CN103045519A

CN103045519A - Lactobacillus casei and application thereof in feed fermentation

Info

Publication number: CN103045519A
Application number: CN 201210594249
Authority: CN
Inventors: 栾锦涛; 陈继伟; 黄玲玲; 张盼盼
Original assignee: Qingdao Haizhongda Biotechnology Development Co Ltd
Current assignee: Qingdao Haizhongda Biotechnology Development Co Ltd
Priority date: 2012-12-31
Filing date: 2012-12-31
Publication date: 2013-04-17

Abstract

The invention relates to Lactobacillus casei Hzd-lc-002, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 6929, and the Lactobacillus casei can be used in fermentation of feed and microecologics and can prevent and cure various animal diseases. The Lactobacillus casei has the advantages that the Lactobacillus casei Hzd-lc-002 has high acid resistance and can grow slowly under a condition with the pH of 2; the Lactobacillus casei Hzd-lc-002 has high temperature resistance and can survive for 10 minutes at a high temperature of 85 DEG C, and the survival rate can still reach 80%; the Lactobacillus casei Hzd-lc-002 can be subjected to high-density culture, and the number of live bacteria can achieve 4.5*1010 per mL; the Lactobacillus casei Hzd-lc-002 has tolerance to various antibiotics, and especially has high tolerance to arsanilic acids and colistin sulfate which are allowed to be used by the country; and the Lactobacillus casei Hzd-lc-002 can accelerate livestock digestive absorption and increase the growth speed, the feed conversion ratio is decreased, and the cultivation cost is reduced; and poult diseases can be prevented, and the death rate is reduced.

Description

A kind of lactobacterium casei and the application in feed fermentation thereof

Technical field

The present invention relates to bacterial strain, particularly a kind of lactobacterium casei and the application in feed fermentation thereof.

Background technology

Probiotic bacterium is the living microorganism food raw material that a class is of value to HUMAN HEALTH, and probiotic bacterium commonly used has Lactobacterium acidophilum and bifidus bacillus two large classes at present, is called as " healthy three beneficial bacterium " with lactobacterium casei.China is also few to the research of lactobacterium casei at present, and the research of lactobacterium casei in fermented feed is short of especially.

Lactobacterium casei is a kind of as probiotic bacterium, can tolerate organic defense mechanism, comprising the bile acide of low pH value and small intestine in the enzyme in the oral cavity, the gastric juice etc.But lactobacterium casei enters large number of viable behind the enteron aisle, plays effects such as regulating intestinal flora balance, promoting digestion absorption.Simultaneously, lactobacterium casei can promote cell fission, produces antibody mediated immunity, the functions such as enhancing body immunity and preventing cancer and inhibition tumor growth; Lactobacterium casei has significant benefit to its host's nutrition, immunity, diseases prevention etc. and gives birth to effect, more and more becomes the focus of people's research, exploitation, to the research of lactobacterium casei and significant with its development functionality goods.

Summary of the invention

For the defective that exists in the prior art, the object of the present invention is to provide a strain to be used for the lactobacterium casei of fermented feed.This lactobacterium casei (Lactobacillus casei) Hzd-lc-002, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 3rd, 2012, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preserving number is: CGMCC No.6929, it can be used in fermented feed and probiotics, can prevent and treat the many animals disease.The present invention shows by test: lactobacterium casei (Lactobacillus casei) Hzd-lc-002 have very strong tolerance to a lot of antibiosis, can with contain antibiotic complete feed etc. and mix feeding; Fermented liquid is quick to growing swine, healthy growth has good promoter action.

Advantage of the present invention is: lactobacterium casei (Lactobacillus casei) Hzd-lc-002 has stronger acid resistance, can be slowly growth under 2 the condition in the pH value; Have stronger heat-resisting ability, keep 10min under 85 ℃ of hot conditionss, survival rate still can reach 80%; Can carry out high-density culture, viable count can reach 4.5 * 10 ¹⁰Individual/mL; Multiple Classes of Antibiotics is had tolerance, and the Pro-gen 90, the colistine sulfate that especially national regulation are allowed to use have stronger tolerance; Can accelerate livestock and poultry and digest and assimilate, accelerate the speed of growth, reduce feedstuff-meat ratio, reduce aquaculture cost; Can prevent the poult disease, reduce mortality ratio.

