°C incubator for 18-48 hours; select and breed Lactobacillus that can grow colonies;
(三)耐高铜选育(3) Breeding for high copper resistance
制备肉汤培养基:10克胰蛋白胨;10克牛肉浸膏;5克酵母浸粉;2克磷酸氢二钾;2克枸椽酸二铵;20克葡萄糖;0.58克七水硫酸镁;0.25克四水硫酸锰;5克乙酸钠;1ml吐温-80;1000毫升蒸馏水;用冰醋酸调pH至5.4,将上述成分与1克五水硫酸铜分别加热溶解后,将二者混合装进Hungates管,趁热充1.5%CO21分钟,封口,高压灭菌,取出晾凉;Prepare broth medium: 10 grams of tryptone; 10 grams of beef extract; 5 grams of yeast extract powder; 2 grams of dipotassium hydrogen phosphate; 2 grams of diammonium citrate; 20 grams of glucose; 0.58 grams of magnesium sulfate heptahydrate; 0.25 5 grams of manganese sulfate tetrahydrate; 5 grams of sodium acetate; 1ml Tween-80; 1000 milliliters of distilled water; adjust the pH to 5.4 with glacial acetic acid, heat and dissolve the above ingredients and 1 gram of copper sulfate pentahydrate respectively, and mix the two into Hungates tubes were filled with 1.5% CO 2 for 1 minute while hot, sealed, autoclaved, and taken out to cool;
按1%接种量将上述能长出菌落的乳酸杆菌培养物接种在上述肉汤培养基中,选育出接种后18-72小时能生长的菌株;Inoculate the above-mentioned Lactobacillus culture that can grow colonies in the above-mentioned broth medium according to the inoculation amount of 1%, and select a strain that can grow 18-72 hours after inoculation;
(四)抗大肠杆菌选育(4) Breeding against Escherichia coli
制作麦康凯培养基,溶解后高压灭菌,无菌操作倾倒平皿;用记号笔在平皿底部划线将平皿分成三个平行的区域,每个区域用接种棒划线接种大肠杆菌(K88、K99和987P)培养液(4.0×108cfu/ml)于培养基表面,然后在距平皿中线约3.5cm的地方挖一宽为0.5cm的沟,补底,沟与划线的培养物垂直;Make MacConkey culture medium, autoclave after dissolving, pour the plate aseptically; draw a line at the bottom of the plate with a marker pen to divide the plate into three parallel areas, and use an inoculation stick to inoculate Escherichia coli (K 88 , K 99 and 987P) culture solution (4.0×10 8 cfu/ml) on the surface of the medium, and then dig a 0.5cm wide groove at a distance of about 3.5cm from the center line of the plate, fill the bottom, and culture the groove and streak vertical;
用上述步骤(三)得到的乳酸杆菌培养物将沟填满(不能溢出),置普通培养箱37℃培养18-24小时后,观察出现大肠杆菌菌落的位置,大肠杆菌的菌落为红色,测定离小沟最近的大肠杆菌菌落与小沟边缘的距离;选育出大肠杆菌菌落离小沟边缘最远的乳酸杆菌;Use the lactobacillus culture obtained in the above step (3) to fill up the ditch (do not overflow), put it in an ordinary incubator at 37 ° C for 18-24 hours, observe the position where E. coli colonies appear, the colonies of E. coli are red, and measure The distance from the nearest E. coli colony to the edge of the minor groove; the Lactobacillus with the farthest E. coli colony from the edge of the minor groove was selected;
(五)将步骤(四)得到的大肠杆菌菌落离小沟边缘最远的乳酸杆菌与大肠杆菌混合培养培养24小时后,观察乳酸杆菌与大肠杆菌的数量变化关系,选育出1株能使大肠杆菌数量下降最多且自身数量较高的乳酸杆菌;(5) after the escherichia coli colony that step (4) obtains is farthest away from the lactic acid bacillus and the escherichia coli of the edge of the small ditch are mixed and cultivated for 24 hours, observe the quantitative change relationship between the lactic acid bacteria and escherichia coli, and select and breed 1 strain that can make Escherichia coli decreased the most and Lactobacillus with a higher number itself;
其选育方法如下:Its breeding method is as follows:
分别取5ml大肠杆菌菌落离小沟边缘最远的乳酸杆菌培养物(108cfu/ml)与15ml无菌普通营养肉汤混合,制成含乳酸杆菌培养物的营养肉汤,接种10%大肠杆菌培养物,大肠杆菌的起始浓度为:107cfu/ml,起始培养液中乳酸杆菌的数量为大肠杆菌的10倍,用0.1N的NaOH调培养液的pH为7.0,置37℃,2.5%CO2培养箱培养24个小时后,取出培养液,培养液经梯度稀释后,取0.3ml稀释液均匀涂布在MRS琼脂培养基上,每个梯度作3个平行样,MRS琼脂培养基置5%CO237℃培养箱培养18-48小时,取菌落数在30-150个的平皿计数,以平均值表示乳酸杆菌的数量,计算每毫升培养液中乳酸杆菌的数量;同样地,在培养24小时后取出培养液稀释100倍后,取0.3ml稀释液置于麦康凯琼脂上,均匀涂布,作3个平行样,将麦康凯琼脂平板置37℃普通培养箱培养18-48个小时,计算每个平板的大肠杆菌菌落数,以平均值表示;最后选育出1株能使大肠杆菌数量下降最多且自身数量较高的乳酸杆菌,该乳酸杆菌经中国科学院微生物研究所鉴定为罗伊氏乳酸杆菌(Lactobacillusreuteri);其鉴定结果如下表:
五、本发明选育的罗伊氏乳酸杆菌Lactobacillus reuteri的生物学特性:Five, the biological characteristics of the Lactobacillus reuteri selected and bred by the present invention:
(一)耐酸存活率:本发明的罗伊氏乳酸杆菌经过pH2.0处理6小时之后的存活率为34.4%;(1) Acid-resistant survival rate: the survival rate of Lactobacillus reuteri of the present invention after being treated at pH 2.0 for 6 hours was 34.4%;
其测试方法如下:Its test method is as follows:
制备肉汤培养基,其组分为:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,溶解后先用冰醋酸调pH至5.6,再用浓盐酸将pH调至2.0。置于Hungates管中,每个管放置20mlMRS肉汤,趁热充1.5%CO21分钟,加塞封口,高压灭菌,晾凉;Prepare broth medium, its component is: 10 gram tryptones, 10 gram beef extracts, 5 gram yeast extract powder, 2 gram dipotassium hydrogen phosphate, 2 gram diammonium citrate, 20 gram glucose, 0.58 gram seven Magnesium sulfate water, 0.25 g manganese sulfate tetrahydrate, 5 g sodium acetate, 1 ml Tween-80, 1000 ml distilled water, after dissolving, first adjust the pH to 5.6 with glacial acetic acid, and then adjust the pH to 2.0 with concentrated hydrochloric acid. Place in Hungates tubes, place 20ml of MRS broth in each tube, fill with 1.5% CO 2 for 1 minute while hot, seal with a stopper, autoclave, and let cool;
将本发明的罗伊氏乳酸杆菌培养物放入凉下来的Hungates管中,每管中加1ml罗伊氏乳酸杆菌培养物,在开始时和第6小时分别取样,进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,每个梯度做3个平行样,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,取平皿中的菌落数50-150的平皿计数,以平均值表示结果;The Lactobacillus reuteri culture of the present invention is put into the Hungates tube that cools down, adds 1ml Lactobacillus reuteri culture in every tube, takes a sample respectively at the beginning and the 6th hour, after carrying out gradient dilution, each Gradient 0.3ml diluted solution was evenly spread on MRS agar (pH5.2), each gradient was made 3 parallel samples, and the plate was placed in a carbon dioxide incubator with 8.5% CO2 concentration at 37°C for 24-48 hour, get the plate count of 50-150 bacterial colonies in the plate, and express the result with the average value;
耐酸存活率的计算公式为:The formula for calculating the acid-resistant survival rate is:
S酸=n6/n0 S acid = n 6 /n 0
S酸为经过pH2.0处理后的乳酸杆菌存活率;S acid is the survival rate of lactobacillus after pH2.0 treatment;
n0为pH2.0处理前涂布的每ml活菌数;n 0 is the number of live bacteria per ml coated before pH2.0 treatment;
n8为pH2.0处理6小时的每ml活菌数,得出本发明的罗伊氏乳酸杆菌经过pH2.0n 8 is the number of live bacteria per ml treated with pH2.0 for 6 hours, and the Lactobacillus reuteri of the present invention is passed through pH2.0
处理6小时之后的存活率为34.4%;The survival rate after 6 hours of treatment was 34.4%;
(二)耐热存活率:本发明选育的罗伊氏乳酸杆菌的耐热存活率为31%;(2) heat-resistant survival rate: the heat-resistant survival rate of the Lactobacillus reuteri selected and bred by the present invention is 31%;
其测试方法如下:Its test method is as follows:
制备肉汤培养基,其组分为:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,溶解后用冰醋酸调pH为6.7,置于Hungtes管中,每个管放置20mlMRS肉汤,趁热充1.5%CO21分钟,加塞封口,高压灭菌,晾凉待用;Prepare broth medium, its component is: 10 gram tryptones, 10 gram beef extracts, 5 gram yeast extract powder, 2 gram dipotassium hydrogen phosphate, 2 gram diammonium citrate, 20 gram glucose, 0.58 gram seven Magnesium sulfate water, 0.25 g manganese sulfate tetrahydrate, 5 g sodium acetate, 1 ml Tween-80, 1000 ml distilled water, adjusted pH to 6.7 with glacial acetic acid after dissolving, placed in Hungtes tubes, each tube placed 20 ml MRS broth, Charge it with 1.5% CO 2 for 1 minute while it is hot, seal it with a stopper, sterilize it under high pressure, and let it cool for later use;
将本发明选育的罗伊氏乳酸杆菌培养物加入Hungtes管中,每管中加1ml罗伊氏乳酸杆菌培养物,放置在37℃培养箱中16小时后,取样进行活菌浓度计数,同时将Hungates管放置在75℃水浴中加热15分钟,取样进行活菌浓度计数。取样进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,每个梯度做3个平行样,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,取平皿中的菌落数50-150的平皿计数,以平均值表示结果;Add the Lactobacillus reuteri culture selected by the present invention into the Hungtes tube, add 1ml of the Lactobacillus reuteri culture in each tube, place it in a 37°C incubator for 16 hours, take a sample to count the viable bacteria concentration, and simultaneously Place the Hungates tube in a water bath at 75°C and heat for 15 minutes, and take samples to count the viable bacteria concentration. After sampling for gradient dilution, take 0.3ml dilution for each gradient and spread evenly on MRS agar (pH5.2), make 3 parallel samples for each gradient, place the plate at 37°C, and culture in carbon dioxide with a concentration of 8.5% CO 2 In the box, cultivate for 24-48 hours, get the plate count of 50-150 bacterial colonies in the plate, and express the result with the average value;
耐热存活率的计算公式为:The formula for calculating the heat-resistant survival rate is:
S热=n0/n1 S heat = n 0 /n 1
S热为经过75℃加热处理后的乳酸杆菌存活率;S heat is the survival rate of lactobacillus after heat treatment at 75°C;
n0为加热处理前每ml的活菌数;n 0 is the number of viable bacteria per ml before heat treatment;
n1为加热处理15分钟后每ml的活菌数,得出本发明选育的罗伊氏乳酸杆菌的耐热存活率为31%的结果;n 1 is the number of viable bacteria per ml after heat treatment for 15 minutes, and the heat-resistant survival rate of Lactobacillus reuteri selected and bred by the present invention is 31%;
