CN107446852B - One lactobacillus plantarum and its application in terms of fermented feed - Google Patents
One lactobacillus plantarum and its application in terms of fermented feed Download PDFInfo
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
Application the invention discloses a lactobacillus plantarum and its in terms of fermented feed belongs to fermented feed technical field.Lactobacillus plantarum (Lactobacillus plantarum) JUN-DY-6 of the invention, China typical culture collection center is preserved on March 23rd, 2017, deposit number is CCTCC M 2017138, and preservation address is that preservation address is China, Wuhan, Wuhan University.Bacterial strain acid spectrum of the invention is wide, and production acid amount is high, has broad-spectrum antibacterial effect, the content of anti-nutritional factors in fermented bean dregs can be effectively reduced, the incidence for reducing diarrhea, that improves nutriment utilizes level, and the storage phase of feed can effectively be extended by preparing feed using the bacterial strain.
Description
Technical field
Application the present invention relates to a lactobacillus plantarum and its in terms of fermented feed belongs to fermented feed technology neck
Domain.
Background technique
With the fast development of aquaculture, from last century the fifties, breeding enterprise is to increase animal productiong amount, dynamic
It joined antibiotic in object diet, and tremendous contribution brought to the development of entire cultivation industry.But over time, resist
Raw element use is more and more frequent, and harm increasingly shows, and causes personages of various circles of society's highest attention.Feed safety is equivalent at present
In food safety concept worldwide oneself become common recognition.The study found that fermented feed can inhibit pathogenic bacteria in alimentary canal
Growth, Animal diseases are played with certain prevention effect, meanwhile, it also has, and palatability is good, nutrient absorption utilization rate is high, can
The advantages that substitute antibiotics use.
Fermentative feedstuff of microbe (Fermented feed) refers under the conditions of artificial controllable, with the agricultural and sideline production of vegetalitas
Product are primary raw material, and by beneficial microorganism metabolism, the macromolecular substances such as par-tial polysaccharide, protein and fat of degrading are raw
At the small-molecule substance of the absorption easy to digest such as organic acid, soluble polypeptide, it is good, full of nutrition, effectively prebiotic to form palatability
The high biological feedstuff of bacterial content, effective component mainly include the beneficial microbe mycoprotein easily absorbed, various biological enzyme
Class, bioactivity widow skin, amino acid, organic acid, active probiotic etc.
Microorganism currently used for fermented feed production specifically includes that bacillus, saccharomycete, aspergillus class fungi and cream
Sour bacterium.Lactic acid bacteria is on the one hand to generate antibiotic in fermented feed to inhibit and kill harmful bacteria to prevent it from growth and breeding,
Reducing or kill harmful substance makes endotoxin content reduction make former host resist different ability enhancing dominant in competition to enhancing,
Carbohydrate breakdown is improved material flavor and palatability by the nutritive value that feed on the other hand can be improved.However, existing lactic acid
The bacterial strain that acid producing ability and antagonistic property are had both in bacterium is less, in addition feed fermentation is usually mixed thallus system, the cream being exogenously introduced
Sour bacterium easily breaks the balance of the mixed microorganism in fermented feed, and the nutritive value of fermented feed is made to improve limitation, shelf-life
It does not grow.
Summary of the invention
The first purpose of the invention is to provide a kind of lactobacillus plantarum (Lactobacillus plantarum) JUN-
DY-6 is preserved in China typical culture collection center on March 23rd, 2017, and deposit number is CCTCC M 2017138,
Preservation address is that preservation address is China, Wuhan, Wuhan University.
A second object of the present invention is to provide the cultural method of the lactobacillus plantarum, the method is to be inoculated in MRS
In culture medium, 30~37 DEG C of 24~48h of culture.
Third object of the present invention is to provide a kind of bacteriostasis method, the method is to be seeded to the lactobacillus plantarum
In liquid, semisolid or solid containing pathogenic bacteria, the miscellaneous bacteria includes in Escherichia coli, staphylococcus aureus or salmonella
At least one.
Fourth object of the present invention is to provide a kind of feed, and the feed contains the lactobacillus plantarum and feed carries
Body.
