CN104560799A - Preparation method of bacteriocin-producing Lactobacillus plantarum subsp. Plantarum Zhang-LL active bacterial preparation - Google Patents
Preparation method of bacteriocin-producing Lactobacillus plantarum subsp. Plantarum Zhang-LL active bacterial preparation Download PDFInfo
- Publication number
- CN104560799A CN104560799A CN201410816266.3A CN201410816266A CN104560799A CN 104560799 A CN104560799 A CN 104560799A CN 201410816266 A CN201410816266 A CN 201410816266A CN 104560799 A CN104560799 A CN 104560799A
- Authority
- CN
- China
- Prior art keywords
- zhang
- fermentation
- lactobacillus plantarum
- freeze
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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
- 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
- C12R2001/25—Lactobacillus plantarum
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a preparation method of a bacteriocin-producing Lactobacillus plantarum subsp. Plantarum Zhang-LL active bacterial preparation, and belongs to the field of microbe application. In the preparation method, by using a bacteriocin-producing Lactobacillus plantarum subsp. Plantarum Zhang-LL CGMCC No. 6936 bacterial strain, the active bacterial preparation is prepared through activation, expanding culture, high-density fermentation, centrifugation, freeze-drying protective agent addition, a prefreezing process, a freeze-drying process and other processes. The preparation method comprises the following steps: firstly, rapeseed peptone is selected as a substituting nitrogen source, so that the culture condition of the lactobacillus plantarum is optimized while the cost of a culture medium is reduced; a 5L of full-automatic fermentation tank is used for performing the high-density fermentation, and under the optimized low-cost culture medium and the optimized culture condition, the viable count of fermented liquid can reach above 1010CFU/mL. In the preparation method, potato pulp is used as a matrix of a freeze-drying protective agent, and an optimal combined formula of the freeze-drying protective agent is determined, so that the survival rate of the Lactobacillus plantarum subsp. Plantarum Zhang-LL reaches above 60% and the viable content can reach above 1011CFU/g. The prepared active bacterial preparation is low in raw material price and relatively high in viable content, can provide a good foundation for developing a novel feed additive for poultry, and can provide a significant economic value.
Description
Technical field
The present invention relates to the preparation method of the viable lactic acid bacteria preparation of microbial technology field, belong to microbe application field, can be used for the production of the probiotics such as feeding live bacterial preparation in poultry animal husbandry.
Background technology
Lactic acid bacteria is a kind of probio be present in mankind's body.Carbohydrate fermentation can be become lactic acid by lactic acid bacteria, thus gains the name.Probiotic lactobacillus is added in food and not only can improve nutritive value of food, and it is with health role, as promoted the absorption of the nutriments such as protein, produce a large amount of benefit materials such as vitamin B complex, improve human body intestinal canal function, recover colony balance in human body intestinal canal, strengthen body immunity, antitumor, anti-ageing, also there is norcholesterol, reducing blood lipid, the effect such as hypotensive.Lactobacillus product in the market mainly comprises yogurt, sour milk beverage, active bacteria formulation etc.
The strain bacteriocinogeny Lactobacillus plantarum plant subspecies Zhang-LL bacterial strain that this patent relates to, confirm that it not only has good fungistatic effect to single listeria spp that increases through overtesting, and tolerance stomach and intestine inverse ring border (tolerance bile and hydrochloric acid in gastric juice), therefore surely can be grown by stomach and intestine smoothly and produce bacteriocin and lactic acid in enteron aisle, there is the effect suppressing some spoilage organisms and pathogenic bacteria.This bacterial strain is prepared into active bacteria formulation and is applied to poultry farming as bio-additive by this patent, can reach the object of prevention poultry intestinal canal disease.
At present, domestic patent about the preparation method of various active bacteria formulation more, such as Beijing City Agriculture and Forestry Institute season seapeak and sumbul Dun Gele " a kind of porcine lactobacillus plantarum plant subspecies lyophilized formulations and preparation method thereof " (CN200910082441.X) that apply for be Lactobacillus plantarum plant subspecies through spreading cultivation, centrifugal, add protective agent, vacuum drying, obtain the Lactobacillus plantarum plant subspecies bacterium powder of high viability; Heilongjiang Bayi Agricultural Reclamation University adds compound sugar in " Lactobacillus plantarum active bacteria formulation containing active material and preparation method thereof " (CN201010283999.7) of green for a long time, Qian Lili and Yao Di application in Lactobacillus plantarum viable bacteria powder, prepares a kind ofly to stablize the active bacteria formulation deposited in greenhouse; " a kind of composite microorganism viable bacteria preparation and its preparation method and application " (ZL200810034746) of Shanghai Eco-well Bioscience Co., Ltd. Zhang Qin and old Lu of building application is a kind of sheet coccus, saccharomycete and bacillus composite microorganism viable bacteria preparation.But about utilizing bacteriocinogeny Lactobacillus plantarum plant subspecies Zhang-LL to prepare active bacteria formulation, and utilize rapeseed protein peptone nitrogenous source as an alternative, the method that potato pulp preserves viable bacteria in biologic product as matrix has no relevant report both at home and abroad.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of bacteriocinogeny Lactobacillus plantarum plant subspecies Zhang-LL active bacteria formulation.