(三)耐贮藏存活率:本发明选育的罗伊氏乳酸杆菌的耐贮藏存活率为85%;(3) storage-resistant survival rate: the storage-resistant survival rate of the Lactobacillus reuteri selected and bred by the present invention is 85%;
其测试方法如下:Its test method is as follows:
制备肉汤培养基,其组分为:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,溶解后用冰醋酸调,pH为6.7,置于Hungates滚管中,每个管放置20mlMRS肉汤,趁热充1.5%CO21分钟,加塞封口,高压灭菌,晾凉待用;Prepare broth medium, its component is: 10 gram tryptones, 10 gram beef extracts, 5 gram yeast extract powder, 2 gram dipotassium hydrogen phosphate, 2 gram diammonium citrate, 20 gram glucose, 0.58 gram seven Magnesium sulfate water, 0.25 g manganese sulfate tetrahydrate, 5 g sodium acetate, 1 ml Tween-80, 1000 ml distilled water, adjusted with glacial acetic acid after dissolving, pH 6.7, placed in Hungates roller tubes, each tube placed 20 ml MRS meat Soup, filled with 1.5% CO 2 for 1 minute while hot, corked and sealed, autoclaved, let cool for later use;
本发明选育的罗伊氏乳酸杆菌培养物置入Hungates滚管中,每管中加1ml罗伊氏乳酸杆菌培养物,放置在37℃培养箱中16小时后,取样进行活菌浓度计数培养。室温下放置30天后,取样进行活菌浓度计数培养。取出1ml进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,每个梯度做3个平行样,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,取平皿中的菌落数50-150个的平皿计数,以平均值表示结果;The culture of Lactobacillus reuteri bred by the present invention is put into Hungates rolling tubes, and 1ml of Lactobacillus reuteri culture is added to each tube, placed in an incubator at 37°C for 16 hours, and samples are taken to count and cultivate the viable bacteria concentration. After standing at room temperature for 30 days, samples were taken to count and culture the viable bacteria concentration. After taking out 1ml for gradient dilution, take 0.3ml dilution for each gradient and spread evenly on MRS agar (pH5.2), make 3 parallel samples for each gradient, and place the plate at 37 ° C, 8.5% CO 2 concentration of carbon dioxide In the incubator, cultivate for 24-48 hours, take the plate count of 50-150 colonies in the plate, and express the result with the average value;
耐贮藏存活率的计算公式为:The formula for calculating the storage-resistant survival rate is:
S贮=n1/n0 S storage =n 1 /n 0
S贮为经过30天贮藏后的乳酸杆菌存活率;S storage is the lactobacillus survival rate after 30 days of storage;
n0为贮藏前每ml的活菌数;n 0 is the number of viable bacteria per ml before storage;
n1为贮藏30天后每ml的活菌数,得出本发明选育的罗伊氏乳酸杆菌的耐贮藏存活率分别为85%的结果; n1 is the number of live bacteria per ml after storage for 30 days, and the result that the storage-resistant survival rate of the Lactobacillus reuteri selected and bred by the present invention is respectively 85% is obtained;
(四)生长量:本发明选育的罗伊氏乳酸杆菌在培养后18小时达到最高生长浓度为109cfu/ml;(4) Growth amount: the Lactobacillus reuteri selected and bred by the present invention reached the highest growth concentration 18 hours after cultivation and was 10 9 cfu/ml;
其测试方法如下:Its test method is as follows:
制备肉汤培养基,其组分为:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,在500ml的锥形瓶里装300ml的MRS肉汤,高压灭菌,晾凉待用;Prepare broth medium, its component is: 10 gram tryptones, 10 gram beef extracts, 5 gram yeast extract powder, 2 gram dipotassium hydrogen phosphate, 2 gram diammonium citrate, 20 gram glucose, 0.58 gram seven Magnesium sulfate water, 0.25 g manganese sulfate tetrahydrate, 5 g sodium acetate, 1 ml Tween-80, 1000 ml distilled water, 300 ml MRS broth in a 500 ml Erlenmeyer flask, autoclaved, let cool for later use;
将本发明选育的罗伊氏乳酸杆菌培养物按5%接种量接种于肉汤培养基中,分别在培养后24小时内每个小时、28、32、36、40、44、48小时取培养液进行梯度稀释后,取0.3ml各梯度稀释液注入装有MRS琼脂(pH6.5)的厌氧Hungates管中,用手轻轻滚匀,培养24-48小时计算滚管中的菌落数,以每管中菌落数在50-150个的管的梯度作为计算用,每个梯度做3个平行,以平均值表示结果,得出本发明选育的罗伊氏乳酸杆菌在培养后18小时达到最高生长浓度为109cfu/ml。The Lactobacillus reuteri culture selected by the present invention was inoculated in the broth medium according to 5% inoculation amount, and was taken every hour, 28, 32, 36, 40, 44, and 48 hours within 24 hours after cultivation. After the culture solution is serially diluted, take 0.3ml of each gradient dilution and inject it into an anaerobic Hungates tube equipped with MRS agar (pH6.5), roll it gently by hand, and cultivate for 24-48 hours to calculate the number of colonies in the tube. Use the gradient of the tube with 50-150 colonies in each tube as calculation, each gradient is done 3 parallels, and the result is expressed with the average value, and the Lactobacillus reuteri selected and bred by the present invention is obtained after 18 hours of cultivation. The highest growth concentration was 10 9 cfu/ml.
本发明还从健康断奶仔猪空肠粘膜中分离选育出一种嗜酸乳酸杆菌Lactobacillusacidophilus,于2002年12月2日保藏于《中国微生物菌种保藏管理委员会普通微生物中心》其保藏号为CGMCC No.0842;The present invention also isolates and breeds a kind of acidophilus Lactobacillus acidophilus from the jejunal mucosa of healthy weaned piglets, which was preserved in "General Microbiology Center of China Microbiological Culture Collection Management Committee" on December 2, 2002, and its preservation number is CGMCC No. 0842;
该嗜酸乳酸杆菌的分离选育步骤如下:The isolation and breeding steps of the Lactobacillus acidophilus are as follows:
一、分离:1. Separation:
用Hungates管制备含分离培养基的无菌厌氧滚管:分离培养基组分为10克胰蛋白胨,5克酵母浸粉,10克葡萄糖,5克阿拉伯糖,5克蔗糖,15克醋酸钠,2克枸椽酸铵,6克磷酸二氢钾,0.30克无水硫酸镁,0.12克硫酸锰,0.03克硫酸亚铁,1克吐温-80,13克琼脂,1000毫升蒸馏水,用醋酸调节pH至5.0;高压蒸汽灭菌,并在Hungates管做成无菌厌氧滚管;Prepare sterile anaerobic roller tubes containing separation medium using Hungates tubes: separation medium components are 10 g tryptone, 5 g yeast extract powder, 10 g glucose, 5 g arabinose, 5 g sucrose, 15 g sodium acetate , 2 g of ammonium citrate, 6 g of potassium dihydrogen phosphate, 0.30 g of anhydrous magnesium sulfate, 0.12 g of manganese sulfate, 0.03 g of ferrous sulfate, 1 g of Tween-80, 13 g of agar, 1000 ml of distilled water, and acetic acid Adjust the pH to 5.0; autoclave, and make sterile anaerobic roller tubes in Hungates tubes;
分离方法为:取健康断奶仔猪空肠粘膜于无菌容器中,先用无菌生理盐水冲洗粘膜上的食糜,将粘膜浸入装有15mlHEPES缓冲液的容器中,摇动5分钟,洗脱粘附在粘膜上的细菌;取其上清液注入装有上述含分离培养基的Hungates无菌厌氧滚管中,轻轻滚动使上清液均匀分布在分离培养基表面;再放入5%-8%CO2培养箱,37℃培养24~72小时,出现白色针尖大小菌落;24-72小时之后,在生物无菌操作下,将上述白色针尖大小菌落分别转移到厌氧无菌Rogosa肉汤里,37℃培养24-72小时,分别得到使肉汤变浑浊的菌株培养物,将得到的使肉汤变浑浊的菌株培养物分别进行革兰氏染色和接触酶试验,分离出呈革兰氏阳性和接触酶试验为阴性的为乳酸杆菌属;The separation method is as follows: take the jejunal mucosa of healthy weaned piglets in a sterile container, first wash the chyme on the mucosa with sterile physiological saline, immerse the mucosa in a container containing 15ml of HEPES buffer, shake it for 5 minutes, and wash off the chyme on the mucosa. Bacteria on the mucous membrane; take its supernatant and inject it into the Hungates sterile anaerobic roller tube containing the above-mentioned separation medium, roll gently to make the supernatant evenly distributed on the surface of the separation medium; then put 5%-8 % CO2 incubator, culture at 37°C for 24-72 hours, white pinpoint-sized colonies appear; 24-72 hours later, under biological aseptic operation, transfer the above-mentioned white pinpoint-sized colonies to anaerobic sterile Rogosa broth , cultivated at 37°C for 24-72 hours, and respectively obtained strain cultures that made the broth turbid, carried out Gram staining and contact enzyme tests on the obtained strain cultures that made the broth turbid, and isolated Gram Lactobacillus is positive and negative in contact enzyme test;
所述的厌氧无菌Rogosa肉汤的组分包括:10克胰蛋白胨,5克酵母浸粉,10克葡萄糖,5克阿拉伯糖,5克蔗糖,15克醋酸钠,2克枸椽酸铵,6克磷酸二氢钾,0.30克无水硫酸镁,0.12克硫酸锰,0.03克硫酸亚铁,1克吐温-80,1000毫升蒸馏水,并用醋酸调节pH至5.4;The component of described anaerobic aseptic Rogosa broth comprises: 10 gram tryptones, 5 gram yeast extract powder, 10 gram glucose, 5 gram arabinose, 5 gram sucrose, 15 gram sodium acetate, 2 gram ammonium citrate , 6 grams of potassium dihydrogen phosphate, 0.30 grams of anhydrous magnesium sulfate, 0.12 grams of manganese sulfate, 0.03 grams of ferrous sulfate, 1 gram of Tween-80, 1000 milliliters of distilled water, and adjust the pH to 5.4 with acetic acid;
二、选育2. Breeding
将上述步骤得到的呈革兰氏阳性和接触酶试验为阴性的乳酸杆菌再进行以下选育:The gram-positive and contact enzyme test obtained by the above steps are negative lactobacillus and then carry out the following selection:
(一)耐酸选育:(1) Breeding for acid resistance:
制备肉汤培养基,其组分为:10克蛋白胨;10克牛肉浸膏;5克酵母浸粉;2克磷酸氢二钾;2克枸椽酸二铵;20克葡萄糖;0.58克七水硫酸镁;0.25克四水硫酸锰;5克乙酸钠;1ml吐温-80;1000毫升蒸馏水;先用冰醋酸调pH至5.2,再将pH用浓盐酸调至2.5;Prepare broth medium, its component is: 10 gram peptones; 10 gram beef extracts; 5 gram yeast extract powder; 2 gram dipotassium hydrogen phosphate; 2 gram diammonium citrate; 20 gram glucose; 0.58 gram heptahydrate Magnesium sulfate; 0.25 g of manganese sulfate tetrahydrate; 5 g of sodium acetate; 1 ml of Tween-80; 1000 ml of distilled water; first adjust the pH to 5.2 with glacial acetic acid, then adjust the pH to 2.5 with concentrated hydrochloric acid;