In one embodiment of the invention, the feed vector include dregs of beans, soya-bean cake, starch, in molasses at least
It is a kind of.
Fifth object of the present invention is to provide the preparation method of the feed, the method is by the lactobacillus plantarum
It is seeded in feed vector, in 30~37 DEG C of 5~7d of standing for fermentation.
It is by the plant cream sixth object of the present invention is to provide a kind of method for extending the fermented feed shelf-life
Bacillus JUN-DY-6 is added in feed vector, is fermented.
7th purpose of the invention is to provide the application of the lactobacillus plantarum.
In one embodiment of the invention, the application includes preparing food, drug, health care product.
The utility model has the advantages that 1, lactobacillus plantarum JUN-DY-6 acid spectrum of the invention is wide, production acid amount is high, and growth rapidly can be with dregs of beans
A variety of organic acids are produced for raw material.And the 28h total acid content that ferments under the conditions of 37 DEG C can reach 14.82g/L；2, using of the invention
Strain fermentation dregs of beans can make high molecular weight protein in fermented bean dregs degrade substantially totally, can effectively reduce anti-nutrition in fermented bean dregs
The content of the factor reduces the incidence of diarrhea, and that improves nutriment utilizes level；3, the feeding prepared using bacterial strain of the invention
Material is conducive to storage, fermented bean dregs wet feed can be made to save under normal temperature conditions 40~60 days, than un-added feed extended shelf-life
2~3 times, and there is no mildew phenomena.
Detailed description of the invention
Fig. 1: lactobacillus plantarum JUN-DY-6 growth curve；
Fig. 2: lactobacillus plantarum JUN-DY-6 growth course pH change curve；
Fig. 3: different process fermented bean dregs PAGE gel electrophoretic analysis；Wherein, Marker indicates reference band, and SM is not
Fermented dregs of beans, 0% indicates to be added to the fermented bean dregs that lactobacillus plantarum is not added with alkali protease, and 0.5% indicates addition
Lactobacillus plantarum is added to the fermented bean dregs of mass fraction 0.5% alkali protease simultaneously.
Biomaterial preservation
One lactobacillus plantarum (Lactobacillus plantarum) JUN-DY-6, in preservation on March 23 in 2017
In China typical culture collection center, deposit number is CCTCC M 2017138, and preservation address is that preservation address is China,
Wuhan, Wuhan University.
Specific embodiment
The measurement of organic acid content: Hitachi's high performance liquid chromatograph, ultraviolet absorption detector, analytical column organic acid are used
Column (Aninex Hpx-87H ion exchange column), 50 DEG C of column temperature, mobile phase 5mM sulfuric acid solution, flow velocity 0.6mL/
Min, sample volume 10 μ L, detection time 27min calculate the content of various organic acids in sample.
Embodiment 1
1: to reach preferable separating effect, fermented bean dregs being accessed into MRS fluid nutrient medium by inoculum concentration 10g/100mL
In, 30 DEG C, 200rpm cultivates 48h, continuous Multiplying culture 2~3 times.
MRS culture medium prescription is as follows: peptone 10.0g, and beef leaches object 10.0g, yeast extract 5.0g, glucose
5.0g, sodium acetate 5.0g, lemon acid diamine 2.0g, Tween-80 1.0g, dipotassium hydrogen phosphate 2.0g, epsom salt 0.2g, seven
Water manganese sulfate 0.05g, calcium carbonate 20.0g, agar 20.0g, distilled water are settled to 1.0L, pH 6.8.
2: strain isolates and purifies: with sterile saline dilute sample step by step, being coated on the separation training prepared
It supports on base (calcium carbonate containing 2%) plate, covers one layer of same culture medium again thereon, interlayer Anaerobic culturel (30 DEG C, 48h),
Then the big single colonie of molten calcium circle is selected, repeatedly scribing line is isolated and purified to obtain pure culture and is named as on isolation medium
JUN-DY-6。
3: the 16SrDNA identification of bacterial strain
Bacterial strain JUN-DY-6 genomic DNA is extracted with bacterial genomes DNA Rapid extraction kit (raw work biology), as
PCR amplification template carries out PCR amplification using 165rDNA primer pair (primer 2 7F and primer 1495).