In described method, Lactobacillus plantarum plant subspecies (Lactobacillus plantarum subsp.plantarum) Zhang-LL, be preserved in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 04th, 2012, Institute of Microorganism, Academia Sinica), its preserving number is: CGMCC No.6936.In MRS culture medium, cultivate 2d, bacterial strain Zhang-LL thalline is rod-short, 0.5 × 1.33 ~ 2.0 μm, single, in pairs or in short catenation, and spore of not sprouting, Gram-positive.
In described method, the bacterium liquid activated is seeded in MRS liquid culture medium with the inoculum concentration of 2% (V/V, volume fraction) after activating for 2 ~ 3 generations by Lactobacillus plantarum plant subspecies Zhang-LL, and cultivate 12h for 37 DEG C, the nutrient solution of acquisition is for subsequent use.
In said method, MRS liquid culture medium: tryptone 10.0g, beef extract 10.0g, yeast leaching powder 5.0g, Triammonium citrate 2.0g, glucose 20.0g, Tween 80 1.0mL, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.5g, manganese sulfate 0.25g, water 1L, pH6.5,121 DEG C of sterilizing 15min
In described method, Lactobacillus plantarum plant subspecies Zhang-LL fermentation medium is on the basis of modified MRS culture medium, carry out substituting and optimizing of nitrogenous source.Culture medium and fermentation condition optimization adopt the multilevel and four factor three level [L of single factor test
9(3
4)] orthogonal test, to measure zymotic fluid viable count for index, analyze and determine culture medium and the fermentation condition of the optimization of high viable count zymotic fluid.Finally determine nitrogenous source (501 rapeseed protein peptone) content 3.0%, fermentation temperature 34 DEG C, zymotic fluid pH 6.5, fermentation inoculum concentration 7% is optimum fermentation culture conditions.Optimize Lactobacillus plantarum plant subspecies fermentation medium one by one 501 rapeseed protein peptone modified MRS culture medium consist of the following composition: 501 rapeseed protein peptone 30.0g/L, Triammonium citrate 2.0g/L, glucose 20.0g/L, Tween 80 1.0mL/L, sodium acetate 5.0g/L, dipotassium hydrogen phosphate 2.0g/L, magnesium sulfate 0.5g/L, manganese sulfate 0.25g/L, pH6.5,121 DEG C of sterilizing 15min.
In described method, apply the fermentation culture conditions after above-mentioned optimization, 5L automatic fermenter is utilized to carry out high density fermentation, by the scale-up medium of Zhang-LL bacterial strain by 7% (V/V, volume fraction) inoculum concentration be seeded in fermentation tank, fermentation temperature is 34 DEG C to utilize fermentation tank automatic temperature control system to ensure, adds the sterilizing Na of 15% (V/V, mass volume ratio concentration) in sweat with stream
2cO
3it is 6.5 that solution controls zymotic fluid pH, and rotating speed is the condition bottom fermentation 16h of 150r/min, obtains zymotic fluid.
In said method, 5L automatic fermenter is produced by Shanghai Gaoji Bioengineering Co., Ltd., and its model is BIOF6005GBN.
In described method, by the zymotic fluid in 5L fermentation tank under 4 DEG C of conditions, the centrifugal 10min of 8000r/min, collects bacterium mud.
In described method; mix collecting the Lactobacillus plantarum plant subspecies bacterium mud obtained with the potato pulp of 1/10 original fermentation liquor volume; adopt single factor test multilevel test; the freeze drying protectant combination formula determining Lactobacillus plantarum plant subspecies Zhang-LL is (V/V; mass volume ratio concentration): 3% skimmed milk+2% sodium glutamate; after mixing, obtain starter culture concentrates.
In said method, potato pulp is by fresh peeling potatoes, stripping and slicing, makes through nine positive board food cooking machines making beating.
In described method by starter culture concentrates in-35 DEG C of refrigerators pre-freeze 13h to fully charge state, obtain pre-freeze leavening, utilize vacuum freeze drier by pre-freeze leavening under the condition of freeze temperature-55 DEG C, vacuum 0.08mBar, freeze-drying 48h, to bone dry state, obtains freeze dried fermenting preparation.