分别将得到的乳酸杆菌培养物按1%接种量接种于上述肉汤培养基中,在试验开始时和第8小时分别用无菌注射器取出1ml进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,计算活菌数,计算存活率;Inoculate the obtained Lactobacillus culture into the above-mentioned broth culture medium according to the inoculum amount of 1%, take out 1ml with a sterile syringe respectively at the beginning of the test and the 8th hour for gradient dilution, and take 0.3ml dilution for each gradient Spread evenly on MRS agar (pH5.2), place the plate in a carbon dioxide incubator with a concentration of 8.5% CO at 37°C, and cultivate for 24-48 hours to calculate the number of viable bacteria and calculate the survival rate;
存活率的计算公式为:The formula for calculating the survival rate is:
S酸=n8/n0 S acid = n 8 /n 0
S酸为经过pH2.5处理后的乳酸杆菌存活率;S acid is the lactobacillus survival rate after pH2.5 treatment;
n0为pH2.5处理前涂布的每ml活菌数;n 0 is the number of viable bacteria per ml coated before pH2.5 treatment;
n8为pH2.5处理8小时的每ml活菌数,选育出在pH2.5处理8小时后的存活率大于18%的乳酸杆菌;n 8 is the number of viable bacteria per ml treated at pH 2.5 for 8 hours, and the survival rate of lactobacilli after 8 hours of pH 2.5 treatment is greater than 18%;
(二)耐胆盐选育(2) Breeding for tolerance to bile salts
制备斜面厌氧琼脂培养基,其组分为:10克胰蛋白胨;10克牛肉浸膏;5克酵母浸粉;2克磷酸氢二钾;2克枸椽酸二铵;20克葡萄糖;0.58克七水硫酸镁;0.25克四水硫酸锰;5克乙酸钠;1ml吐温-80;1000毫升蒸馏水;3克猪胆盐;1000毫升蒸馏水,用冰醋酸调pH至5.2,将上述培养基溶解完后,分装在Hungates管,每管装15ml左右,充1.5%二氧化碳1分钟,加盖封口后,高压灭菌,取出后摆成斜面培养基,冷却后待用;Prepare slant anaerobic agar medium, its component is: 10 gram tryptones; 10 gram beef extracts; 5 gram yeast extract powder; 2 gram dipotassium hydrogen phosphate; 2 gram diammonium citrate; 20 gram glucose; Magnesium sulfate heptahydrate; 0.25 grams of manganese sulfate tetrahydrate; 5 grams of sodium acetate; 1ml Tween-80; 1000 milliliters of distilled water; 3 grams of pig bile salt; After dissolving, divide into Hungates tubes, each tube contains about 15ml, fill with 1.5% carbon dioxide for 1 minute, cover and seal, autoclave, take it out and place it as a culture medium on a slant, and cool it for later use;
将上述步骤选育出的耐酸性强的乳酸杆菌培养物用无菌生理盐水稀释1000倍后,取0.2ml稀释液接种,轻轻摇荡使乳酸杆菌均匀分散在培养基表面,置37℃培养箱培养18-48小时,选育出能长出菌落的乳酸杆菌;Dilute the acid-resistant Lactobacillus culture bred by the above steps 1000 times with sterile saline, inoculate with 0.2ml of the diluted solution, shake gently to disperse the Lactobacillus evenly on the surface of the medium, and place it in a 37°C incubator Cultivate for 18-48 hours to select and breed lactobacilli that can grow colonies;
(三)耐高铜选育(3) Breeding for high copper resistance
制备肉汤培养基,其组分为:10克胰蛋白胨;10克牛肉浸膏;5克酵母浸粉;2克磷酸氢二钾;2克枸椽酸二铵;20克葡萄糖;0.58克七水硫酸镁;0.25克四水硫酸锰;5克乙酸钠;1ml吐温-80;1000毫升蒸馏水;用冰醋酸调pH至5.4,将上述成分与1克五水硫酸铜分别加热溶解后,将二者混合装进Hungates管,趁热充1.5%CO21分钟,封口,高压灭菌,取出晾凉后待用;Prepare broth medium, its component is: 10 gram tryptones; 10 gram beef extracts; 5 gram yeast extract powder; 2 gram dipotassium hydrogen phosphate; 2 gram diammonium citrate; 20 gram glucose; Magnesium sulfate water; 0.25 grams of manganese sulfate tetrahydrate; 5 grams of sodium acetate; 1ml Tween-80; 1000 milliliters of distilled water; adjust the pH to 5.4 with glacial acetic acid, heat and dissolve the above ingredients and 1 gram of copper sulfate pentahydrate respectively, and then Mix the two into a Hungates tube, fill it with 1.5% CO 2 for 1 minute while it is hot, seal it, sterilize it under high pressure, take it out and let it cool for later use;
将步骤(二)选育出的能长出菌落的乳酸杆菌培养物按1%接种量分别接种在上述肉汤培养基中,选育出接种后18-72小时能生长的菌株;Inoculate the lactobacillus cultures capable of growing colonies selected in step (2) into the above-mentioned broth culture medium according to 1% inoculum size, and select and breed bacterial strains that can grow in 18-72 hours after inoculation;
(四)抗大肠杆菌选育(4) Breeding against Escherichia coli
制作麦康凯培养基,溶解后高压灭菌,无菌操作倾倒平皿。用记号笔在平皿底部划线将平皿分成三个平行的区域,每个区域用接种棒划线接种大肠杆菌(K88、K99和987P)培养液(4.0×108cfu/ml)于培养基表面,然后在距平皿中线约3.5cm的地方挖一宽为0.5cm的沟,补底,沟与划线的培养物垂直;Make MacConkey Medium, autoclave after dissolving, and pour the plates aseptically. Draw a line on the bottom of the plate with a marker pen to divide the plate into three parallel areas, each area is streaked with an inoculation stick to inoculate E. coli (K 88 , K 99 and 987P) culture solution (4.0×10 8 cfu/ml) in the base surface, and then dig a 0.5cm wide ditch about 3.5cm away from the center line of the plate, fill the bottom, and the ditch is perpendicular to the cultured line;
用将上述步骤(三)选育出接种后18-72小时能生长的菌株培养物将沟填满(不能溢出),置普通培养箱37℃培养18-24小时后,观察出现大肠杆菌菌落的位置,大肠杆菌的菌落为红色,测定离小沟最近的大肠杆菌菌落与小沟边缘的距离,选育出大肠杆菌菌落离小沟边缘最远的乳酸杆菌;Fill up the ditch with the strain culture that can grow 18-72 hours after inoculation by the above-mentioned step (3), put it in an ordinary incubator at 37°C and cultivate it for 18-24 hours, and observe the occurrence of Escherichia coli colonies position, the colony of Escherichia coli is red, measure the distance from the nearest Escherichia coli colony to the edge of the minor ditch, and select and breed the lactic acid bacteria whose Escherichia coli colony is the farthest from the edge of the minor ditch;
(五)将步骤(四)选育出的大肠杆菌菌落离小沟边缘最远的乳酸杆菌与大肠杆菌混合培养24小时后,观察乳酸杆菌与大肠杆菌的数量变化关系,选育出1株能使大肠杆菌数量下降最多且自身数量较高的乳酸杆菌,该乳酸杆菌经中国科学院微生物研究所鉴定为嗜酸乳酸杆菌(Lactobacillus acidophilus);(5) After the escherichia coli colony selected in step (4) is farthest away from the lactic acid bacilli and escherichia coli on the edge of the small ditch and mixed culture for 24 hours, observe the quantitative change relationship between lactic acid bacteria and escherichia coli, and select and breed a strain capable of Lactobacillus that reduces the number of Escherichia coli the most and has a high number of itself, which was identified as Lactobacillus acidophilus by the Institute of Microbiology, Chinese Academy of Sciences;
其具体方法如下:The specific method is as follows:
分别取5ml乳酸杆菌培养物(108cfu/ml)与15ml无菌普通营养肉汤混合,制成含乳酸杆菌培养物的营养肉汤,接种10%大肠杆菌培养,大肠杆菌的起始浓度为:107cfu/ml,起始培养液中乳酸杆菌的数量为大肠杆菌的10倍,用0.1N的NaOH调培养液的pH为7.0,置37℃,2.5%CO2培养箱培养24个小时后,取出培养液,培养液经梯度稀释后,取0.3ml稀释液均匀涂布MRS琼脂培养基上,每个梯度的培养液作3个平行样,MRS琼脂培养基置5%CO237℃培养箱培养18-48小时,取菌落数在30-150个的平皿计数,以平均值表示乳酸杆菌的数量,计算每毫升培养液中乳酸杆菌的数量。同样地,在培养24小时后取出培养液稀100倍后,取0.3ml稀释液置于麦康凯琼脂上,均匀涂布,做3个平行样,将麦康凯琼脂平板置37℃,普通培养箱培养18-48个小时,计算每个平板的大肠杆菌菌落数,以平均值表示;选育出1株能使大肠杆菌数量下降最多且自身数量较高的乳酸杆菌,该乳酸杆菌经中国科学院微生物研究所鉴定为嗜酸乳酸杆菌(Lactobacillus acidophilus);Take 5ml of lactic acid bacillus culture (10 8 cfu/ml) and mix them with 15ml of sterile common nutrient broth to make a nutrient broth containing lactic acid bacillus culture, inoculate 10% Escherichia coli for culture, and the initial concentration of Escherichia coli is : 10 7 cfu/ml, the number of Lactobacillus in the initial culture solution is 10 times that of Escherichia coli, the pH of the culture solution is adjusted to 7.0 with 0.1N NaOH, and cultured in a 37°C, 2.5% CO 2 incubator for 24 hours Afterwards, take out the culture solution, after the culture solution has been diluted in a gradient, take 0.3ml of the diluted solution and spread it evenly on the MRS agar medium, make 3 parallel samples of the culture solution of each gradient, and place the MRS agar medium in 5% CO 2 at 37°C Cultivate in the incubator for 18-48 hours, count the plates with 30-150 colonies, express the number of lactobacilli with the average value, and calculate the number of lactobacilli in each milliliter of culture solution. Similarly, after 24 hours of cultivation, take out the culture solution and dilute it 100 times, take 0.3ml of the diluted solution and place it on MacConkey agar, spread it evenly, make 3 parallel samples, and place the MacConkey agar plate at 37°C in an ordinary incubator Cultivate for 18-48 hours, calculate the number of E. coli colonies on each plate, and express it as the average value; select and breed a strain of Lactobacillus that can reduce the number of E. coli the most and have a higher number of itself. Identified by the Institute as Lactobacillus acidophilus;
其鉴定结果见下表:
五、其生物学特性5. Its biological characteristics
(一)耐酸存活率:本发明选育的嗜酸乳酸杆菌经过pH2.0处理6小时之后的存活率为69.5%;(1) Acid-resistant survival rate: the survival rate of Lactobacillus acidophilus selected and bred by the present invention is 69.5% after being treated at pH 2.0 for 6 hours;
其测试方法如下:Its test method is as follows:
制备肉汤培养基,其组分为:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,溶解后先用冰醋酸调pH至5.6,再用浓盐酸将pH调至2.0。置于Hungates管中,每个管放置20mlMRS肉汤,趁热充1.5%CO21分钟,加塞封口,高压灭菌,晾凉后待用;Prepare broth medium, its component is: 10 gram tryptones, 10 gram beef extracts, 5 gram yeast extract powder, 2 gram dipotassium hydrogen phosphate, 2 gram diammonium citrate, 20 gram glucose, 0.58 gram seven Magnesium sulfate water, 0.25 g manganese sulfate tetrahydrate, 5 g sodium acetate, 1 ml Tween-80, 1000 ml distilled water, after dissolving, first adjust the pH to 5.6 with glacial acetic acid, and then adjust the pH to 2.0 with concentrated hydrochloric acid. Place in Hungates tubes, place 20ml of MRS broth in each tube, fill with 1.5% CO 2 for 1 minute while hot, stopper and seal, autoclave, and let cool before use;
在晾凉后的Hungates管中,按每管中加1ml嗜酸乳酸杆菌培养物,在开始时和第6小时分别取样,进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,每个梯度做3个平行样,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,取平皿中的菌落数50-150的平皿计数,以平均值表示结果;In the cooled Hungates tube, add 1ml of Lactobacillus acidophilus culture to each tube, take samples at the beginning and the 6th hour respectively, and after gradient dilution, take 0.3ml dilution for each gradient on MRS agar (pH5 .2) Apply evenly on top, make 3 parallel samples for each gradient, place the plate in a carbon dioxide incubator with 8.5% CO2 concentration at 37°C, cultivate for 24-48 hours, take the number of colonies in the plate 50-150 The plate count of , expresses the result with the average value;
耐酸存活率的计算公式为:The formula for calculating the acid-resistant survival rate is:
S酸=n6/n0 S acid = n 6 /n 0
S酸为经过pH2.0处理后的乳酸杆菌存活率;S acid is the survival rate of lactobacillus after pH2.0 treatment;
n0为pH2.0处理前涂布的每ml活菌数;n 0 is the number of live bacteria per ml coated before pH2.0 treatment;
n8为pH2.0处理6小时的每ml活菌数,得出本发明选育的嗜酸乳酸杆菌经过n 8 is the number of live bacteria per ml that pH2.0 is processed for 6 hours, and the acidophilus Lactobacillus selected and bred in the present invention passes through
pH2.0处理6小时之后的存活率为69.5%;The survival rate after pH 2.0 treatment for 6 hours was 69.5%;
(二)耐热存活率:本发明选育的嗜酸乳酸杆菌的耐热存活率为62%;(2) heat-resistant survival rate: the heat-resistant survival rate of the Lactobacillus acidophilus selected and bred by the present invention is 62%;
其测试方法如下:Its test method is as follows:
制备肉汤培养基,其组分:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,溶解后用冰醋酸调pH为6.7,置于Hungates管中,每个管放置20mlMRS肉汤,趁热充1.5%CO21分钟,加塞封口,高压灭菌,晾凉后待用;Prepare broth medium, its component: 10 grams of tryptone, 10 grams of beef extract, 5 grams of yeast extract powder, 2 grams of dipotassium hydrogen phosphate, 2 grams of diammonium citrate, 20 grams of glucose, 0.58 grams of heptahydrate Magnesium sulfate, 0.25 g of manganese sulfate tetrahydrate, 5 g of sodium acetate, 1 ml of Tween-80, 1000 ml of distilled water, adjusted to pH 6.7 with glacial acetic acid after dissolving, placed in Hungates tubes, 20 ml of MRS broth in each tube, while Hot charge with 1.5% CO 2 for 1 minute, stopper and seal, autoclave, let cool before use;
在Hungates管中,按每管中加1ml嗜酸乳酸杆菌培养物,放置在37℃培养箱中16小时后,取样进行活菌浓度计数,同时将Hungates管放置在75℃水浴中加热15分钟,取样进行活菌浓度计数。取样进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,每个梯度做3个平行样,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,取平皿中的菌落数50-150的平皿计数,以平均值表示结果;In the Hungates tubes, add 1ml of Lactobacillus acidophilus culture to each tube, place in a 37°C incubator for 16 hours, take a sample to count the viable bacteria concentration, and place the Hungates tubes in a 75°C water bath to heat for 15 minutes. Sampling was performed to count viable bacteria. After sampling for gradient dilution, take 0.3ml dilution for each gradient and spread evenly on MRS agar (pH5.2), make 3 parallel samples for each gradient, place the plate at 37°C, and culture in carbon dioxide with a concentration of 8.5% CO 2 In the box, cultivate for 24-48 hours, get the plate count of 50-150 bacterial colonies in the plate, and express the result with the average value;
耐热存活率的计算公式为:The formula for calculating the heat-resistant survival rate is:
S热=n1/n0 S heat = n 1 /n 0
S热为经过75℃加热处理后的乳酸杆菌存活率;S heat is the survival rate of lactobacillus after heat treatment at 75°C;
n0为加热处理前每ml的活菌数;n 0 is the number of viable bacteria per ml before heat treatment;
n1为加热处理15分钟后每ml的活菌数,得出本发明选育的嗜酸乳酸杆菌的耐热存活率为62%; n1 is the number of viable bacteria per ml after heat treatment for 15 minutes, and the heat-resistant survival rate of the Lactobacillus acidophilus selected and bred by the present invention is 62%;
(三)耐贮藏存活率:本发明选育的罗伊氏乳酸杆菌的耐贮藏存活率为59%;(3) storage-resistant survival rate: the storage-resistant survival rate of the Lactobacillus reuteri selected and bred by the present invention is 59%;
其测试方法如下:Its test method is as follows:
制备肉汤培养基,其组分为:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,溶解后用冰醋酸调pH为6.7,置于Hungates滚管中,每个管放置20mlMRS肉汤,趁热充1.5%CO21分钟,加塞封口,高压灭菌,晾凉后待用;Prepare broth medium, its component is: 10 gram tryptones, 10 gram beef extracts, 5 gram yeast extract powder, 2 gram dipotassium hydrogen phosphate, 2 gram diammonium citrate, 20 gram glucose, 0.58 gram seven Magnesium sulfate water, 0.25g manganese sulfate tetrahydrate, 5g sodium acetate, 1ml Tween-80, 1000ml distilled water, adjust the pH to 6.7 with glacial acetic acid after dissolving, place in Hungates roller tubes, place 20ml MRS broth in each tube , filled with 1.5% CO 2 for 1 minute while hot, plugged and sealed, autoclaved, and left to cool before use;
在Hungates滚管中,按每管中加1ml嗜酸乳酸杆菌培养物,放置在37℃培养箱中16小时后,取样进行活菌浓度计数培养。室温下放置30天后,取样进行活菌浓度计数培养。取出1ml进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,每个梯度做3个平行样,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,取平皿中的菌落数50-150个的平皿计数,以平均值表示结果;In the Hungates roller tube, add 1ml of Lactobacillus acidophilus culture to each tube, place it in a 37°C incubator for 16 hours, then take a sample to count and culture the viable bacteria concentration. After standing at room temperature for 30 days, samples were taken to count and culture the viable bacteria concentration. After taking out 1ml for gradient dilution, take 0.3ml dilution for each gradient and spread evenly on MRS agar (pH5.2), make 3 parallel samples for each gradient, and place the plate at 37 ° C, 8.5% CO 2 concentration of carbon dioxide In the incubator, cultivate for 24-48 hours, take the plate count of 50-150 colonies in the plate, and express the result with the average value;
耐贮藏存活率的计算公式为:The formula for calculating the storage-resistant survival rate is:
S贮=n1/n0 S storage =n 1 /n 0
S贮为经过30天贮藏后的乳酸杆菌存活率;S storage is the lactobacillus survival rate after 30 days of storage;
n0为贮藏前每ml的活菌数;n 0 is the number of viable bacteria per ml before storage;
n1为贮藏30天后每ml的活菌数,得出本发明选育的罗伊氏乳酸杆菌的耐贮藏存活率为59%; n1 is the number of live bacteria per ml after storage for 30 days, and the storage-resistant survival rate of the Lactobacillus reuteri selected and bred by the present invention is 59%;
(四)生长量:本发明选育的嗜酸乳酸杆菌在培养后18小时达到最高生长浓度为1011cfu/ml;(4) Growth amount: the Lactobacillus acidophilus selected and bred by the present invention reached the highest growth concentration in 18 hours after cultivation and was 10 cfu/ml;
其测试方法如下:Its test method is as follows:
制备肉汤培养物,其组分为:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,在500ml的锥形瓶里装300ml的MRS肉汤,高压灭菌,晾凉后待用;Prepare broth culture, its component is: 10 grams of tryptone, 10 grams of beef extract, 5 grams of yeast extract powder, 2 grams of dipotassium hydrogen phosphate, 2 grams of diammonium citrate, 20 grams of glucose, 0.58 grams of seven Magnesium sulfate water, 0.25 grams of manganese sulfate tetrahydrate, 5 grams of sodium acetate, 1 ml of Tween-80, 1000 milliliters of distilled water, 300 ml of MRS broth in a 500 ml conical flask, autoclaved, and set aside after cooling;
将嗜酸乳酸杆菌培养物按5%接种量分别接种于锥形瓶中,分别在培养后24小时内每个小时、28、32、36、40、44、48小时取培养液进行梯度稀释后,取0.3ml各梯度稀释液注入装有MRS琼脂(pH6.5)的厌氧Hungates管中,用手轻轻滚匀,培养24-48小时计算滚管中的菌落数,以每管中菌落数在50-150个的管的梯度作为计算用,每个梯度做3个平行,以平均值表示结果,本发明选育的嗜酸乳酸杆菌在培养后18小时达到最高生长浓度为1011cfu/ml。The culture of Lactobacillus acidophilus was respectively inoculated into conical flasks according to the inoculum amount of 5%, and the culture solution was taken every hour, 28, 32, 36, 40, 44, and 48 hours within 24 hours after cultivation for gradient dilution. , take 0.3ml of each gradient dilution and inject it into the anaerobic Hungates tube equipped with MRS agar (pH6.5), roll it evenly by hand, and cultivate for 24-48 hours to calculate the number of colonies in the rolling tube, and take the number of colonies in each tube The gradient of 50-150 tubes is used as calculation, and each gradient is done 3 parallels, and the result is expressed with the average value. The acidophilus lactobacillus selected and bred by the present invention reaches the highest growth concentration in 18 hours after cultivation and is 10 11 cfu/ ml.