PCR reaction process is as follows: 95 DEG C of denaturation 5min；95 DEG C/30s, 55 DEG C/30s, 72 DEG C/2min, 35 circulations, 72
DEG C keep 5min.PCR product is obtained into band brighter at about 1500bp through electrophoretic analysis, with Q-quick Gel
Extraction Kit (Promeg company) carries out gel extraction purifying, the sequencing of PCR purified product Song Sheng work biotech firm.It will survey
The sequence of bacterial strain and Genbank database after sequence carries out sequence analysis, show that bacterial strain JUN-DY-6 is lactobacillus plantarum
(Lactobacillus plantarum)。
Embodiment 2
1, the preparation of indicator bacteria bacteria suspension: the pathogenic bacteria glycerol tube such as Escherichia coli, salmonella and staphylococcus aureus
It is inoculated in culture medium, is cultivated 16-18 hours in 37 DEG C of shaking table 200r/min, bacterium is dense up to 1.2 × 1010CFU/mL。
2, purebred lactobacillus plantarum the preparation of Bacillus acidi lactici bacteria suspension: is inoculated in 100mL MRS liquid in aseptic condition
In body culture medium, 37 DEG C of 24~48h of stationary culture, bacterium is dense up to 1.5 × 1010CFU/mL。
3, the preparation of double-layer plate takes the plate of diameter about 90mm, pours into the nutrient agar 18 of heating and melting respectively
~20mL makes it uniformly spread out cloth in plate, and place makes to solidify on level table, as bottom.Separately take semisolid nutrient agar rouge
After the appropriate heating and melting of culture medium (agar content 1%), let cool to 48-50 DEG C, indicator bacteria is added in every 50~100mL culture medium
0.1~0.2mL of bacteria suspension is separately added into 5mL in every 1 plate, it is made uniformly to spread out cloth on bottom, as bacterium layer.It places
It is spare with equidistant uniform placement Oxford cup 4 in every 1 plate on level table after cooling.
Wherein nutrient agar formula is as follows: tryptone 10g, yeast extract 5g, NaCl 10g deionization
Water, which is settled to 1L. solid medium, need to add 15~20g agar, and semisolid culturemedium need to add 10g agar.
4, bacteriostasis property is evaluated: being taken step 2 fermented liquid supernatant, is dripped respectively in the Oxford cup in each double-layer plate and fill 200 μ
After L, 37 DEG C of culture 18h, each antibacterial circle diameter (or area) is measured to make an appraisal.Typical plate is chosen to take a picture.
Biocidal property: inhibition zone<10mm is that fungistatic effect is faint, and 10mm<inhibition zone<15mm is that moderate is antibacterial, inhibition zone>
15mm is that height is antibacterial.
Remarks: 1) inhibition zone is bigger illustrates that fungistatic effect is better；2) inhibition zone refers to the entire diameter of circular non-opaque circle.
Table 1 is bacteriostasis (unit mm) of the lactobacillus plantarum JUN-DY-6 to different microorganisms
2 lactobacillus plantarum JUN-DY-6 of table counts the antibacterial result of pathogenic bacteria
As shown in table 1-2, bacterial strain JUN-DY-6 is significantly antibacterial to Escherichia coli, staphylococcus aureus and salmonella
Effect, antibacterial circle diameter up to 12~22mm, 12.77~15.9mm, 12.4~16.3mm, average value 18.0mm, 14.8mm,
13.9mm, standard deviation is less than 0.1.
Embodiment 3
Referring to " Berger bacterial identification manual " and " lactic acid bacteria taxonomic identification and experimental method ", and to bacterial strain JUN-DY-6
Carry out 16sRNA sequencing.Wherein the results are shown in Table 4 for bacterial strain bio-chemical characteristics, and carbon source through fermentation test result is as shown in table 3, knot
Physiology and biochemistry and carbon source through fermentation experimental result are closed, and 16S information is compared.According to cell microscopic morphology, Physiology and biochemistry number
According to 16S rRNA gene sequence data, bacterial strain is accredited as lactobacillus plantarum (Lactobacillus plantarum).