In described method, through colony counting, the Lactobacillus plantarum plant subspecies Zhang-LL zymotic fluid viable count obtained can reach 10
10more than CFU/mL, the viable count of freeze drying viable microorganism preparation can reach 10
11more than CFU/g, the survival rate of bacterial strain is to more than 60%.Nitrogenous source during this kind of active bacteria formulation utilizes rapeseed protein peptone replacing whole modified MRS culture medium to fill a prescription, namely instead of the tryptone in modified MRS culture medium, beef extract and yeast leaching powder, not only reduce the cost of culture medium, but also significantly increase the number of viable of zymotic fluid lactic acid bacteria.Therefore, utilize potato pulp can reduce the cost of active bacteria formulation further as protection matrix, for the production of poultry biological feed stuff additive and industrialization provide new approaches.
The preparation method of the bacteriocinogeny Lactobacillus plantarum plant subspecies Zhang-LL active bacteria formulation that said method obtains and active bacteria formulation product thereof, and patent bacterial classification belongs to scope.
Detailed description of the invention
Experimental technique in following embodiment, if no special instructions, is conventional method.
Percentage composition in following embodiment, if no special instructions, is the percentage composition of weight to volume.
Embodiment 1, Lactobacillus plantarum plant subspecies Zhang-LL active bacteria formulation and preparation method thereof
1, the nitrogenous source in Lactobacillus plantarum plant subspecies low-cost fermentation culture medium is determined
Under the condition of fermentation temperature 37 DEG C, zymotic fluid inoculum concentration 2%, fermentation time 16h, zymotic fluid pH 6.5, choosing fermentation medium is modified MRS culture medium, the nitrogenous source in modified MRS is substituted respectively with 501 rapeseed protein peptones, 502 rapeseed protein peptones, 503 rapeseed protein peptones, carry out fermentation test, measure the number of viable of Zhang-LL bacterial strain in different fermentations culture medium zymotic fluid, to determine the nitrogenous source kind of the suitableeest fermentation medium, result of the test is in table 1.
Table 1 nitrogenous source kind is on the impact of zymotic fluid Zhang-LL bacterial strain viable count
As shown in Table 1, when nitrogenous source in culture medium is 501 rapeseed protein peptone, zymotic fluid viable count reaches maximum, is 2.38 × 10
9cFU/mL, therefore selected 501 rapeseed protein peptones are the alternative nitrogenous source in culture medium.
Choose optimum fermentation medium nitrogenous source to test further, the nitrogenous source in modified MRS culture medium is substituted with 501 rapeseed protein peptones respectively.Choosing fermentation medium is modified MRS culture medium, substitute tryptone and dusty yeast, 501 rapeseed protein peptones with 501 rapeseed protein peptones respectively to substitute tryptone and beef extract, 501 rapeseed protein peptones and substitute whole nitrogenous sources in modified MRS culture medium, carry out fermentation test, measure the number of viable of Zhang-LL bacterial strain in different fermentations culture medium zymotic fluid, to determine the nitrogenous source composition in the suitableeest fermentative medium formula, result of the test is in table 2.
Table 2 501 rapeseed protein peptone to substitute in MRS different nitrogen sources to the impact of zymotic fluid Zhang-LL bacterial strain viable count
As shown in Table 2,501 rapeseed protein peptones are by when alternative for the whole nitrogenous sources (tryptone, beef extract, dusty yeast) in modified MRS culture medium, and the viable count in zymotic fluid is maximum, can reach 2.83 × 10
9cFU/mL.
2, single factor experiment determination Lactobacillus plantarum plant subspecies optimal conditions of fermentation
(1) content of nitrogenous source is determined
Choosing under optimum fermentation medium condition, determine the impact of the different content of nitrogenous source on zymotic fluid Zhang-LL bacterial strain viable count, carry out fermented and cultured test, measure the number of viable in different fermentations culture medium zymotic fluid, to determine the nitrogenous source content of the suitableeest fermentation medium, result of the test is in table 3.
Table 3 rapeseed protein peptone content is on the impact of zymotic fluid Zhang-LL bacterial strain viable count
As shown in Table 3, when the content of 501 rapeseed protein peptones is 2.5%, zymotic fluid viable count, reaches maximum, is 2.21 × 10
9cFU/mL; And the content of 501 rapeseed protein peptones is when being 3.0% and 3.5%, zymotic fluid number of viable reduces.This may be due to the increase along with rapeseed protein peptone consumption, and wherein residual free gossypol content also increases, and has certain toxic action, inhibit the growth of Zhang-LL bacterial strain to thalline.Therefore the consumption determining the suitableeest fermentation medium 501 rapeseed protein peptone is 2.5%.