本发明还从健康断奶仔猪结肠粘膜中分离选育出一种发酵乳酸杆菌(Lactobacillus fermentum),于2002年12月2日保藏于《中国微生物菌种保藏管理委员会普通微生物中心》其保藏号为CGMCC No.0843;The present invention also isolates and breeds a fermenting Lactobacillus (Lactobacillus fermentum) from the colonic mucosa of healthy weaned piglets, which was preserved in the "General Microbiology Center of China Microbiological Culture Collection Management Committee" on December 2, 2002, and its preservation number is CGMCC No.0843;
该发酵乳酸杆菌的分离选育的步骤如下:The step of the separation and selection of this lactobacillus fermentum is as follows:
一、分离1. Separation
用Hungates管制备含分离培养基的无菌厌氧滚管:分离培养基组分为10克胰蛋白胨,5克酵母浸粉,10克葡萄糖,5克阿拉伯糖,5克蔗糖,15克醋酸钠,2克枸椽酸铵,6克磷酸二氢钾,0.30克无水硫酸镁,0.12克硫酸锰,0.03克硫酸亚铁,1克吐温-80,13克琼脂,1000毫升蒸馏水,用醋酸调节pH至5.0;高压蒸汽灭菌,并在Hungates管做成无菌厌氧滚管;Prepare sterile anaerobic roller tubes containing separation medium using Hungates tubes: separation medium components are 10 g tryptone, 5 g yeast extract powder, 10 g glucose, 5 g arabinose, 5 g sucrose, 15 g sodium acetate , 2 g of ammonium citrate, 6 g of potassium dihydrogen phosphate, 0.30 g of anhydrous magnesium sulfate, 0.12 g of manganese sulfate, 0.03 g of ferrous sulfate, 1 g of Tween-80, 13 g of agar, 1000 ml of distilled water, and acetic acid Adjust the pH to 5.0; autoclave, and make sterile anaerobic roller tubes in Hungates tubes;
分别取来自健康断奶仔猪结肠的粘膜于无菌容器中,先用无菌生理盐水冲洗粘膜上的食糜,将粘膜浸入装有15mlHEPES缓冲液的容器杯中,摇动5分钟,洗脱粘附在粘膜上的细菌;取其上清液注入装有上述含分离培养基的Hungates无菌厌氧滚管中,轻轻滚动使上清液均匀分布在分离培养基表面;再放入5%-8%CO2培养箱,37℃培养24~72小时,出现白色针尖大小菌落;24-72小时之后,在生物无菌操作下,将上述白色针尖大小菌落分别转移到厌氧无菌Rogosa肉汤里,37℃培养24-72小时,分别得到使肉汤变浑浊的菌株培养物;Take the mucous membranes from the colon of healthy weaned piglets in sterile containers, wash the chyme on the mucous membranes with sterile physiological saline, immerse the mucous membranes in a container cup filled with 15ml HEPES buffer solution, shake for 5 minutes, and wash off the adhering Bacteria on the mucous membrane; take its supernatant and inject it into the Hungates sterile anaerobic roller tube containing the above-mentioned separation medium, roll gently to make the supernatant evenly distributed on the surface of the separation medium; then put 5%-8 % CO2 incubator, culture at 37°C for 24-72 hours, white pinpoint-sized colonies appear; 24-72 hours later, under biological aseptic operation, transfer the above-mentioned white pinpoint-sized colonies to anaerobic sterile Rogosa broth , cultivated at 37°C for 24-72 hours to obtain strain cultures that make the broth turbid;
所述的厌氧无菌Rogosa肉汤的组分包括:10克胰蛋白胨,5克酵母浸粉,10克葡萄糖,5克阿拉伯糖,5克蔗糖,15克醋酸钠,2克枸椽酸铵,6克磷酸二氢钾,0.30克无水硫酸镁,0.12克硫酸锰,0.03克硫酸亚铁,1克吐温-80,1000毫升蒸馏水,并用醋酸调节pH至5.4;The component of described anaerobic aseptic Rogosa broth comprises: 10 gram tryptones, 5 gram yeast extract powder, 10 gram glucose, 5 gram arabinose, 5 gram sucrose, 15 gram sodium acetate, 2 gram ammonium citrate , 6 grams of potassium dihydrogen phosphate, 0.30 grams of anhydrous magnesium sulfate, 0.12 grams of manganese sulfate, 0.03 grams of ferrous sulfate, 1 gram of Tween-80, 1000 milliliters of distilled water, and adjust the pH to 5.4 with acetic acid;
将上述步骤得到的使肉汤变浑浊的菌株培养物分别进行革兰氏染色和接触酶试验,分离出呈革兰氏阳性和接触酶试验为阴性的乳酸杆菌;Carry out Gram staining and contact enzyme test respectively with the bacterial strain culture that makes broth become turbid that above-mentioned step obtains, isolate the lactobacillus that is Gram-positive and contact enzyme test is negative;
二、选育2. Breeding
将上述步骤得到的呈革兰氏阳性和接触酶试验为阴性的乳酸杆菌进行以下选育:The gram-positive and contact enzyme test obtained by the above steps are negative lactobacilli for the following selection:
(一)耐酸选育:(1) Breeding for acid resistance:
制备肉汤培养基,其组分为:10克胰蛋白胨;10克牛肉浸膏;5克酵母浸粉;2克磷酸氢二钾;2克枸椽酸二铵;20克葡萄糖;0.58克七水硫酸镁;0.25克四水硫酸锰;5克乙酸钠;1ml吐温-80;1000毫升蒸馏水;先用冰醋酸调pH至5.2,再将pH用浓盐酸调至2.5;Prepare broth medium, its component is: 10 gram tryptones; 10 gram beef extracts; 5 gram yeast extract powder; 2 gram dipotassium hydrogen phosphate; 2 gram diammonium citrate; 20 gram glucose; Magnesium sulfate water; 0.25 g manganese sulfate tetrahydrate; 5 g sodium acetate; 1 ml Tween-80; 1000 ml distilled water; first adjust the pH to 5.2 with glacial acetic acid, then adjust the pH to 2.5 with concentrated hydrochloric acid;
分别将上述乳酸杆菌培养物按1%接种量接种在肉汤培养基中,在试验开始时和第8小时分别用无菌注射器取出培养物进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,计算活菌数,计算存活率,得出在pH2.5处理8小时后的存活率大于18%的乳酸杆菌;The above-mentioned Lactobacillus cultures were inoculated in the broth medium at a 1% inoculum amount, and after the culture was taken out with a sterile syringe at the beginning of the test and at the 8th hour for gradient dilution, 0.3ml of the dilution was taken for each gradient. Uniform coating on MRS agar (pH5.2), plate is placed in 37 DEG C, 8.5% CO Concentration in the carbon dioxide incubator of concentration, carry out cultivation 24-48 hour, count viable bacteria number, calculate survival rate, draw at pH2. 5 Lactobacillus whose survival rate is greater than 18% after 8 hours of treatment;
存活率的计算公式为:The formula for calculating the survival rate is:
S酸=n8/n0 S acid = n 8 /n 0
S酸为经过pH2.5处理后的乳酸杆菌存活率;S acid is the lactobacillus survival rate after pH2.5 treatment;
n0为pH2.5处理前涂布的每ml活菌数;n 0 is the number of viable bacteria per ml coated before pH2.5 treatment;
n8为pH2.5处理8小时的每ml活菌数。n 8 is the number of viable bacteria per ml treated with pH 2.5 for 8 hours.
选育出在pH2.5处理8小时后的存活率大于18%的乳酸杆菌;Breeding of Lactobacillus with a survival rate greater than 18% after pH 2.5 treatment for 8 hours;
(二)耐胆盐选育(2) Breeding for tolerance to bile salts
制备斜面厌氧琼脂培养基,其组分为:10克胰蛋白胨;10克牛肉浸膏;5克酵母浸粉;2克磷酸氢二钾;2克枸椽酸二铵;20克葡萄糖;0.58克七水硫酸镁;0.25克四水硫酸锰;5克乙酸钠;1ml吐温-80;1000毫升蒸馏水;3克猪胆盐;1000毫升蒸馏水,用冰醋酸调pH至5.2,将上述培养基溶解完后,分装在Hungates管,每管装15ml左右,充1.5%二氧化碳1分钟,加盖封口后,高压灭菌,取出后摆成斜面培养基,冷却后待用;Prepare slant anaerobic agar medium, its component is: 10 gram tryptones; 10 gram beef extracts; 5 gram yeast extract powder; 2 gram dipotassium hydrogen phosphate; 2 gram diammonium citrate; 20 gram glucose; Magnesium sulfate heptahydrate; 0.25 grams of manganese sulfate tetrahydrate; 5 grams of sodium acetate; 1ml Tween-80; 1000 milliliters of distilled water; 3 grams of pig bile salt; After dissolving, divide into Hungates tubes, each tube contains about 15ml, fill with 1.5% carbon dioxide for 1 minute, cover and seal, autoclave, take it out and place it as a culture medium on a slant, and cool it for later use;
取上述831株耐酸性强的乳酸杆菌培养物0.1ml,用无菌生理盐水稀释1000倍后,取0.2ml稀释液接种,轻轻摇荡使乳酸杆菌均匀分散在培养基表面,置37℃培养箱培养18-48小时。选择123株能长出菌落的乳酸杆菌。Take 0.1ml of the above-mentioned 831 strains of acid-resistant Lactobacillus culture, dilute 1000 times with sterile saline, inoculate with 0.2ml of the diluted solution, shake gently to disperse the Lactobacillus evenly on the surface of the medium, and place in a 37°C incubator Incubate for 18-48 hours. Select 123 lactobacilli that can grow colonies.