Table 3: the sugar fermentating test result of lactobacillus plantarum JUN-DY-6.
Note: "+" indicates that strong fermentation, " d " expression are slightly fermented, and "-" indicates azymic
Table 4: the Physicochemical test result of lactobacillus plantarum JUN-DY-6.
Note: "-" represents feminine gender, and "+" represents the positive.
Embodiment 4
Fermented bean dregs, specific steps are produced using lactobacillus plantarum (Lactobacillus plantarum) JUN-DY-6
It is as follows:
By air-dried dregs of beans 100g, water 50g, the lactobacillus plantarum bacterium solution (OD 7~8) of molasses 3g and 3~10mL logarithmic phase
It is uniformly mixed and is placed in the valve bag through aseptic process, sealing is placed in 37 DEG C of incubators the 48h that ferments.It takes within every four hours primary
Sample.Every batch of experiment does three in parallel.Every sub-sampling takes 4g fermented bean dregs in 50ml centrifuge tube, and 40ml deionized water is added and stirs
It mixes after mixing stands 1h and takes supernatant, be placed in liquid phase bottle through 0.22 μm of membrane filtration for efficient liquid phase chromatographic analysis, as long as
Analyze the type and content of organic acid in sample.3g dregs of beans is weighed after fermentation in 100ml beaker, and 30mL is added,
0.2%NaOH stirs 15min at room temperature, is centrifuged 7min in 6000r/min, abandons rouge layer and precipitating, carefully leave and take supernatant 2mL,
5 times of dilution keeps sample.Take the 30 diluted sample supernatants of μ L, add 10 μ 4 × buffer of L, boiling water bath 10min, 13000rpm from
Heart 5min supernatant loading carries out the degradation situation of macromolecular substances in PAGE gel electrophoretic analysis fermented bean dregs.As a result
As shown in figure 3, SM is the protein content in thick dregs of beans, 0% is the protein content in the fermented bean dregs for add lactobacillus plantarum.By
Figure is it is found that production of the lactobacillus plantarum for fermented bean dregs can obvious degradation macromolecular substances therein.
Embodiment 5
Using lactobacillus plantarum (Lactobacillus plantarum) JUN-DY-6 and add alkali protease production hair
Ferment dregs of beans:
By air-dried dregs of beans 100g, water 50g, molasses 3g, the plant cream bar of alkali protease 0.5g and 3~10mL logarithmic phase
Bacterium bacterium solution (OD 7~8) is uniformly mixed and is placed in the valve bag through aseptic process, and sealing, which is placed in 37 DEG C of incubators, ferments
48h.Take a sample within every four hours.Every batch of experiment does three in parallel.Sampling and processing mode are same as above.
Table 5 is the kinds of organic acids and highest content (g/L) that different fermentations technique produces fermented bean dregs
Note: G1 indicates only addition lactobacillus plantarum (Lactobacillus plantarum) JUN-DY-6 group；G2 expression adds
Add lactobacillus plantarum (Lactobacillus plantarum) JUN-DY-6 and alkali protease group.Production when being 28h or so
Acid value.
By in table it is found that with lactobacillus plantarum (Lactobacillus plantarum) JUN-DY-6 produce fermented bean dregs,
In 48h the bacterium can mass propagation produce acid rapidly, and acid spectrum is wide that produce acid amount high.Its highest when being used cooperatively with alkali protease
Producing acid amount can reach 14.82g/L.It is found by PAGE gel electrophoresis (Fig. 3), addition lactobacillus plantarum JUN-DY-6 can
By the protein degradation in dregs of beans about 50%, and production fermented bean dregs not only produce acid and measure when the bacterium and a small amount of protease are used cooperatively
Height, and high molecular weight protein is degraded totally substantially.