(2) fermentation temperature is determined
The 501 rapeseed protein peptones with 2.5% substitute whole nitrogenous sources (the tryptone 10.0g in modified MRS culture medium, beef extract 10.0g, yeast leaching powder 5.0g) as fermentation medium, under zymotic fluid inoculum concentration 2%, fermentation time 16h, zymotic fluid pH 6.5 condition, choose fermentation temperature 30 DEG C, 34 DEG C, 37 DEG C, 40 DEG C, carry out fermentation test, to determine the suitableeest fermentation temperature, result of the test is in table 4.
Table 4 fermentation temperature is on the impact of zymotic fluid Zhang-LL bacterial strain viable count
Fermentation temperature is one of key factor affecting strain growth breeding.Fermentation temperature is different, and the upgrowth situation of bacterial strain is obviously different.As shown in Table 4, when fermentation temperature is 34 DEG C, zymotic fluid viable count is the highest, is 2.40 × 10
9cFU/mL; When fermentation temperature is 30 DEG C, zymotic fluid viable count takes second place, and is 2.04 × 10
9cFU/mL; When fermentation temperature is 37 DEG C, zymotic fluid viable count is relatively low, is 1.62 × 10
9cFU/mL; When fermentation temperature is 40 DEG C, zymotic fluid viable count is minimum, is 1.05 × 10
9cFU/mL.Zhang-LL bacterial strain shows the most vigorous growth vigor 34 DEG C time, along with the rising of temperature or the vigor on the contrary that reduces decline to some extent, when illustrating that this bacterial strain optimum growth temperature is near 34 DEG C thus, zymotic fluid viable count is the highest, therefore to choose the suitableeest fermentation temperature be 34 DEG C.
(3) zymotic fluid pH is determined
The 501 rapeseed protein peptones with 2.5% substitute whole nitrogenous sources (the tryptone 10.0g in modified MRS culture medium, beef extract 10.0g, yeast leaching powder 5.0g) as fermentation medium, under the condition of fermentation temperature 37 DEG C, fermentation time 16h, zymotic fluid pH 6.5, choose under pH is respectively 5.5,6.0,6.5,7.0,7.5 conditions, carry out fermentation test, to determine the suitableeest zymotic fluid pH.
Table 5 pH value is on the impact of zymotic fluid Zhang-LL bacterial strain viable count
As shown in Table 5, as zymotic fluid pH6.5, zymotic fluid viable count is the highest, is 2.10 × 10
9cFU/mL; Along with the reduction of pH, zymotic fluid viable count also reduces thereupon; Along with the increase of pH, viable bacteria also reduces zymotic fluid thereupon.Therefore, controlling the suitableeest zymotic fluid pH is 6.5, and guarantee zymotic fluid viable count reaches maximum.(4) inoculum concentration of fermenting is determined
The 501 rapeseed protein peptones with 2.5% substitute whole nitrogenous sources (the tryptone 10.0g in modified MRS culture medium, beef extract 10.0g, yeast leaching powder 5.0g) as fermentation medium, under the condition of fermentation temperature 37 DEG C, fermentation time 16h, zymotic fluid pH 6.5, choose fermentation inoculum concentration 2%, 4%, 6%, 8%, carry out fermentation test, to determine the suitableeest fermentation inoculum concentration.
Table 6 inoculum concentration is on the impact of zymotic fluid Zhang-LL bacterial strain viable count
As shown in Table 6, when zymotic fluid inoculum concentration is 6% time, zymotic fluid viable count is the highest, is 2.46 × 10
9cFU/mL; When zymotic fluid inoculum concentration is 4% time, zymotic fluid viable count takes second place, and is 2.37 × 10
9cFU/mL; When zymotic fluid inoculum concentration is 2% time, zymotic fluid viable count is relatively low, is 2.12 × 10
8cFU/mL; When zymotic fluid inoculum concentration is 8% time, zymotic fluid viable count is minimum, is 2.05 × 10
9cFU/mL.Illustrate that Zhang-LL bacterial strain the suitableeest fermentation inoculum concentration is 6% thus, zymotic fluid viable count is the highest, therefore to choose the suitableeest fermentation inoculum concentration be 6%.
(5) optimization of orthogonal test Lactobacillus plantarum plant subspecies fermentation condition
On above-mentioned single factor experiment result basis, root root single factor experiment result, selects nitrogenous source, fermentation temperature, pH, fermentation inoculum concentration to be influence factor, designs four factor three level [L
9(3
4)] orthogonal test (see table 7), to measure zymotic fluid Zhang-LL bacterial strain viable count for index, analyze the optimization of fermentation conditions determining high viable count zymotic fluid.By to the range analysis of result of the test and variance analysis determination optimal conditions of fermentation (see table 8).