(三)耐高铜选育(3) Breeding for high copper resistance
肉汤:10克胰蛋白胨;10克牛肉浸膏;5克酵母浸粉;2克磷酸氢二钾;2克枸椽酸二铵;20克葡萄糖;0.58克七水硫酸镁;0.25克四水硫酸锰;5克乙酸钠;1ml吐温-80;1000毫升蒸馏水;用冰醋酸调pH至5.4,将上述成分与1克五水硫酸铜分别加热溶解后,将二者混合装进Hungates管,趁热充1.5%CO21分钟,封口,高压灭菌,取出凉下来后,按1%接种量分别接种上述123株乳酸杆菌培养物在上述肉汤中,选择51株接种后18-72小时能生长的菌株。Broth: 10 grams of tryptone; 10 grams of beef extract; 5 grams of yeast extract powder; 2 grams of dipotassium hydrogen phosphate; 2 grams of diammonium citrate; 20 grams of glucose; 0.58 grams of magnesium sulfate heptahydrate; 0.25 grams of tetrahydrate Manganese sulfate; 5 g of sodium acetate; 1 ml of Tween-80; 1000 ml of distilled water; adjust the pH to 5.4 with glacial acetic acid, heat and dissolve the above ingredients and 1 g of copper sulfate pentahydrate respectively, mix the two into a Hungates tube, Fill with 1.5% CO 2 while hot for 1 minute, seal, autoclave, take it out and cool down, inoculate the above 123 strains of Lactobacillus cultures in the above broth according to the inoculation amount of 1%, and select 51 strains 18-72 hours after inoculation strains capable of growing.
(四)抗大肠杆菌选育(4) Breeding against Escherichia coli
制作麦康凯培养基,溶解后高压灭菌,无菌操作倾倒平皿。用记号笔在平皿底部划线将平皿分成三个平行的区域,每个区域用接种棒划线接种大肠杆菌(K88、K99和987P)培养液(4.0×108cfu/ml)于培养基表面,然后在距平皿中线约3.5cm的地方挖一宽为0.5cm的沟,补底,沟与划线的培养物垂直。用上述51株乳酸杆菌培养物将沟填满(不能溢出),置普通培养箱37℃培养18-24小时后,观察出现大肠杆菌菌落的位置,大肠杆菌的菌落为红色,测定离小沟最近的大肠杆菌菌落与小沟边缘的距离。选择出现大肠杆菌菌落离小沟边缘最远的2株乳酸杆菌。Make MacConkey Medium, autoclave after dissolving, and pour the plates aseptically. Draw a line on the bottom of the plate with a marker pen to divide the plate into three parallel areas, each area is streaked with an inoculation stick to inoculate E. coli (K 88 , K 99 and 987P) culture solution (4.0×10 8 cfu/ml) in the Base surface, then dig a 0.5cm wide ditch at a distance of about 3.5cm from the center line of the petri dish, fill the bottom, and the ditch is perpendicular to the culture that was marked. Fill the ditch with the above 51 strains of Lactobacillus culture (do not overflow), put it in an ordinary incubator at 37°C for 18-24 hours, and observe the position where E. coli colonies appear. The colonies of E. coli are red, and they are closest to the small ditch. The distance between the E. coli colony and the edge of the minor groove. Select the 2 strains of Lactobacillus whose Escherichia coli colonies are farthest from the edge of the minor ditch.
(五)乳酸杆菌与大肠杆菌混合培养的选育。将上述2株乳酸杆菌与大肠杆菌混合培养24小时后,观察乳酸杆菌与大肠杆菌的数量变化关系。方法如下:(5) Breeding of mixed culture of Lactobacillus and Escherichia coli. After the above two strains of Lactobacillus and Escherichia coli were mixed and cultured for 24 hours, the relationship between the quantity changes of Lactobacillus and Escherichia coli was observed. Methods as below:
分别取5ml乳酸杆菌培养物(108cfu/ml)与15ml无菌普通营养肉汤混合,制成含乳酸杆菌培养物的营养肉汤,接种10%大肠杆菌培养,大肠杆菌的起始浓度为:107cfu/ml,起始培养液中乳酸杆菌的数量为大肠杆菌的10倍,用0.1N的NaOH调培养液的pH为7.0,置37℃,2.5%CO2培养箱培养24个小时后,取出培养液,培养液经梯度稀释后,取0.3ml稀释液均匀涂布MRS琼脂培养基上,每个梯度的培养液作3个平行样,MRS琼脂培养基置5%CO237℃培养箱培养18-48小时,取菌落数在30-150个的平皿计数,以平均值表示乳酸杆菌的数量,计算每毫升培养液中乳酸杆菌的数量。同样地,在培养24小时后取出培养液稀100倍后,取0.3ml稀释液置于麦康凯琼脂上,均匀涂布,作3个平行样,将麦康凯琼脂平板置37℃,普通培养箱培养18-48个小时,计算每个平板的大肠杆菌菌落数,以平均值表示。选育出1株能使大肠杆菌数量下降最多且自身数量较高的乳酸杆菌,该乳酸杆菌经中国科学院微生物研究所鉴定为发酵乳酸杆菌(Lactobacillus fermentum)。Take 5ml of lactic acid bacillus culture (10 8 cfu/ml) and mix them with 15ml of sterile common nutrient broth to make a nutrient broth containing lactic acid bacillus culture, inoculate 10% Escherichia coli for culture, and the initial concentration of Escherichia coli is : 10 7 cfu/ml, the number of Lactobacillus in the initial culture solution is 10 times that of Escherichia coli, the pH of the culture solution is adjusted to 7.0 with 0.1N NaOH, and cultured in a 37°C, 2.5% CO 2 incubator for 24 hours Afterwards, take out the culture solution, after the culture solution has been diluted in a gradient, take 0.3ml of the diluted solution and spread it evenly on the MRS agar medium, make 3 parallel samples of the culture solution of each gradient, and place the MRS agar medium in 5% CO 2 at 37°C Cultivate in the incubator for 18-48 hours, count the plates with 30-150 colonies, express the number of lactobacilli with the average value, and calculate the number of lactobacilli in each milliliter of culture solution. Similarly, after 24 hours of cultivation, take out the culture solution and dilute it 100 times, take 0.3ml of the diluted solution and place it on MacConkey agar, spread it evenly, make 3 parallel samples, put the MacConkey agar plate at 37°C, and put it in an ordinary incubator. Cultivate for 18-48 hours, calculate the number of E. coli colonies on each plate, and express it as the average value. A strain of Lactobacillus that can reduce the number of Escherichia coli the most and has a higher number was selected and bred. This Lactobacillus was identified as Lactobacillus fermentum by the Institute of Microbiology, Chinese Academy of Sciences.
四、鉴定结果
五、生物学特性5. Biological characteristics
(一)耐酸存活率。本发明选育出来的发酵乳酸杆菌经过pH2.0处理6小时之后的存活率为80.9%。方法如下:(1) Acid-resistant survival rate. The survival rate of the lactobacillus fermentum selected and bred by the present invention is 80.9% after being treated at pH 2.0 for 6 hours. Methods as below:
肉汤组分:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,溶解后先用冰醋酸调pH至5.6,再用浓盐酸将pH调至2.0。置于Hungates管中,每个管放置20mlMRS肉汤,趁热充1.5%CO21分钟,加塞封口,高压灭菌,凉下来后每管中加1ml发酵乳酸杆菌培养物,在开始时和第6小时分别取样。进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,每个梯度做3个平行样,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,取平皿中的菌落数50-150的平皿计数,以平均值表示结果。Broth components: 10 grams of tryptone, 10 grams of beef extract, 5 grams of yeast extract powder, 2 grams of dipotassium hydrogen phosphate, 2 grams of diammonium citrate, 20 grams of glucose, 0.58 grams of magnesium sulfate heptahydrate, 0.25 grams Manganese sulfate tetrahydrate, 5 grams of sodium acetate, 1 ml of Tween-80, 1000 ml of distilled water, after dissolving, adjust the pH to 5.6 with glacial acetic acid, and then adjust the pH to 2.0 with concentrated hydrochloric acid. Place in Hungates tubes, place 20ml of MRS broth in each tube, fill with 1.5% CO 2 while hot for 1 minute, stopper and seal, autoclave, add 1ml of Lactobacillus fermentum culture to each tube after cooling down, at the beginning and the first Samples were taken at 6 hours. After performing gradient dilution, take 0.3ml of dilution for each gradient and spread evenly on MRS agar (pH5.2), make 3 parallel samples for each gradient, and place the plate in a carbon dioxide incubator with 8.5% CO2 concentration at 37°C , cultured for 24-48 hours, counted the number of colonies in the plate with 50-150 plate counts, and expressed the results as the average value.
耐酸存活率的计算公式为:The formula for calculating the acid-resistant survival rate is:
S酸=n6/n0 S acid = n 6 /n 0
S酸为经过pH2.0处理后的乳酸杆菌存活率;S acid is the survival rate of lactobacillus after pH2.0 treatment;
n0为pH2.0处理前每ml活菌数;n 0 is the number of viable bacteria per ml before pH2.0 treatment;
n8为pH2.0处理6小时的每ml活菌数。n 8 is the number of viable bacteria per ml treated with pH 2.0 for 6 hours.
(二)耐热存活率。本发明选育出来的发酵乳酸杆菌的耐热存活率为45%。方法如下:(2) Heat-resistant survival rate. The heat-resistant survival rate of the fermenting lactobacillus bred by the invention is 45%. Methods as below:
肉汤组分:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,溶解后用冰醋酸调pH为6.7,置于Hungtes管中,每个管放置20mlMRS肉汤,趁热充1.5%CO21分钟,加塞封口,高压灭菌,凉下来后每管中加1ml发酵乳酸杆菌培养物,放置在37℃培养箱中16小时后,取样进行活菌浓度计数,同时将Hungates管放置在75℃水浴中加热15分钟,取样进行活菌浓度计数。取样进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,每个梯度做3个平行样,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,取平皿中的菌落数50-150的平皿计数,以平均值表示结果。Broth components: 10 grams of tryptone, 10 grams of beef extract, 5 grams of yeast extract powder, 2 grams of dipotassium hydrogen phosphate, 2 grams of diammonium citrate, 20 grams of glucose, 0.58 grams of magnesium sulfate heptahydrate, 0.25 grams Manganese sulfate tetrahydrate, 5g sodium acetate, 1ml Tween-80, 1000ml distilled water, adjust the pH to 6.7 with glacial acetic acid after dissolving, put in Hungtes tubes, put 20ml MRS broth in each tube, fill with 1.5% CO while hot 2 1 minute, stopper and seal, autoclave, add 1ml fermented Lactobacillus culture to each tube after cooling down, place in 37°C incubator for 16 hours, take samples to count the concentration of viable bacteria, and place the Hungates tube at 75 ℃ in a water bath for 15 minutes, and samples were taken to count the concentration of viable bacteria. After sampling for gradient dilution, take 0.3ml dilution for each gradient and spread evenly on MRS agar (pH5.2), make 3 parallel samples for each gradient, place the plate at 37°C, and culture in carbon dioxide with a concentration of 8.5% CO 2 In the box, culture was carried out for 24-48 hours, the number of colonies in the plate was 50-150, and the result was expressed as the average value.