Description of drawings:

Fig. 1 lactobacterium casei (Lactobacillus casei) Hzd-lc-002 colonial morphology photo;

Fig. 2 lactobacterium casei (Lactobacillus casei) Hzd-lc-002 microscopically aspect graph;

Fig. 3 lactobacterium casei Hzd-lc-002 growth curve chart;

The phylogenetic tree that the 16SrDNA of the bacterial strain that communicates of including among Fig. 4 lactobacterium casei (Lactobacillus casei) Hzd-lc-002 and the Genbank is constructed;

Lactobacterium casei among Fig. 5 embodiment 3 (Lactobacillus casei) Hzd-lc-002 high temperature tolerance test-results;

Fig. 6 lactobacterium casei (Lactobacillus casei) Hzd-lc-002 16SrDNA electrophorogram.

Embodiment

Method among the following embodiment if no special instructions, is ordinary method.

Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.

Embodiment 1

The acquisition of lactobacterium casei (Lactobacillus casei) Hzd-lc-002 and culture condition thereof detect

Screening and the evaluation of lactobacterium casei (Lactobacillus casei) Hzd-lc-002

1) screening of lactobacterium casei (Lactobacillus casei) Hzd-lc-002

From the old Pericarppium arachidis hypogaeae that villages and small towns, Shandong Pingdu south man of peasant household piles up, choose some fermentation sign of surface as sample, sterilized water soaks, and adopts gradient dilution to be coated with dull and stereotyped partition method, with Pericarppium arachidis hypogaeae solution gradient dilution 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5After, absorption 0.1mL is applied on the ATB isolation medium bacterial strain is separated, and fills N in 37 ℃ of anaerobic box ₂Cultivate 5-7 d, colonial morphology is that oyster white, smooth, colony diameter are that single bacterium colony about 1 mm is repeatedly rule and separated until purifying on the picking ATB isolation medium, as shown in Figure 1.

ATB basic medium: peptone 1%, yeast extract 0.5%, glucose 1%, MgSO ₄7H ₂O 0.02%, MnSO ₄4 H ₂O 0.005%, cysteine hydrochloride 0.5 g/L, and tomato juice 25%, liquid nutrient medium is regulated pH value to 4.8, and solid medium is regulated pH value to 5.0, and to the agar powder that wherein adds 2.0%, 121 ℃ of sterilization 20 min.

ATB isolation medium: add respectively cycloheximide 50 mg/L in the ATB basic medium, vancomycin 50 mg/L.

2) evaluation of bacterial classification

Behind the bacterial strain purifying on the ATB isolation medium flat board colonial morphology be that oyster white, smooth, single colony diameter are that primary dcreening operation bacterial strain about 1mm is identified.

The strain cell gramstaining is positive, and single thalline is shaft-like, paired or one-tenth chain alignment, as shown in Figure 2.

Catalase test, indole test, gelatin liquification test, nitrate reduction test are all feminine gender, and fermentation does not produce H ₂S can utilize glucose fermentation to produce acid but aerogenesis not.

The solid culture feature: bacterial strain is cultured continuously 5 d under 37 ℃ of anaerobism culture condition, the smooth oyster white bacterium colony about MRS solid culture primary surface formation diameter 1 mm of improvement.

Modified MRS culture medium: peptone 1%, yeast extract 0.5 %, extractum carnis 1%, glucose 2%, sodium acetate, anhydrous 0.3%, dibasic ammonium citrate 0.2%, tween 80 0.1%, dipotassium hydrogen phosphate 0.2%, MgSO ₄7H ₂O 0.02%, MnSO ₄4 H ₂O 0.005%, and cysteine hydrochloride 0.05% is regulated Medium's PH Value to 6.8, adds 2.0% agar powder in the solid medium, 121 ℃ of sterilization 20 min.

The liquid culture feature: picking list bacterium colony in the MRS liquid nutrient medium of improvement of clarification 37 ℃ leave standstill cultivation.Poor growth and homogeneous in the bacterial strain liquid medium within, and obvious demixing phenomenon is arranged.

The 16SrDNA sequence of lactobacterium casei Hzd-lc-002 is shown in SEQ ID No.1.Institute is surveyed the 16SrDNA sequence contribute by BLAST contrast, Mega, as shown in Figure 4, identify that it is lactobacterium casei.

Can find out according to Fig. 3, lactobacterium casei Hzd-lc-002 begins to enter logarithmic phase behind 12h, and 12-44h is logarithmic phase, is stationary phase behind the 44h.Measuring viable count during 44h is 4.5 * 10 ¹⁰Individual/mL.

Embodiment 2

The acid resistance test of lactobacterium casei Hzd-lc-002

With lactobacterium casei Hzd-lc-002 by 5% inoculum size in above-mentioned modified MRS culture medium, leave standstill cultivation in 37 ℃ of lower anaerobic culture boxes, measure its OD every 4h ₆₆₀Value, as shown in table 1.Test-results surface lactobacterium casei Hzd-lc-002 has very strong acid resistance, so can adapt to preferably the environment behind the fermentation and acid.