Table 7 optimization of fermentation conditions Orthogonal Experiment and Design factor level table
Table 8 optimization of fermentation conditions [L
9(3
4)] orthogonal experiments
Note: in table, data are the mean value of n=3 mensuration
As shown in Table 8, with the number of viable of zymotic fluid Zhang-LL bacterial strain for test index, then R
a> R
b> R
d> R
c, namely nitrogenous source content is the main factor affecting number of viable, and fermentation temperature takes second place, and zymotic fluid pH value and fermentation inoculum concentration affect less.By k in table 8
a3> k
a2> k
a1, k
b2> k
b1> k
b3, k
c2> k
c1> k
c3, k
d3> k
d2> k
d1known, the optimum combination optimizing batch fermentation conditions is A
3b
2c
2d
3, namely nitrogenous source (501 rapeseed protein peptone) is 3.0%, fermentation temperature is 34 DEG C, pH is 6.5, fermentation inoculum concentration is 7%.(6) demonstration test
In order to determine that optimum level combines further, according to theoretical optimum combination A
3b
2c
2d
3maximum combination A is reached with viable count in orthogonal test
3b
2c
1d
3, demonstration test is done in two kinds of different combinations.Ferment under two conditions, optimum combination A
3b
2c
2d
3condition bottom fermentation liquid Zhang-LL bacterial strain viable count is 5.35 × 10
9cFU/mL, and at combination A
3b
2c
1d
3under condition, zymotic fluid Zhang-LL bacterial strain viable count is 4.82 × 10
9cFU/mL.It can thus be appreciated that the optimum combination optimizing batch fermentation conditions is A
3b
2c
2d
3, when namely nitrogenous source is 3.0%, fermentation temperature is 34 DEG C, pH is 6.5, fermentation inoculum concentration is 7%, zymotic fluid viable count reaches maximum.(7) 5L fermentation tank ferments automatically
On above-mentioned assay optimization fermentation medium and optimization of fermentation conditions basis, 5L automatic fermenter is utilized to carry out high density fermentation, by Zhang-LL bacterial strain by 7% (V/V, volume fraction) inoculum concentration culture transferring in 501 rapeseed protein peptone modified MRS culture medium, controlling fermentation temperature is 34 DEG C, with the sterilizing Na of 15% (W/V, mass volume ratio concentration) in sweat
2cO
3solution controls zymotic fluid pH6.5, and speed of agitator is 150r/min, and fermentation 16h, final zymotic fluid Zhang-LL bacterial strain viable count can reach 10
10more than CFU/mL.4, the collection of thalline and protectant interpolation
After fresh potato peeling, break into slurry through food cooking machine, make potato pulp; Get Lactobacillus plantarum plant subspecies Zhang-LL bacterial strain fermentation liquor and be placed in centrifugal bottle, with 4 DEG C, the centrifugal 10min of 8000r/min, abandon supernatant, collect bacterium mud; Bacterium mud is mixed with the potato pulp (code name is M) of 1/10 original fermentation liquor volume, then adds the freeze drying protectant of various combination respectively, after mixing, obtain starter culture concentrates; The freeze drying protectant of various combination is as shown in table 9.Zhang-LL bacterial strain number of viable before adopting pour plate cultivation to detect freeze-drying and after freeze-drying, each dilution factor establishes 3 repetitions, determines the more excellent combination formula of freeze drying protectant, the results are shown in Table 9.
The freeze drying protectant of table 9 various combination is on the impact of zymotic fluid bacterial classification survival rate
As shown in Table 9, when potato pulp is as protection matrix, choose 3% skimmed milk+2% sodium glutamate as protective agent, Lactobacillus plantarum plant subspecies Zhang-LL survival rate is the highest, is 61.98%, and after freeze-drying, the viable count of sample can reach 2.56 × 10
11cFU/g; When to choose 3% skimmed milk be freeze drying protectant, protected effect takes second place, and is 53.72%; During with potato pulp+5% skimmed milk for protective agent, Strain survival rate is minimum is 47.11%.Therefore determine that Lactobacillus plantarum plant subspecies Zhang-LL freeze drying protectant optimum combination formula is potato pulp+3% skimmed milk+2% sodium glutamate, after its freeze-drying, the Zhang-LL bacterial strain viable count of sample can reach 10
11more than CFU/g, survival rate can reach more than 60%.
