CN106497828B - A kind of application of Lactobacillus casei and its lyophilized preparation in pannage - Google Patents
A kind of application of Lactobacillus casei and its lyophilized preparation in pannage Download PDFInfo
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- CN106497828B CN106497828B CN201610895694.9A CN201610895694A CN106497828B CN 106497828 B CN106497828 B CN 106497828B CN 201610895694 A CN201610895694 A CN 201610895694A CN 106497828 B CN106497828 B CN 106497828B
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- lactobacillus casei
- zlc018
- pig
- freeze
- lactobacillus
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/245—Lactobacillus casei
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
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Abstract
The invention discloses the application of a kind of Lactobacillus casei (Lactobacillus caseii) and its lyophilized preparation in pannage, disclose the Optimal Medium and freeze drying protectant of bacterial strain of the present invention.The microbial preservation number of Lactobacillus casei of the invention is CGMCC No.9041.Bacterial strain of the present invention is obtained from the separation of health pig enteron aisle, breeding, and hereditary basis is clear, has good production acid and biocidal property.The culture medium and freeze drying protectant of bacterial strain of the present invention can be improved the culture efficiency and freeze-drying survival rate of the bacterium, enhance its resistance.The lyophilized preparation of bacterial strain of the present invention adds in pannage, can promote the intestinal health of pig, reduce the diarrhea rate of pig, improves the production performance of pig, reduces the harmful bacteria discharge in excrement.
Description
Technical field
The invention belongs to microorganism fields, are related to a kind of Lactobacillus casei (Lactobacillus casei) bacterial strain, the present invention
Further relate to application of its lyophilized preparation in pannage.
Background technique
In pig production, the digestive organs and physiological function of piglet are still complete without development, and gastric acid secretion is insufficient, endogenous
Property digestive enzyme activity is lower, and the microecological balance in enteron aisle is not yet set up, and harmful bacteria is easy to take advantage of a weak point, and leads to piglet
Diarrhea influences the growth and survival rate of piglet.Therefore, organic acid can be produced to piglet supplement, can inhibit harmful bacteria (such as large intestine bar
Bacterium and staphylococcus aureus etc.), the feeding lactobacillus of piglet healthy growth can be promoted, be an important solution route.
Lactobacillus casei (Lactobacillus casei) be population in Lactobacillus (Lactobacillus) it
One, it is that the Ministry of Agriculture announces " catalogue of feed additive varieties (2013) " and is allowed for the lactic acid bacteria of animal-breeding.Related cheese
What lactobacillus was applied in feed applies for a patent: " one plant of Lactobacillus casei and its application in feed " (application number
201210277454.4), Lactobacillus casei L-1 is that isolated, strains ferment generation phosphatide is screened from soil
Enzyme A2, using the bacterial strain as fermentation strain, using the oil crops of the phosphatide containing plant or its processed side product as fermentation substrate, with sulphur
Sour ammonium, sodium chloride, cornstarch prepare feed addictive by solid state fermentation as energy donor, in hog feed.
" a kind of Lactobacillus casei fermentation liquid application " in feed (application No. is 201310162105.2), discloses Lactobacillus casei
Application of the 1yT-7 fermentation liquid in feed, the rape cake that Lactobacillus casei fermentation liquid is passed through spray drying and is added after detoxification,
It is made containing Lactobacillus casei 109Viable bacteria/gram feed addictive, in pig starter feed.It " a kind of Lactobacillus casei and its is raising
Application in material fermentation " (application number 201210594249.0), the Lactobacillus casei being related to (Hzd-lc-002) is located away from old
In peanut shell, anaerobism mode culture is taken, fermentation liquid is for raising growing and fattening pigs and meat duck.
Above-mentioned patented technology prepares feed by the modes such as spray drying after solid state fermentation, liquid state fermentation, liquid state fermentation and adds
Add agent, be applied in animal feed, be not involved with pig source Lactobacillus casei, is not involved with the product acid activity of strain, inhibits
Content in terms of harmful bacteria situation and freeze-drying preparation survival rate.
Summary of the invention
The first purpose of this invention is to provide one plant of cheese cream bar that separation, breeding obtain from health pig enteron aisle
Bacterium；
Second object of the present invention is to provide the culture medium for being suitble to above-mentioned Lactobacillus casei growth；
Third object of the present invention is to provide the lyophilized preparation of the Lactobacillus casei and the preparation method of preparation；
The object of the invention is also to provide purposes of the Lactobacillus casei in pannage.