Embodiment 5
Using lactobacillus plantarum (Lactobacillus plantarum) JUN-DY-6 and add alkali protease production hair
The shelf stability experiment of ferment dregs of beans:
Dregs of beans 100g, water 50g, molasses 3g that will be air-dried, (enzyme-activity unit of alkali protease be alkali protease 0.5g
15000~20000U/g) and 3~10mL logarithmic phase lactobacillus plantarum bacterium solution (OD 7~8) be uniformly mixed be placed on through sterile
In the valve bag of processing, sealing is placed in 37 DEG C of incubators the 48h that ferments.It is pair so that the fermented bean dregs of lactobacillus plantarum not to be added
According to.
Fermented bean dregs wet feed is placed in room temperature after fermentation and is stored, routine observation its whether occur mildew (occur
Mildew).Pass through lactobacillus plantarum JUN-DY-6 as the result is shown and the fermented bean dregs wet feed for adding the production of alkali protease can
It saves 40~60 days and does not go mouldy at room temperature.And control group (other ingredients are all the same in addition to not adding lactobacillus plantarum)
It can only be saved under wet feed room temperature about 20 days.The production of fermented bean dregs, the storage of wet feed are carried out with commercialized lactobacillus plantarum
Phase was at 30 days or so.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of lactobacillus plantarum (Lactobacillus plantarum) JNU-DY-6, is preserved on March 23rd, 2017
China typical culture collection center, deposit number are CCTCC NO:M 2017138, and preservation address is that preservation address is China,
Wuhan, Wuhan University.
2. a kind of method of the inhibition pathogenic bacteria for non-disease Clinics and Practices purposes, which is characterized in that by claim 1 institute
The lactobacillus plantarum JNU-DY-6 stated is seeded in the liquid containing pathogenic bacteria, semisolid or solid.
3. according to the method described in claim 2, it is characterized in that, the pathogenic bacteria include Escherichia coli, Staphylococcus aureus
At least one of bacterium or salmonella.
4. a kind of feed, which is characterized in that the feed contains lactobacillus plantarum JNU-DY-6 and feed described in claim 1
Carrier.
5. feed according to claim 4, which is characterized in that the feed vector includes dregs of beans, soya-bean cake, starch, molasses
At least one of.
6. a kind of method for preparing feed described in claim 5, which is characterized in that the lactobacillus plantarum is seeded to feed and is carried
In body, in 30~37 DEG C of 5~7d of standing for fermentation.
7. according to the method described in claim 6, it is characterized in that, also adding protease before fermentation；The additive amount of protease is
75~100U/g feed vector.
8. a kind of method for extending the fermented feed shelf-life, which is characterized in that by lactobacillus plantarum JNU- described in claim 1
DY-6 is added in feed vector, is fermented.
9. application of the lactobacillus plantarum JNU-DY-6 in field of food described in claim 1.
10. the food containing lactobacillus plantarum JNU-DY-6 described in claim 1.
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| CN109287847A (en) * | 2018-09-27 | 2019-02-01 | 江南大学 | A kind of preparation method of the fermented bean dregs rich in phenyllactic acid |
| CN109198162A (en) * | 2018-10-29 | 2019-01-15 | 江南大学 | A kind of method that bacterium enzyme cooperative fermentation prepares dregs of beans |
| WO2021063163A1 (en) * | 2019-09-30 | 2021-04-08 | 江南大学 | Method for preparing feed by bacterium-enzyme synergistic fermentation |
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| CN110583852A (en) * | 2019-09-30 | 2019-12-20 | 江南大学 | Bacterium-enzyme synergistic fermentation method for camellia seed meal |
| CN111557377B (en) * | 2020-05-12 | 2022-09-27 | 江南大学 | Method for preparing fruit and vegetable waste fermented feed |
| CN111676153B (en) * | 2020-05-30 | 2022-05-31 | 青岛玛斯特生物技术有限公司 | Lactobacillus plantarum and application thereof in aquatic product fermented feed |
| CN113261613A (en) * | 2021-06-23 | 2021-08-17 | 贵州理工学院 | Method for preserving wet white spirit vinasse and preventing mildew and feed |
| CN113355265B (en) * | 2021-06-30 | 2022-11-08 | 广东温氏大华农生物科技有限公司 | Lactobacillus plantarum and application thereof |
| CN117660250B (en) * | 2023-12-08 | 2024-07-23 | 华中农业大学 | Low-temperature lactobacillus and application thereof |
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