耐热存活率的计算公式为:The formula for calculating the heat-resistant survival rate is:
S热=n1/n0 S heat = n 1 /n 0
S热为经过75℃加热处理后的乳酸杆菌存活率;S heat is the survival rate of lactobacillus after heat treatment at 75°C;
n0为加热处理前每ml的活菌数;n 0 is the number of viable bacteria per ml before heat treatment;
n1为加热处理15分钟后每ml的活菌数。n 1 is the number of viable bacteria per ml after heat treatment for 15 minutes.
(三)耐贮藏存活率。本发明选育出来的罗伊氏乳酸杆菌的耐贮藏存活率分别为78%。方法如下:(3) Storage-resistant survival rate. The storage-resistant survival rate of the Lactobacillus reuteri bred by the invention is 78%. Methods as below:
肉汤组分:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,溶解后用冰醋酸调pH为6.7,置于Hungates滚管中,每个管放置20mlMRS肉汤,趁热充1.5%CO21分钟,加塞封口,高压灭菌,凉下来后每管中加1ml发酵乳酸杆菌培养物,放置在37℃培养箱中16小时后,取样进行活菌浓度计数培养。室温下放置30天后,取样进行活菌浓度计数培养。取出1ml进行梯度稀释后,每个梯度取0.3ml稀释液在MRS琼脂(pH5.2)上均匀涂布,每个梯度作3个平行样,平皿放置在37℃,8.5%CO2浓度的二氧化碳培养箱中,进行培养24-48小时,取平皿中的菌落数50-150个的平皿计数,以平均值表示结果。Broth components: 10 grams of tryptone, 10 grams of beef extract, 5 grams of yeast extract powder, 2 grams of dipotassium hydrogen phosphate, 2 grams of diammonium citrate, 20 grams of glucose, 0.58 grams of magnesium sulfate heptahydrate, 0.25 grams Manganese sulfate tetrahydrate, 5g sodium acetate, 1ml Tween-80, 1000ml distilled water, adjust the pH to 6.7 with glacial acetic acid after dissolving, put in Hungates roller tubes, put 20ml MRS broth in each tube, fill 1.5% while hot CO 2 for 1 minute, stopper and seal, autoclave, add 1ml culture of Lactobacillus fermentum to each tube after cooling down, place in 37°C incubator for 16 hours, take samples for counting culture of viable bacteria concentration. After standing at room temperature for 30 days, samples were taken to count and culture the viable bacteria concentration. After taking out 1ml for gradient dilution, take 0.3ml dilution for each gradient and spread evenly on MRS agar (pH5.2), make 3 parallel samples for each gradient, place the plate at 37°C, 8.5% CO2 concentration of carbon dioxide In the incubator, culture was carried out for 24-48 hours, and the number of colonies in the plate was counted in the range of 50-150, and the results were expressed as the average value.
耐贮藏存活率的计算公式为:The formula for calculating the storage-resistant survival rate is:
S贮=n1/n0 S storage =n 1 /n 0
S贮为经过30天贮藏后的乳酸杆菌存活率;S storage is the lactobacillus survival rate after 30 days of storage;
n0为贮藏前每ml的活菌数;n 0 is the number of viable bacteria per ml before storage;
n1为贮藏30天后每ml的活菌数。n 1 is the number of viable bacteria per ml after storage for 30 days.
(四)生长量。本发明选育的发酵乳酸杆菌在培养后24小时达到最高生长浓度为1011cfu/ml。方法如下:(4) The amount of growth. The fermenting lactobacillus selected and bred by the present invention reaches the highest growth concentration of 10 11 cfu/ml 24 hours after cultivation. Methods as below:
肉汤组分:10克胰蛋白胨,10克牛肉浸膏,5克酵母浸粉,2克磷酸氢二钾,2克枸椽酸二铵,20克葡萄糖,0.58克七水硫酸镁,0.25克四水硫酸锰,5克乙酸钠,1ml吐温-80,1000毫升蒸馏水,在500ml的锥形瓶里装300ml的MRS肉汤,高压灭菌,凉下来接种5%的发酵乳酸杆菌培养物。分别在培养后24小时内每个小时、28、32、36、40、44、48小时取培养液进行梯度稀释后,取0.3ml各梯度稀释液注入装有MRS琼脂(pH6.5)的厌氧Hungates管中,轻轻滚匀,培养24-48小时计算滚管中的菌落数,以每管中菌落数在50-150个的管的梯度作为计算用,每个梯度做3个平行,以平均值表示结果。Broth components: 10 grams of tryptone, 10 grams of beef extract, 5 grams of yeast extract powder, 2 grams of dipotassium hydrogen phosphate, 2 grams of diammonium citrate, 20 grams of glucose, 0.58 grams of magnesium sulfate heptahydrate, 0.25 grams Manganese sulfate tetrahydrate, 5 g of sodium acetate, 1 ml of Tween-80, 1000 ml of distilled water, 300 ml of MRS broth in a 500 ml Erlenmeyer flask, autoclaved, cooled down to inoculate 5% culture of Lactobacillus fermentum. Take the culture solution every hour, 28, 32, 36, 40, 44, and 48 hours within 24 hours after culturing for gradient dilution, and then take 0.3ml of each gradient dilution and inject it into an analyte equipped with MRS agar (pH6.5). In the oxygen Hungates tube, roll gently, culture for 24-48 hours to calculate the number of colonies in the rolling tube, use the gradient of the tube with 50-150 colonies in each tube as the calculation, and do 3 parallels for each gradient, Results are expressed as mean values.
本发明提供的格氏乳酸杆菌的用途为:该格氏乳酸杆菌与在仔猪十二指肠发挥益生作用的罗伊氏乳酸杆菌、在空肠中发挥益生作用的嗜酸乳酸杆菌和在结肠中发挥益生作用的发酵乳酸杆菌组合,用于制备有效防治仔猪断奶腹泻和促进生长的复合益生菌制剂,应用于仔猪断奶后日粮中。The purposes of the Lactobacillus gasseri provided by the present invention are: the Lactobacillus gasseri and the Lactobacillus reuteri which exert a probiotic effect in the duodenum of piglets, the Lactobacillus acidophilus which exerts a probiotic effect in the jejunum, and the Lactobacillus acidophilus which exerts a probiotic effect in the jejunum and the colon. The combination of probiotic Lactobacillus fermentum is used to prepare a compound probiotic preparation that can effectively prevent piglets from weaning diarrhea and promote growth, and is used in post-weaning diets of piglets.
本发明提供的罗伊氏乳酸杆菌的用途为:该罗伊氏乳酸杆菌与在仔猪胃中发挥益生作用的格氏乳酸杆菌、在空肠中发挥益生作用的嗜酸乳酸杆菌和在结肠中发挥益生作用的发酵乳酸杆菌组合,用于制备有效防治仔猪断奶腹泻和促进生长的复合益生菌制剂,应用于仔猪断奶后日粮中。The purposes of the Lactobacillus reuteri provided by the present invention are: the Lactobacillus reuteri and the Lactobacillus gasseri which exert a probiotic effect in the stomach of piglets, the Lactobacillus acidophilus which exert a probiotic effect in the jejunum, and the Lactobacillus acidophilus which exert a probiotic effect in the colon The active Lactobacillus fermentum combination is used to prepare a compound probiotic preparation that can effectively prevent piglets from weaning diarrhea and promote growth, and is used in post-weaning diets of piglets.
本发明提供的嗜酸乳酸杆菌的用途为:该嗜酸乳酸杆菌与在仔猪胃中发挥益生作用的格氏乳酸杆菌、在十二指肠中发挥益生作用的罗伊氏乳酸杆菌和在结肠中发挥益生作用的发酵乳酸杆菌组合,用于制备有效防治仔猪断奶腹泻和促进生长的复合益生菌制剂,应用于仔猪断奶后日粮中。The purpose of the Lactobacillus acidophilus provided by the present invention is: the Lactobacillus acidophilus and the Lactobacillus gasseri which exert a probiotic effect in the stomach of piglets, the Lactobacillus reuteri which exert a probiotic effect in the duodenum and the Lactobacillus reuteri which exert a probiotic effect in the colon The combination of fermented Lactobacillus exerting probiotic effect is used to prepare a compound probiotic preparation that can effectively prevent weaning diarrhea and promote growth of piglets, and is applied to post-weaning diets of piglets.
本发明提供的发酵乳酸杆菌的用途为:该发酵乳酸杆菌与在仔猪胃中发挥益生作用的格氏乳酸杆菌、在十二指肠中发挥益生作用的罗伊氏乳酸杆菌和在空肠中中发挥益生作用的嗜酸乳酸杆菌组合,用于制备有效防治仔猪断奶腹泻和促进生长的复合益生菌制剂,应用于仔猪断奶后日粮中。The use of the fermented Lactobacillus provided by the present invention is: the fermented Lactobacillus and the Lactobacillus gasseri which exert a probiotic effect in the stomach of piglets, the Lactobacillus reuteri which exert a probiotic effect in the duodenum, and the Lactobacillus reuteri which exert a probiotic effect in the jejunum. A combination of Lactobacillus acidophilus with probiotic effect is used to prepare a compound probiotic preparation that can effectively prevent piglets from weaning diarrhea and promote growth, and is used in post-weaning diets of piglets.