The present invention separates from health pig enteron aisle, selects one plant of pig source Lactobacillus casei (Lactobacillus
Casei), it is named as Lactobacillus casei ZLC018, by producing a series of experiments such as sour, antibacterial in vitro, it was demonstrated that it is with prebiotic spy
Property.By Optimal Medium, freeze drying protectant and lyophilized technique, the culture efficiency and freeze-drying survival rate of bacterial strain, preparation are improved
Lyophilized preparation be added in nursery-stage pig feed, can reduce the diarrhea rate of pig, improve daily gain, significantly reduce harmful bacteria in excrement
Quantity.The bacterial strain was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 06 18th, 2014
(abbreviation CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postal service are compiled
Code: 100101.Deposit number: CGMCC No.9041, biological deposits survival information: survival.
Lactobacillus casei ZLC018 of the present invention has characteristics that
1. at 37 DEG C, after cultivating 18h, into growth stationary phase；For 24 hours, viable count reaches 6.0 × 10 for culture9CFU/mL；
2. at 37 DEG C, for 24 hours, pH reaches 3.69, and Lactic Acid from Fermentation Broth content reaches 105.92mmoL/L for culture；48h is cultivated,
PH reaches 3.66, and Lactic Acid from Fermentation Broth content reaches 220.75mmoL/L；
3. handling 4h in the simulated gastric fluid of pH=2.0, survival rate 77.62%；It is handled in the simulated intestinal fluid of pH=6.8
4h, survival rate 95.56%；
4. inhibited to harmful bacteria.The fermented liquid is to 106The Escherichia coli of CFU/mL, Staphylococcus aureus
Bacterium and salmonella have an inhibiting effect, and antibacterial circle diameter is up to 18~24.2mm.
A kind of composition of preferred culture medium of the Lactobacillus casei ZLC018 are as follows: glucose 80g, soy peptone 10g,
Beef extract 10g, yeast extract 20g, anhydrous sodium acetate 5g, diammonium hydrogen citrate 2g, K2HPO4·3H2O 2g、MgSO4·7H2O
0.6g、MnSO4·H2O 0.2g, calcium carbonate 0.5g, Tween-80 1mL, distilled water 1000mL.Using the culture medium prescription, can make
The culture efficiency of the bacterium greatly improves, and viable count is not less than 6.0 × 109CFU/mL, effect are better than MRS culture medium.
The composition of a kind of preferred freeze drying protectant of the Lactobacillus casei ZLC018 are as follows: skimmed milk power 25g, trehalose
10g, L-cysteine 1g, lactose 5g, sodium glutamate 3g, dextrin 20, glycerol 1g, sodium carboxymethylcellulose 0.4g, distilled water
100mL.Using the protection agent prescription, the freeze-drying survival rate of the bacterium can be made to reach 94.6%.
The preparation method of the Lactobacillus casei ZLC018 lyophilized preparation, comprises the technical steps that:
1. seed liquor prepare: take widened method step by step, by the Lactobacillus casei ZLC018 saved at -70 DEG C with
1% inoculum concentration is inoculated in fluid nutrient medium, is primary seed solution after cultivating 18h at 37 DEG C；Primary seed solution again with
1% inoculum concentration is inoculated in fluid nutrient medium, cultivates 18h at 37 DEG C, is secondary seed solution, for use；
2. expanding culture: the seed liquor of Lactobacillus casei ZLC018 is inoculated in fluid nutrient medium with 1% inoculum concentration,
At 37 DEG C culture 22~for 24 hours；
3. being centrifugated thallus: by step, 2. middle expand cultivates fermentation liquid centrifuge separation obtained, obtains bacterium mud；
4. protectant preparation: required distilled water is filtered with the sterile filters that filter sizes are 0.22 μm to sterile barrel
In, protective agent ingredient is successively weighed, is mixed；
5. emulsification: prepared protective agent and bacterium mud being uniformly mixed, using high-shearing dispersion emulsifying machine, revolving speed 3000r/
Min emulsifies 30min, and bacteria suspension is made；
6. vacuum freeze drying: the bacteria suspension ambient-temp-stable 30min that will have been emulsified is placed in refrigerator tray, thickness 0.5~
1.5cm, pre-freeze 2h, then vacuum freeze drying 20h under conditions of condensation temperature is -30 DEG C, vacuum degree 0.37Pa, is frozen
Dry powder；
7. collecting freeze-dried powder, crush, packing, aluminium foil bag vacuum packaging, cryo-conservation.