5, pre-freeze and freeze-drying
By each sample of packing in-35 DEG C of refrigerators after pre-freeze 13h, utilize vacuum freeze drier by pre-freeze active bacteria formulation in-55 DEG C, under the condition of vacuum 0.08mBar, freeze-drying 48h, to bone dry state, obtains freeze drying viable microorganism preparation.The number of viable of Zhang-LL bacterial strain before adopting pour plate cultivation to detect freeze-drying and after freeze-drying, each dilution factor establishes 3 repetitions, the results are shown in Table 9.
In sum, by activation, expand the Lactobacillus plantarum plant subspecies Zhang-LL inoculation of cultivation in 2L 501 rapeseed protein peptone modified MRS culture medium, rapeseed protein peptone modified MRS culture medium consists of the following composition: 501 rapeseed protein peptone 30.0g/L, Triammonium citrate 2.0g/L, glucose 20.0g/L, Tween 80 1.0mL/L, sodium acetate 5.0g/L, dipotassium hydrogen phosphate 2.0g/L, magnesium sulfate 0.5g/L, manganese sulfate 0.25g/L; Utilize 5L automatic fermenter to carry out high density fermentation, Zhang-LL bacterial strain is pressed the inoculum concentration culture transferring of 7% (V/V, volume fraction) in fermentation tank, control temperature 34 DEG C, zymotic fluid pH 6.5, speed of agitator is 150r/min, fermentation 16h, obtains zymotic fluid; By zymotic fluid in 4 DEG C, the centrifugal 10min of 8000r/min, collect and obtain bacterium mud; The potato pulp of bacterium mud with 1/10 original fermentation liquor volume is mixed, adds the protective agent of 3% skimmed milk and 2% sodium glutamate, after mixing, obtain starter culture concentrates; Starter culture concentrates after pre-freeze 13h, is obtained pre-freeze active bacteria formulation in-35 DEG C of refrigerators; Utilize vacuum freeze drier by pre-freeze active bacteria formulation in-55 DEG C, under the condition of vacuum 0.08mBar, freeze-drying 48h, to bone dry state, obtains Lactobacillus plantarum plant subspecies Zhang-LL bacterial strain active bacteria formulation.
The viable count of the Lactobacillus plantarum plant subspecies Zhang-LL active bacteria formulation adopting the fermentation condition of above-mentioned optimization and freeze-dry process to obtain can reach 10
11more than CFU/g, the survival rate of bacterial classification can reach more than 60%.The active bacteria formulation that this method obtains is cheap, and viable count is high, for development of new poultry biological feed stuff additive provides practical basis, has wide market application foreground.
Claims (2)
1. Lactobacillus plantarum plant subspecies (Lactobacillus plantarum subsp.plantarum) Zhang-LL CGMCC No.6936.
2. the method utilizing bacteriocinogeny Lactobacillus plantarum plant subspecies Zhang-LL to prepare active bacteria formulation, it is characterized in that: Lactobacillus plantarum plant subspecies (Lactobacillus plantarum subsp.plantarum) Zhang-LL rapeseed protein peptone modified MRS fermentation medium consists of the following composition: 501 rapeseed protein peptone 30.0g/L, Triammonium citrate 2.0g/L, glucose 20.0g/L, Tween 80 1.0mL/L, sodium acetate 5.0g/L, dipotassium hydrogen phosphate 2.0g/L, magnesium sulfate 0.5g/L, manganese sulfate 0.25g/L; Lactobacillus plantarum plant subspecies Zhang-LL scale-up medium is pressed 7% inoculum concentration culture transferring in rapeseed protein peptone modified MRS fermentation medium, 5L automatic fermenter is utilized to carry out high density fermentation, control fermentation temperature 34 DEG C, zymotic fluid pH 6.5, speed of agitator 150r/min, fermentation 16h, obtains zymotic fluid; By zymotic fluid in 4 DEG C, the centrifugal 10min of 8000r/min, collect and obtain bacterium mud; The potato pulp of bacterium mud with 1/10 original fermentation liquor volume is mixed, then adds 3% skimmed milk and 2% sodium glutamate as freeze drying protectant, after mixing, obtain starter culture concentrates; By starter culture concentrates in-35 DEG C of refrigerators after pre-freeze 13h, utilize vacuum freeze drier by pre-freeze active bacteria formulation in-55 DEG C, under the condition of vacuum 0.08mBar, freeze-drying 48h, to bone dry state, obtains Lactobacillus plantarum plant subspecies Zhang-LL active bacteria formulation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410816266.3A CN104560799A (en) | 2014-12-25 | 2014-12-25 | Preparation method of bacteriocin-producing Lactobacillus plantarum subsp. Plantarum Zhang-LL active bacterial preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410816266.3A CN104560799A (en) | 2014-12-25 | 2014-12-25 | Preparation method of bacteriocin-producing Lactobacillus plantarum subsp. Plantarum Zhang-LL active bacterial preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104560799A true CN104560799A (en) | 2015-04-29 |