The experiment of table 1 lactobacterium casei Hzd-lc-002 acid resistance

Embodiment 3

The test of lactobacterium casei Hzd-lc-002 high temperature tolerance

The lactobacterium casei Hzd-lc-002 that 1200mL is fermented is respectively charged into 100mL in control group (CK, 4 ℃), 75 ℃, 85 ℃, 95 ℃ three high-temperature gradients of experimental group, respectively do 3 groups parallel, under the temperature that requires, keep 10min, as seen from Figure 5, lactobacterium casei Hzd-lc-002 still can keep 80% survival rate under 85 ℃ high temperature, also can reach 52.1% survival rate under 95 ℃.

Embodiment 4

The antibiotic resistance test of lactobacterium casei Hzd-lc-002

The agar dilution that this test is strictly recommended according to U.S. clinical laboratory standard committee (NCCLS) is measured lactobacterium casei Hzd-lc-002 allows interpolation to country 7 kinds of antibiotic tolerances.

The antibiotic resistance test of table 2 lactobacterium casei Hzd-lc-002

The microbiotic title	Antibiotic concentration (ppm)	National regulation addition (ppm)
			Duomycin	500	20-75
Zinc-bacitracin	1000	4-100
			Pro-gen 90	2000	50-100
Olaquindox	800	50-100
			Colistine sulfate	1600	2-40
Kitasamycin	50	5-330
			Salinomycin.	500	10-75

Found that lactobacterium casei hzd-lc-002 7 kinds of conventional medicines that permission is added to the feed Chinese Home have tolerance in various degree: wherein the tolerance of Ponazuril is the strongest, is colistine sulfate secondly; Tolerance to Zinc-bacitracin, olaquindox, duomycin, Salinomycin. is also very strong; But the patience to kitasamycin is relatively poor, below the highest addition of national regulation.

Embodiment 5

From slant medium in the triangular flask of the ATB substratum of picking list bacterium colony in the embodiment 1 that 250mL is housed, 37 ℃ of anaerobism, leave standstill cultivation.OD in liquid A TB substratum ₆₀₀Value reaches at 2.0 o'clock, and under aseptic condition, the amount by 5% is inoculated in the MRS liquid culture medium among the embodiment 1.In anaerobic culture box, 37 ℃ leave standstill and carry out application test after cultivating 44h.

The application test of lactobacterium casei hzd-lc-002 fermented liquid in growing and fattening pigs

Test period: on November 1,1 day to 2011 August in 2011, amount to 90 days.

The source of test pig and grouping: Qingdao Shuan He live pig Specialty Co-operative Organization

Address: Jimo City's dragon's fountain town East River Bei Cun

Control group adopts the complete feed of buying on the market, and test group is that identical complete feed and lactobacterium casei hzd-lc-002 fermented liquid is combined with

Test-results:

Table 3: the application test of lactobacterium casei hzd-lc-002 fermented liquid in growing and fattening pigs

Adopt automatic diet machine for food.Control group is used the 1500kg complete feed altogether, and feedstuff-meat ratio is 2.85:1; Test group is used complete feed 1520kg altogether, and lactobacterium casei hzd-lc-002 fermented liquid amounts to 8100mL, and feedstuff-meat ratio has reduced than control group, is 2.56:1.

Embodiment 6

The application test of lactobacterium casei hzd-lc-002 fermented liquid in meat duck (10 age in days)

Test period: on July 28,1 day to 2011 May in 2011, amount to 90 days.

The source of test pig and grouping: all from Juxian County at sunshine, Shandong Province; Control group adopts the fowl feed of buying on the market, and test group is that identical fowl feed and lactobacterium casei hzd-lc-002 fermented liquid is combined with

Test site: Rizhao City Juxian County, Shandong Province duckery.

Test-results:

Table 4: the application test of lactobacterium casei hzd-lc-002 fermented liquid in meat duck (10 age in days)

Test-results shows: add lactobacterium casei hzd-lc-002 fermented liquid in the feed and greatly reduce young duck mortality ratio.

Sequence table

Claims

1. an a kind of lactobacterium casei (Lactobacillus casei) is characterized in that described lactobacterium casei is lactobacterium casei (Lactobacillus casei) Hzd-lc-002, and preserving number is: CGMCC No.6929.

2. the application of lactobacterium casei according to claim 1 (Lactobacillus casei) Hzd-lc-002 in fermented feed.