Studies have shown that Lactobacillus casei ZLC018 of the present invention is easily cultivated, is grown, and have stronger production it is sour, it is acidproof and
With bacteriostasis.Application effect of the Lactobacillus casei ZLC018 of the present invention in pannage: it is added in child care swine rations dry
Lactobacillus paracasei ZLC018 lyophilized preparation, makes the viable count in daily ration reach 4.0 × 109CFU/kg, the diarrhea rate decline of child care pig
7.5 percentage points, survival rate improves 3.33 percentage points, and daily gain improves 12.71%, and feed-weight ratio reduces by 7.34%, in pig manure
Harmful bacteria quantity sharp fall, in which: Escherichia coli quantity decline 46.29%, staphylococcus aureus quantity decline
30.82%.After feeding Lactobacillus casei ZLC018 lyophilized preparation, the health status of child care pig is improved, it is latent to be conducive to its production
The performance of energy.
Detailed description of the invention
Fig. 1 Lactobacillus casei ZLC018 thallus, bacterium colony and fermentation liquid picture, Fig. 1 a is MRS culture medium, at 37 DEG C, training
Thalli morphology (Gram's staining) after supporting 48h, Fig. 1 b is MRS culture medium, the colonial morphology at 37 DEG C, after cultivating 48h；Figure
1c is the fermentation liquid picture of Optimal Medium culture for 24 hours.
Fig. 2 Lactobacillus casei ZLC018 incubation OD600nmChange curve.
Fig. 3 Lactobacillus casei ZLC018 incubation pH change curve.
Specific embodiment
The separation of embodiment 1, screening and identification pig source Lactobacillus casei ZLC018
From the child care pig slaughter of Beijing scale pig farm selection health, enteron aisle is taken, scrapes intestinal mucosa and sterile saline
It mixes.1mL suspension is taken, is diluted to 10 respectively with sterile saline-5、10-6、10-7, respectively take 0.1mL to be respectively coated on MRS solid
On body culture medium, SL solid medium and LBS solid medium, 37 DEG C of Anaerobic culturels.The doubtful lactobacillus of picking is milky white after 48h
Color bacterium colony carries out anaerobism pure culture.After 48h, the bacterium colony after half of picking culture carries out H2O2Enzyme test and Gram's staining mirror
Inspection, all Gram-positives, H2O2Enzyme test is negative, thalli morphology is that rod-shaped bacterial strain can primarily determine as lactobacillus.So
Remaining half of bacterium colony is subjected to scribing line culture afterwards, until being determined as pure bacterium, classification volume then is carried out to isolated bacterial strain
Number, and carry out freezen protective.
To isolated bacterial strain, by growth curve, sour, acidproof, bile tolerance and the specificity analysis such as antibacterial are produced, is filtered out
One plant of best lactobacillus of overall characteristic, and morphological feature, physiological and biochemical property and 16S rRNA have been carried out to the bacterial strain
Gene sequencing, identification are as follows: Lactobacillus casei (Lactobacillus casei).The physicochemical characteristics of the bacterial strain are shown in Table 1, DNA
Sequence is shown in sequence table SEQ ID No.1.
The thallus and colonial morphology of bacterial strain are shown in Fig. 1.On MRS solid medium, which is creamy white, is regular, convex
It rises, wet, opaque, neat in edge.Thallus is in the shape of a rod, and 0.65~1.2 × 2~6 μm, in pairs or at catenation；Gram
Stained positive.
The physiological and biochemical property of 1 Lactobacillus casei ZLC018 of table
Note: "+" indicates that >=90% bacterial strain is positive in table, and "-" indicates that >=90% bacterial strain is negative, and " d " indicates 80%~90%
Bacterial strain is positive.