Family
ID=53077880
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410816266.3A Pending CN104560799A (en) | 2014-12-25 | 2014-12-25 | Preparation method of bacteriocin-producing Lactobacillus plantarum subsp. Plantarum Zhang-LL active bacterial preparation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104560799A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106188252A (en) * | 2016-07-18 | 2016-12-07 | 北京农学院 | Polypeptide and Lactobacillus plantarum Extracellular metabolism and their application and the method for induction Lactobacillus plantarum bacteriocinogeny and authentication method |
| CN106834186A (en) * | 2017-03-06 | 2017-06-13 | 北京农学院 | A kind of Lactobacillus plantarum probiotics and its preparation method and application |
| CN107988288A (en) * | 2017-12-11 | 2018-05-04 | 常熟理工学院 | A kind of method of high density fermentation production Propionibacterium bacteriocin |
| CN109022331A (en) * | 2018-09-12 | 2018-12-18 | 北京农学院 | A kind of compound micro-ecological preparation and its preparation and application |
| CN113817661A (en) * | 2021-09-04 | 2021-12-21 | 芜湖职业技术学院 | Culture method for improving antibacterial activity of lactobacillus plantarum |
| CN114507619A (en) * | 2022-01-28 | 2022-05-17 | 河北一然生物科技股份有限公司 | Liquid composite probiotic preparation, preparation method thereof and application thereof in aspects of improving diarrhea and protecting liver |
| CN115404181A (en) * | 2022-07-25 | 2022-11-29 | 福建华闽晟业生物科技有限公司 | Application of Jujun grass juice as lactobacillus plantarum fermentation medium |
| CN116694539A (en) * | 2023-08-02 | 2023-09-05 | 东北农业大学 | Lactobacillus plantarum J26 as direct vat set starter, and preparation method and application thereof |
| CN117089504A (en) * | 2023-10-20 | 2023-11-21 | 善恩康生物科技（苏州）有限公司 | Method for fermenting lactobacillus plantarum |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531562A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin |
| CN104531561A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | (Lactobacillus planetarium subsp. plantarum)Zhang-LL bacteriocin and Nisin composite bacteriostatic agent and its use in chilled meat |
| CN104560790A (en) * | 2014-12-15 | 2015-04-29 | 北京农学院 | Functional lactobacillus plantarum and preparation method of compound bacterial powder of functional lactobacillus plantarum |
| CN104726360A (en) * | 2014-11-14 | 2015-06-24 | 北京农学院 | Construction and application of shuttle vector between Escherichia coli and Lactobacilli |
-
2014
- 2014-12-25 CN CN201410816266.3A patent/CN104560799A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726360A (en) * | 2014-11-14 | 2015-06-24 | 北京农学院 | Construction and application of shuttle vector between Escherichia coli and Lactobacilli |
| CN104531562A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin |
| CN104531561A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | (Lactobacillus planetarium subsp. plantarum)Zhang-LL bacteriocin and Nisin composite bacteriostatic agent and its use in chilled meat |
| CN104560790A (en) * | 2014-12-15 | 2015-04-29 | 北京农学院 | Functional lactobacillus plantarum and preparation method of compound bacterial powder of functional lactobacillus plantarum |
Non-Patent Citations (2)
| Title |
|---|
| 凌代文，东秀珠编: "《乳酸细菌分类鉴定及实验方法》", 31 March 1999 * |
| 赵爽 等: "泡菜直投式乳酸菌发酵剂的制备", 《食品工业科技》 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106188252A (en) * | 2016-07-18 | 2016-12-07 | 北京农学院 | Polypeptide and Lactobacillus plantarum Extracellular metabolism and their application and the method for induction Lactobacillus plantarum bacteriocinogeny and authentication method |
| CN106188252B (en) * | 2016-07-18 | 2019-07-19 | 北京农学院 | The method and identification method of polypeptide and lactobacillus plantarum Extracellular metabolism and their application and induction lactobacillus plantarum bacteriocinogeny |
| CN106834186B (en) * | 2017-03-06 | 2020-07-28 | 北京农学院 | Lactobacillus plantarum microecological preparation and preparation method and application thereof |
| CN106834186A (en) * | 2017-03-06 | 2017-06-13 | 北京农学院 | A kind of Lactobacillus plantarum probiotics and its preparation method and application |
| CN107988288A (en) * | 2017-12-11 | 2018-05-04 | 常熟理工学院 | A kind of method of high density fermentation production Propionibacterium bacteriocin |