本发明提供的含乳酸杆菌的用于断奶仔猪饲料中的复合添加剂,含在仔猪胃中发挥益生作用的格氏乳酸杆菌、在仔猪十二指肠发挥益生作用的罗伊氏乳酸杆菌、在空肠中发挥益生作用的嗜酸乳酸杆菌和在结肠发挥益生作用的发酵乳酸杆菌;The compound additive containing Lactobacillus for weaned piglet feed provided by the present invention contains Lactobacillus gasseri which exerts a probiotic effect in the stomach of piglets, Lactobacillus reuteri which exerts a probiotic effect in the duodenum of piglets, and Lactobacillus reuteri which exerts a probiotic effect in the duodenum of piglets, Lactobacillus acidophilus that plays a prebiotic role in the human body and Lactobacillus fermentum that plays a prebiotic role in the colon;
将分离选育出的4种乳酸杆菌按不同的数量比例关系添加到断奶仔猪曰粮中,以生长性能和腹泻发病情况为指标优化4种乳酸杆菌的组合,试验选择4种乳酸杆菌的6个水平,水平分别为:2.0×104、4×104、6×104、8×104、1×105、1.2×105。选择均匀设计表中6个水平4因素的设计表安排动物试验。每个处理仔猪饲喂基础日粮加不同组合的乳酸杆菌制剂,发酵培养后离心得到的乳酸杆菌分别用稀释剂稀释后,乳酸杆菌复合制剂以0.1%的比例添加到日粮中。21日龄断奶仔猪的体重在6.0-10kg之间,试验设6个处理,每个处理6个重复,每个重复3头仔猪,试验期21天。记录每周的日增重、曰采食量和腹泻情况。经过均匀设计4.0统计软件统计分析和综合比较得到四株乳酸杆菌在仔猪日粮中的合理比例为:格氏乳酸杆菌:4.0-5.0×104cfu/g;罗伊氏乳酸杆菌:6.0-8.0×104cfu/g;嗜酸乳酸杆菌:1.0-1.2×105cfu/g;发酵乳酸杆菌:2.0-4.0×104cfu/g,组成优质高效的益生素产品。The 4 kinds of Lactobacillus isolated and bred were added to the diet of weaned piglets according to different quantitative ratios, and the combination of the 4 Lactobacilli was optimized based on the growth performance and the incidence of diarrhea. Level, the levels are: 2.0×10 4 , 4×10 4 , 6×10 4 , 8×10 4 , 1×10 5 , 1.2×10 5 . Choose the design table with 6 levels and 4 factors in the uniform design table to arrange animal experiments. Piglets in each treatment were fed basic diet plus different combinations of Lactobacillus preparations, and the Lactobacillus obtained by centrifugation after fermentation was diluted with diluent respectively, and the Lactobacillus compound preparation was added to the diet at a ratio of 0.1%. The body weight of the 21-day-old weaned piglets was between 6.0-10kg. The experiment consisted of 6 treatments, 6 repetitions for each treatment, 3 piglets for each repetition, and the test period was 21 days. Daily weight gain, daily feed intake and diarrhea were recorded weekly. After uniform design 4.0 statistical software statistical analysis and comprehensive comparison, the reasonable ratio of the four strains of Lactobacillus in piglet diets is: Lactobacillus grizzar: 4.0-5.0×10 4 cfu/g; Lactobacillus reuteri: 6.0-8.0 ×10 4 cfu/g; Lactobacillus acidophilus: 1.0-1.2×10 5 cfu/g; Lactobacillus fermentum: 2.0-4.0×10 4 cfu/g, forming high-quality and efficient prebiotic products.
具体实施方式Detailed ways
【实施例1】【Example 1】
选择12头21±2日龄断奶的健康杜长大商品杂交仔猪,体重为7.65±1.10公斤,随机分成2组,在代谢笼上单笼饲养,猪代谢室的室温控制在25-27℃,处理组的6头仔猪每天饮水口服液态益生乳酸杆菌制剂,每毫升饮水中各株乳酸杆菌制剂的浓度分别为:格氏乳酸杆菌:4.0×104cfu/ml;罗伊氏乳酸杆菌:6.0×104cfu/ml;嗜酸乳酸杆菌:1.0×105cfu/ml;发酵乳酸杆菌:4.0×104cfu/ml。对照组的6头仔猪饮用自来水作为对照,7天后用大肠杆菌K99、K88和987P的混合培养液(108)口服攻毒,一日二次,共一天,测定攻毒后腹泻发病率和腹泻指数,观察益生菌对仔猪的保护作用,试验期14天。结果表明饮用乳酸杆菌复合制剂组的仔猪的腹泻率比对照组下降69.1%,腹泻指数下降66。Select 12 healthy Du long commercial hybrid piglets weaned at 21 ± 2 days old, with a body weight of 7.65 ± 1.10 kg, randomly divide them into 2 groups, and raise them in single cages on metabolic cages. The room temperature of the pig metabolic room is controlled at 25-27°C. The 6 piglets in the treatment group drank orally liquid probiotic Lactobacillus preparations every day, and the concentrations of each strain of Lactobacillus preparations per milliliter of drinking water were: Lactobacillus gasseri: 4.0×10 4 cfu/ml; Lactobacillus reuteri: 6.0× 10 4 cfu/ml; Lactobacillus acidophilus: 1.0×10 5 cfu/ml; Lactobacillus fermentum: 4.0×10 4 cfu/ml. The 6 piglets in the control group drank tap water as a control, and 7 days later, they were orally challenged with a mixed culture solution (10 8 ) of Escherichia coli K 99 , K 88 and 987P, twice a day, for a total of one day, and the incidence of diarrhea after challenge was determined and diarrhea index, to observe the protective effect of probiotics on piglets, and the test period was 14 days. The results showed that the diarrhea rate of the piglets drinking the lactobacillus compound preparation group decreased by 69.1% compared with the control group, and the diarrhea index decreased by 66%.
【实施例2】[Example 2]
试验选择36头杜长大断奶仔猪,体重为7.64±1.09kg,按体重和性别随机分成2组,每组为一个处理,每组18头,每组小猪随机分成6个小组,每小组3头仔猪一个栏圈,栏圈面积为2.0×2.0米,高层网上饲养,每个栏圈为一个重复,每个处理三圈阉公猪,三圈母猪。试验期间仔猪饲养在全封闭式的保育仔猪舍内,舍内的温度保持在24-27℃。自由采食,自由饮水。基础日粮不含有任何抗生素,仔猪的免疫按猪常规兽医传染病的免疫程序进行。处理1为基础日粮添加0.1%乳酸杆菌复合制剂;乳酸杆菌复合制剂中4种菌的比例:格氏乳酸杆菌:5.0×104cfu/g;罗伊氏乳酸杆菌:8.0×104cfu/g;嗜酸乳酸杆菌:1.2×105cfu/g;发酵乳酸杆菌:2.0×104cfu/g。处理2为基础日粮添加100mg/kg喹乙醇。试验期21天,在试验开始、7天、14天和21天时称仔猪体重,记录每周的日增重、饲料采食量、腹泻指数以及腹泻发病率。试验结果表明益生乳酸杆菌制剂与喹乙醇相比,试验全期可提高仔猪日增重27.6%,提高日采食量18.2%,腹泻率下降19%,腹泻指数下降60。In the experiment, 36 weaned piglets from Dudu were selected, with a body weight of 7.64±1.09kg. They were randomly divided into 2 groups according to body weight and gender, each group was a treatment, and each group had 18 piglets. Each group of piglets was randomly divided into 6 groups, each group 3 One piglet has one pen, the pen area is 2.0×2.0 meters, raised on the high-rise net, each pen is a repetition, and each treatment has three pens for barrows and three pens for sows. During the test period, the piglets were kept in a fully enclosed nursery piglet house, and the temperature in the house was kept at 24-27°C. Free access to food and water. The basal diet did not contain any antibiotics, and the piglets were immunized according to the immunization procedures for routine veterinary infectious diseases in pigs. Treatment 1 added 0.1% Lactobacillus compound preparation to the basal diet; the ratio of the four strains in the Lactobacillus compound preparation: Lactobacillus gasseri: 5.0×10 4 cfu/g; Lactobacillus reuteri: 8.0×10 4 cfu/g g; Lactobacillus acidophilus: 1.2×10 5 cfu/g; Lactobacillus fermentum: 2.0×10 4 cfu/g. Treatment 2 added 100mg/kg olaquindox to the basal diet. The test period was 21 days, and the piglets were weighed at the beginning of the test, 7 days, 14 days and 21 days, and the weekly daily gain, feed intake, diarrhea index and diarrhea incidence were recorded. The test results show that compared with the olaquindox, the probiotic Lactobacillus preparation can increase the daily gain of piglets by 27.6%, increase the daily feed intake by 18.2%, reduce the diarrhea rate by 19%, and reduce the diarrhea index by 60%.
【实施例3】[Example 3]
试验选择72头杜长大断奶仔猪,体重为8.42±1.15kg,按体重和性别随机分成3组,每组为一个处理,每组24头,每组小猪随机分成6个小组,每小组4头仔猪一个栏圈,栏圈面积为2.0×2.0米,高层网上饲养,每个栏圈为一个重复,每个处理三圈阉公猪,三圈母猪。试验期间仔猪饲养在全封闭式的保育仔猪舍内,舍内的温度保持在24-27℃。自由采食,自由饮水。基础日粮不含有任何抗生素,仔猪的免疫按猪常规兽医传染病的免疫程序进行。处理1为对照组,仔猪饲喂基础日粮,基础日粮营养水平按照NRC(1998)的推荐量配制。处理2为基础日粮添加0.1%乳酸杆菌复合制剂;乳酸杆菌复合制剂中4种菌的比例:格氏乳酸杆菌:4.5×104cfu/g;罗伊氏乳酸杆菌:7.0×104cfu/g;嗜酸乳酸杆菌:1.1×105cfu/g;发酵乳酸杆菌:3.0×104cfu/g。处理3为基础日粮添加100mg/kg金霉素。试验期21天,在试验开始、7天、14天和21天时称仔猪体重,记录每周的日增重、饲料采食量、腹泻指数以及腹泻发病率。试验结果表明益生乳酸杆菌制剂与对照组、金霉素相比,分别可提高仔猪日增重20.3%和9%,提高日采食量15.9%和5.2%,腹泻率下降46%和13%,腹泻指数下降69和16。In the experiment, 72 weaned piglets from Dudu were selected, with a body weight of 8.42±1.15kg. They were randomly divided into 3 groups according to body weight and sex, each group was a treatment, and each group had 24 piglets. Each group of piglets was randomly divided into 6 groups, each group 4 One piglet has one pen, the pen area is 2.0×2.0 meters, raised on the high-rise net, each pen is a repetition, and each treatment has three pens for barrows and three pens for sows. During the test period, the piglets were kept in a fully enclosed nursery piglet house, and the temperature in the house was kept at 24-27°C. Free access to food and water. The basal diet did not contain any antibiotics, and the piglets were immunized according to the immunization procedures for routine veterinary infectious diseases in pigs. Treatment 1 was the control group, and the piglets were fed basal rations, and the nutritional level of the basal rations was prepared according to the recommended amount of NRC (1998). Treatment 2 added 0.1% Lactobacillus compound preparation to the basal diet; the ratio of the four strains in the Lactobacillus compound preparation: Lactobacillus gasseri: 4.5×10 4 cfu/g; Lactobacillus reuteri: 7.0×10 4 cfu/g g; Lactobacillus acidophilus: 1.1×10 5 cfu/g; Lactobacillus fermentum: 3.0×10 4 cfu/g. Treatment 3 added 100mg/kg aureomycin to the basal diet. The test period was 21 days, and the piglets were weighed at the beginning of the test, 7 days, 14 days and 21 days, and the weekly daily gain, feed intake, diarrhea index and diarrhea incidence were recorded. The test results show that compared with the control group and aureomycin, the probiotic Lactobacillus preparation can increase the daily gain of piglets by 20.3% and 9%, increase the daily feed intake by 15.9% and 5.2%, and reduce the diarrhea rate by 46% and 13%. Diarrhea index dropped 69 and 16.
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