Strain ZLC018 is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation information is such as
Under:
Strain name: Lactobacillus casei Lactobacillus casei
Preservation title: Lactobacillus casei ZLC018
Preservation date: on 06 18th, 2014
Biological deposits survival information: survival
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica
Postcode: 100101
Deposit number: CGMCC No.9041
The biological nature of the measurement of embodiment 2 Lactobacillus casei ZLC018
2.1 growth curve
Using OD600nmIt is index with pH value, determines the growth characteristics of Lactobacillus casei ZLC018.As a result see Fig. 2 cheese
Lactobacillus ZLC018 incubation OD600nmChange curve, Fig. 3 Lactobacillus casei ZLC018 incubation pH change curve.
By Fig. 2, Fig. 3 it is found that Lactobacillus casei ZLC018 is during 4~12h, bacterium solution OD600nmValue increases sharply, and pH value is fast
Speed decline；During 12~18h, bacterium solution OD600nmIt is gradually reduced with pH value variation, illustrates to have reached logarithmic growth latter stage；18h with
OD afterwards600nmIt is unobvious with pH value variation, show that the bacterium enters growth stationary phase.
2.2 produce acidity test
The seed liquor for the Lactobacillus casei ZLC018 for activating 2 times is inoculated in this with 1% inoculum concentration (volume ratio, similarly hereinafter)
In the fluid nutrient medium of invention, cultivated at 37 DEG C, respectively in 12h, for 24 hours, 36h, 48h, 72h, 96h, 120h sampling, using cream
Acidohydrogenase method measures Lactic Acid from Fermentation Broth content, and detection kit builds up Bioengineering Research Institute purchased from Nanjing.It the results are shown in Table
2。
2 Lactobacillus casei ZLC018 of table cultivates different time Lactic Acid from Fermentation Broth content
2.3 acid resistance test
2.3.1 the preparation of simulated gastric fluid: sodium chloride 0.2g, pepsin 1g, with the dilute hydrochloric acid solution of pH2.0 or pH3.0
100mL after completely dissolution, with filtering with microporous membrane degerming, for use.
2.3.2 the seed liquor for the Lactobacillus casei ZLC018 for activating 2 times is inoculated in liquid of the invention with 1% inoculum concentration
It in body culture medium, is cultivated at 37 DEG C for 24 hours, takes 5mL bacterium solution, 6000r/min is centrifuged 5min, abandons supernatant, is separately added into and has gone out
The simulated gastric fluid 5mL of the different pH of the physiological saline (pH 6.5) and filtration sterilization of bacterium, 37 DEG C of heat preservations are sampled in 0h, 2h, 4h
Plate count is carried out, survival rate is calculated, the results are shown in Table 3.
Survival rate (%) of the 3 Lactobacillus casei ZLC018 of table in simulated gastric fluid
As shown in Table 3, Lactobacillus casei ZLC018 acts on 4h, survival rate difference in the simulated gastric fluid of pH2.0 and pH3.0
For 77.62% and 85.11%.Illustrate that Lactobacillus casei ZLC018 has stronger acid resistance, adapts to the gastric acid environment of animal.
2.4 resistance to simulated intestinal fluid tests
2.4.1 the preparation of simulated intestinal fluid: potassium dihydrogen phosphate 0.68g adds distilled water to dissolve, molten with 0.4% sodium hydroxide
Liquid adjusts pH value, until volume is 100mL, pH6.8.Then plus 1g trypsase, filtering with microporous membrane degerming is used after completely dissolution,
For use.
2.4.2 the seed liquor for the Lactobacillus casei ZLC018 for activating 2 times is inoculated in liquid of the invention with 1% inoculum concentration
In body culture medium, 37 DEG C of cultures for 24 hours, take 5mL bacterium solution, and 6000r/min is centrifuged 5min, abandon supernatant, are separately added into sterilized life
Simulated intestinal fluid (pH6.8) 5mL of salt water (pH 6.5) and filtration sterilization are managed, 37 DEG C of heat preservations carry out flat in 0h, 2h, 4h sampling
Plate bacterium colony counts, and the results are shown in Table 4.
Survival rate (%) of the 4 Lactobacillus casei ZLC018 of table in simulated intestinal fluid
As shown in Table 4, Lactobacillus casei ZLC018 acts on 4h in simulated intestinal fluid, and viable count does not have significant changes, deposits
Motility rate is 95.56%, illustrates that Lactobacillus casei ZLC018 is not influenced by simulated intestinal fluid, adapts to the intestinal environment of animal.