| CN107988288B (en) * | 2017-12-11 | 2020-05-19 | 常熟理工学院 | Method for producing propionibacterium bacteriocin through high-density fermentation |
| CN109022331A (en) * | 2018-09-12 | 2018-12-18 | 北京农学院 | A kind of compound micro-ecological preparation and its preparation and application |
| CN113817661A (en) * | 2021-09-04 | 2021-12-21 | 芜湖职业技术学院 | Culture method for improving antibacterial activity of lactobacillus plantarum |
| CN114507619A (en) * | 2022-01-28 | 2022-05-17 | 河北一然生物科技股份有限公司 | Liquid composite probiotic preparation, preparation method thereof and application thereof in aspects of improving diarrhea and protecting liver |
| CN114507619B (en) * | 2022-01-28 | 2024-01-12 | 河北一然生物科技股份有限公司 | Liquid composite probiotic preparation, preparation method thereof and application thereof in aspects of improving diarrhea and protecting liver |
| CN115404181A (en) * | 2022-07-25 | 2022-11-29 | 福建华闽晟业生物科技有限公司 | Application of Jujun grass juice as lactobacillus plantarum fermentation medium |
| CN116694539A (en) * | 2023-08-02 | 2023-09-05 | 东北农业大学 | Lactobacillus plantarum J26 as direct vat set starter, and preparation method and application thereof |
| CN116694539B (en) * | 2023-08-02 | 2023-11-17 | 东北农业大学 | Lactobacillus plantarum J26 as direct vat set starter, and preparation method and application thereof |
| CN117089504A (en) * | 2023-10-20 | 2023-11-21 | 善恩康生物科技（苏州）有限公司 | Method for fermenting lactobacillus plantarum |
| CN117089504B (en) * | 2023-10-20 | 2023-12-26 | 善恩康生物科技（苏州）有限公司 | Method for fermenting lactobacillus plantarum |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104560799A (en) | Preparation method of bacteriocin-producing Lactobacillus plantarum subsp. Plantarum Zhang-LL active bacterial preparation | |
| CN103820363B (en) | A kind of preparation and application of faecium bacterium powder | |
| CN102864095B (en) | Lactobacillus plantarum used for silage alfalfa and use method thereof | |
| CN103627656B (en) | A kind of clostridium butyricum and bacillus coagulans mixed culture solid state fermentation method | |
| CN102505003B (en) | High-density fermenting method of bacillus coagulans for livestock and poultry, as well as preparation prepared by method and application thereof | |
| CN102643767B (en) | Lactobacillus plantarum and application thereof in fermenting and ensiling sweet potato stem and leaf | |
| CN102640885B (en) | Applications of Lactobacillus plantarum in complete-strain Triticumspp silage | |
| CN105341149A (en) | Yoghourt fermenting agent and preparation method thereof | |
| CN103911323A (en) | Bacillus licheniformis, bacillus subtilis and lactobacillus plantarum preparation and preparation | |
| CN102864093B (en) | Lactobacillus plantarum suitable for silage straw and use method thereof | |
| CN106754579A (en) | A kind of bacillus coagulans and application thereof | |
| CN106173225B (en) | Method for preparing protein feed additive by solid state fermentation of plant protein feed | |
| CN109666599B (en) | Lactobacillus reuteri high-density fermentation medium, fermentation method and application | |
| CN106260540A (en) | A kind of biological feedstuff for creep feed and creep feed | |
| CN107974421A (en) | A kind of lactobacillus acidophilus and its screening technique and application, a kind of microbial inoculum | |
| CN104593290A (en) | Pickled vegetable composite ferment with antioxidation function and application thereof | |
| CN112126599B (en) | High-density culture method of lactobacillus helveticus, preparation of high-activity bacterium powder and application of high-density culture method | |
| CN104164459A (en) | Method utilizing fermentation to improve gamma-aminobutyric acid content of brown rice | |
| CN106889306A (en) | A kind of feed fermentation agent and the application on feed fermentation | |
| CN103952336B (en) | Bacillus licheniformis, bacillus subtilis and lactobacillus casei preparation and preparation | |
| CN103911327A (en) | Bacillus licheniformis probiotic preparation and preparation method thereof | |
| CN105132321A (en) | Enterococcus faecium, culture medium thereof for high-density solid-state fermentation, and high-density solid-state fermentation method | |
| CN102586137B (en) | Method for preparing bile salt hydrolase lactobacillus powder starter | |
| CN102994421B (en) | Lactic acid bacteria suitable for ensiling oat and applications thereof | |
| CN106497842A (en) | The preparation method and application of a kind of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150429 |