2.5 bacteriostatic test
Bacteriostatic test is carried out using Odontothrips loti.
2.5.1 Lactobacillus casei ZLC018 fermentation liquid is prepared, without thallus supernatant and thallus suspension: by activation 2 times do
The seed liquor of Lactobacillus paracasei ZLC018 is inoculated in fluid nutrient medium of the invention with 1% inoculum concentration, is cultivated at 37 DEG C
For 24 hours, fermentation liquid is obtained.Take 5mL fermentation liquid in sterile centrifugation tube, 6000r/min is centrifuged 5min, and obtained supernatant is used
0.22 μm of sterile filters filtering, obtains no thallus supernatant；Obtained bacterium mud is centrifuged to be washed 2 times with sterile saline, then
It is suspended with 5mL sterile saline, thallus suspension is made.
2.5.2 it prepares indicator bacteria plate: 2 Escherichia coli, staphylococcus aureus and salmonellas will be activated with 1%
Inoculum concentration be inoculated in MH fluid nutrient medium respectively, at 37 DEG C cultivate 18h after, bacteria suspension of 1 Maxwell than turbid standard is made,
MH agar medium by 1% volume ratio with temperature lower than 50 DEG C is uniformly mixed, and every plate (specification is diameter 90mm) is inclined
Infuse 15mL, cooling obtained indicator bacteria plate.
2.5.3 the Oxford cup of sterilizing is placed on indicator bacteria plate, the Lactobacillus casei ZLC018 fermentation liquid of preparation, nothing
Thallus supernatant and thallus suspension are separately added into Oxford cup, every 200 μ L of hole, and the culture medium for not connecing with physiological saline and bacterium is pair
According to 4 DEG C of refrigerators spread 5h, and 16~18h is cultivated under the conditions of 30 DEG C, measure antibacterial circle diameter.It the results are shown in Table 5.
The 5 antibacterial result of Lactobacillus casei ZLC018 (mm) of table
Oxford cup internal diameter 6mm, outer diameter 8mm, high 10mm.
Embodiment 3 prepares Lactobacillus casei preparation
The active stabilization of lactic acid bacteria freeze drying preparation is easily stored, feature easy to use.Lyophilized preparation can raised directly
It is added in material, the liquid preparation that different viable bacteria contents can also be mixed and made into from drinking water is drunk for animal, acceptable and feed
Raw material or complete feed mixing carry out solid and expand numerous culture or solid state fermentation.And in the production process of lyophilized preparation, culture medium
Formula, freeze drying protectant and lyophilized technique are important technical links, directly affect Viable detection, lyophilized preparation viable count and
The unit cost of production.The present invention has carried out technological improvement to these three sport technique segments, achieves remarkable result.
3.1 improve the culture medium of Lactobacillus casei ZLC018
For the biological characteristics of Lactobacillus casei ZLC018, it is formulated based on MRS culture medium, using orthogonal experiment plan
Count L16(45) method, screening is optimized to glucose therein, soy peptone, yeast extract and beef extract dosage.Finally
Filter out the optimal culture medium prescription of effect are as follows: glucose 80g, soy peptone 10g, beef extract 10g, yeast extract 20g, nothing
Water sodium acetate 5g, diammonium hydrogen citrate 2g, K2HPO4·3H2O 2g、MgSO4·7H2O 0.6g、MnSO4·H2O 0.2g, carbonic acid
Calcium 0.5g, Tween-80 1mL, distilled water 1000mL.The above ingredient is mixed, is dissolved by heating, pH6.5, packing, 121 DEG C of height are corrected
Pressure sterilizing 15min.With the Lactobacillus casei of this culture medium culture, viable count is not less than 6.0 × 109CFU/mL, effect are better than MRS
Culture medium.It the results are shown in Table 6.
The viable count (CFU/mL) of 6 Lactobacillus casei ZLC018 of table in different medium
MRS culture medium prescription: glucose 20g, peptone 10g, beef extract 10g, yeast extract 5g, anhydrous sodium acetate 5g,
Diammonium hydrogen citrate 2g, K2HPO4·3H2O 2g、MgSO4·7H2O 0.58g、MnSO4·H2O 0.19g, Tween-80 1mL,
Distilled water 1000mL.
The freeze drying protectant of 3.2 optimization Lactobacillus casei ZLC018
Using Response Surface Method, the freeze drying protectant ingredient of Lactobacillus casei freeze-dried powder is optimized.With
Plackett-Burman design, the substances such as discovery skimmed milk power, trehalose and L-cysteine are to Lactobacillus casei ZLC018's
Survival rate has a significant impact, and approaches peak response region using steepest hill climbing, is analyzed by center combination design and response phase method
Determine the compound proportion of optimal freeze drying protectant, it may be assumed that skimmed milk power 25g, trehalose 10g, L-cysteine 1g, lactose 5g, paddy
Propylhomoserin sodium 3g, dextrin 20g, glycerol 1g, sodium carboxymethylcellulose 0.4g, distilled water 100mL.With the formula, cheese cream bar can be made
The freeze-drying survival rate of bacterium ZLC018 reaches 94.6%.
The preparation method of 3.3 Lactobacillus casei lyophilized preparations, comprises the technical steps that:
1. seed liquor prepare: take widened method step by step, by the Lactobacillus casei ZLC018 saved at -70 DEG C with
1% inoculum concentration is inoculated in fluid nutrient medium, is primary seed solution after cultivating 18h at 37 DEG C；Primary seed solution again with
1% inoculum concentration is inoculated in fluid nutrient medium, cultivates 18h at 37 DEG C, is secondary seed solution, for use；
2. expanding culture: the seed liquor of Lactobacillus casei ZLC018 is inoculated in fluid nutrient medium with 1% inoculum concentration,
At 37 DEG C culture 22~for 24 hours；
3. being centrifugated thallus: by step, 2. middle expand cultivates fermentation liquid centrifuge separation obtained, obtains bacterium mud；
4. protectant preparation: required distilled water is filtered with the sterile filters that filter sizes are 0.22 μm to sterile barrel
In, protective agent ingredient is successively weighed, is mixed；
5. emulsification: prepared protective agent and bacterium mud being uniformly mixed, using high-shearing dispersion emulsifying machine, revolving speed 3000r/
Min emulsifies 30min, and bacteria suspension is made；
6. vacuum freeze drying: the bacteria suspension ambient-temp-stable 30min that will have been emulsified is placed in refrigerator tray, thickness 0.5~
1.5cm, pre-freeze 2h, then vacuum freeze drying 20h under conditions of condensation temperature is -30 DEG C, vacuum degree 0.37Pa, is frozen
Dry powder；
7. collecting freeze-dried powder, crush, packing, aluminium foil bag vacuum packaging, cryo-conservation.
4 Lactobacillus casei ZLC018 lyophilized preparation tolerance test of embodiment
In the simulated gastric fluid (pH2.0) of 225mL, the solution of simulated intestinal fluid (pH6.8) and 225mL containing Pig cholate of 225mL
In (cholate ultimate density is 0.3%), it is separately added into the Lactobacillus casei ZLC018 lyophilized preparation of 25g, is uniformly mixed, is placed in and turns
In fast 75r/min shaking table, 37 DEG C of heat preservations.In 0h, 2h, 4h and 6h, separately sampled carry out plate count calculates survival
Rate the results are shown in Table 7.
7 Lactobacillus casei ZLC018 lyophilized preparation of table is survived in simulated gastric fluid, simulated intestinal fluid and 0.3% pig gall salting liquid
Rate (%)
As shown in Table 7, Lactobacillus casei ZLC018 lyophilized preparation is in simulated gastric fluid (pH2.0) and simulated intestinal fluid (pH6.8)
In, 0h to 2h viable count slightly declines, and then increases；Viable count continues to increase in pig gall salting liquid (0.3%), illustrates cheese
Protective ingredient in lactobacillus ZLC018 lyophilized preparation can be improved tolerance of the thallus to simulated gastric fluid, simulated intestinal fluid and Pig cholate
Ability can be improved thallus in the survival rate of animal gastrointestinal tract.
5 Lactobacillus casei ZLC018 lyophilized preparation of embodiment is applied in nursery-stage pig feed
5.1 test materials and method
Test carries out on Beijing pig farm.
Child care pig 180 similar in selection health, weight and age in days, be randomly divided into 3 groups (control group, antibiotic group and
Lactic acid bacteria group), every group of 6 repetitions, 10 pigs of each repetition.It is freely eaten, free water, net bed raising, room temperature 24~
28 DEG C, feeding management routinely carries out.Experimental period is 30d.
Control group fed basal diet；Antibiotic group adds Bacitracin Zinc and colistine sulfate in basal diet, often
Bacitracin Zinc containing 40mg and 8mg colistine sulfate in kilogram of feed；Lactic acid bacteria group adds Lactobacillus casei in basal diet
ZLC018 freeze-dried powder, living bacteria count reaches 4.0 × 10 in every kilogram of feed9CFU.The composition and trophic level of daily ration are shown in Table 8.
8 child care swine rations of table composition and trophic level (air bells dry basis)
Note: 1) compound premix provides Cu 150mg for every kilogram of feed；Fe 150mg；Zn 140mg；Mn 40mg；Se
0.3mg；I 0.6mg；VA 8000IU；VD31800IU；VE 24IU；VK 1.8mg；VB11.6mg；VB26mg；VB6
3.2mg；VB120.05mg；Biotin 0.3mg；Folic acid 1.2mg；Niacin 24mg；Pantothenic acid 18mg；Choline chloride 0.4g；L- relies ammonia
Hydrochlorate 2.2g；Threonine salt 0.4g；Calcium 5g and total phosphorus 3g.
2) moisture, thick protein, calcium and total phosphorus are measured value.
5.2 test result
As shown in Table 9, child care pig average daily gain can be improved by antibiotic and lactic acid bacteria being added in child care swine rations, be reduced
Feed-weight ratio.Compared with the control group, 8.58% and 12.71% is respectively increased in antibiotic group and lactic acid bacteria group average daily gain；Material weight
Than reducing by 4.52% and 7.34% respectively.Compared with antibiotic group, the average daily gain of lactic acid bacteria group improves 3.80%, material weight
Than reducing 2.96%.The using effect of Lactobacillus casei preparation is better than antibiotic usage effect.
Influence of the Lactobacillus casei ZLC018 lyophilized preparation to child care pig production performance is added in 9 diet of table
Note: colleague shoulder mark lowercase difference person indicates significant difference (P < 0.05).
By table 10 as it can be seen that feeding Lactobacillus casei reduces the death rate and diarrhea rate of child care pig, divide compared with the control group
3.33 percentage points and 7.5 percentage points are not reduced.
Influence of the 10 Lactobacillus casei ZLC018 of table to the child care pig death rate and diarrhea rate
As shown in Table 11, compared with the control group, after Lactobacillus casei ZLC018 lyophilized preparation is added in daily ration, child care pig
Harmful bacteria quantity sharp fall in excrement, Escherichia coli quantity have dropped 46.29% (P < 0.05), Staphylococcus aureus
Bacterium number amount has dropped 30.82% (P < 0.05)；The ratio of lactic acid bacteria and Escherichia coli improves (P < 0.05).
Influence of the Lactobacillus casei ZLC018 lyophilized preparation to flora in child care swine excrement is added in 11 daily ration of table
Note: colleague shoulder mark lowercase difference person indicates significant difference (P < 0.05).
In conclusion after adding Lactobacillus casei ZLC018 lyophilized preparation in nursery-stage pig feed, the intestinal health of pig,
The diarrhea rate of pig reduces, and survival rate improves；Daily gain improves, and feed-weight ratio reduces；Escherichia coli quantity in pig manure
It is significantly reduced with staphylococcus aureus quantity, lactic acid bacteria increases with Escherichia coli ratio.These main performance index are advantageous
In the healthy growth of child care pig and the raising of productivity effect, be conducive to the improvement of pig farm breeding environment.
Claims (9)
1. Lactobacillus casei (Lactobacillus caseii ) ZLC018 bacterial strain, microbial preservation number are as follows: CGMCC
No.9041。
2. a kind of Lactobacillus casei lyophilized preparation, including Lactobacillus casei ZLC018 described in claim 1 and freeze drying protectant.
3. Lactobacillus casei lyophilized preparation as claimed in claim 2, which is characterized in that the composition of the freeze drying protectant
Are as follows: 25 g of skimmed milk power, 10 g of trehalose, 1 g of L-cysteine, 5 g of lactose, 3 g of sodium glutamate, 20 g of dextrin, glycerol 1g,
0.4 g of sodium carboxymethylcellulose, 100 mL of distilled water.
4. the preparation method of Lactobacillus casei lyophilized preparation described in claim 2 or 3, which is characterized in that the preparation method
Include the following steps:
1. prepared by seed liquor: taking widened method step by step, the Lactobacillus casei ZLC018 that saves at -70 DEG C is connect with 1%
Kind amount is inoculated in fluid nutrient medium, is primary seed solution after 18 h are cultivated at 37 DEG C；Primary seed solution is again with 1% inoculation
Amount is inoculated in fluid nutrient medium, and 18 h are cultivated at 37 DEG C, is secondary seed solution, for use；
2. expanding culture: the seed liquor of Lactobacillus casei ZLC018 being inoculated in fluid nutrient medium with 1% inoculum concentration, 37
22~24 h are cultivated at DEG C；
3. being centrifugated thallus: by step, 2. middle expand cultivates fermentation liquid centrifuge separation obtained, obtains bacterium mud；
4. protectant preparation: required distilled water is filtered with the sterile filters that filter sizes are 0.22 μm into sterile barrel,
Protective agent ingredient is successively weighed, is mixed；
5. emulsification: prepared protective agent and bacterium mud being uniformly mixed, using high-shearing dispersion emulsifying machine, 3000 r/min of revolving speed
30 min are emulsified, bacteria suspension is made；
6. vacuum freeze drying: 30 min of bacteria suspension ambient-temp-stable that will have been emulsified is placed in refrigerator tray, thickness is 0.5~1.5
Cm, 2 h of pre-freeze, then 20 h of vacuum freeze drying under conditions of condensation temperature is -30 DEG C, vacuum degree 0.37Pa, is frozen
Dry powder；
7. collecting freeze-dried powder, crush, packing, aluminium foil bag vacuum packaging, cryo-conservation.
5. the feed addictive containing Lactobacillus casei described in claim 1.
6. the feed containing Lactobacillus casei described in claim 1.
7. feed as claimed in claim 6 is pannage.
8. Lactobacillus casei described in claim 1 is preparing the application in pannage.
9. application according to claim 8, which is characterized in that add Lactobacillus casei ZLC018 freeze-drying in basal diet
Powder, living bacteria count reaches 4.0 × 10 in every kilogram of feed9 CFU。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| AU2017100632A4 (en) * | 2017-05-29 | 2017-07-06 | Bgi Shenzhen | An oral liquid probiotic formulation and method of preventing and/or treating arthritis |
| CN107164269B (en) * | 2017-06-06 | 2020-03-24 | 北京市农林科学院 | Lactobacillus paracasei, preparation and application of lactobacillus paracasei in pig feed |
| CN110804571B (en) * | 2019-12-02 | 2021-03-05 | 华中农业大学 | Compound lactobacillus preparation and application thereof in preparing feed additive |
| CN112042765A (en) * | 2020-09-09 | 2020-12-08 | 甘肃华羚生物技术研究中心有限公司 | Yogurt powder freeze-dried yogurt crisp product and preparation method thereof |
| CN112369500A (en) * | 2020-11-03 | 2021-02-19 | 青岛普罗百世生物科技有限公司 | Lactobacillus casei and application thereof in weaned pig feed |
| CN112266890A (en) * | 2020-11-09 | 2021-01-26 | 中国科学院东北地理与农业生态研究所 | Lactobacillus casei and application thereof |
| CN112625983B (en) * | 2021-03-05 | 2021-05-28 | 山东中科嘉亿生物工程有限公司 | Lactobacillus casei L.Casei21 with treatment effect on diarrhea and application thereof |
| CN113088468B (en) * | 2021-04-09 | 2022-06-03 | 中国农业大学 | Lactobacillus casei Ma. GLRGJ1 and application thereof |
| CN114874952B (en) * | 2022-06-14 | 2024-04-09 | 广西壮族自治区兽医研究所 | Lactobacillus casei HJD and application thereof |
| CN116240150B (en) * | 2023-04-10 | 2024-06-11 | 山东威曼宠物食品有限公司 | Lactobacillus casei King99 and microbial inoculum for improving pet soft stool, preparation method and application |
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