CN110923186A - Lactobacillus plantarum proliferation agent - Google Patents

Lactobacillus plantarum proliferation agent Download PDF

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Publication number
CN110923186A
CN110923186A CN201911418565.0A CN201911418565A CN110923186A CN 110923186 A CN110923186 A CN 110923186A CN 201911418565 A CN201911418565 A CN 201911418565A CN 110923186 A CN110923186 A CN 110923186A
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lactobacillus plantarum
fermentation
culture
proliferation agent
supernatant
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张楠
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Nanchang Norway Pharmaceutical Technology Co ltd
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Nanchang Norway Pharmaceutical Technology Co ltd
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    • 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
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • 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
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • 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

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Abstract

The invention discloses a lactobacillus plantarum proliferation agent, which is prepared from the following components in parts by weight: and (3) lactobacillus plantarum LP45 supernatant. Preferably, the fermentation supernatant is obtained by fermentation in a medium containing betaine. The preparation method of the proliferation agent provided by the invention is simple, the cost is low, and the used bacterial strain is probiotics and can improve other health-care probiotic effects; the proliferation agent prepared by the invention has the growth promoting effect on the lactobacillus plantarum in milk, and the preferred embodiment has the capability of highly efficiently and remarkably promoting the lactobacillus plantarum to be enhanced, so that the application potential of the lactobacillus plantarum in the practical product development of the application space is greatly improved.

Description

Lactobacillus plantarum proliferation agent
Technical Field
The invention relates to a lactobacillus plantarum proliferation agent, belonging to the technical field of microorganisms.
Background
The lactobacillus plantarum is one of lactobacillus, the optimal growth temperature is 30-35, the lactobacillus plantarum is anaerobic or facultative anaerobic, the strain is a straight or bent rod, the strain is single or sometimes paired or chained, the optimal pH value is about 6.5, and the lactobacillus plantarum belongs to homofermentation lactobacillus. The bacterium is different from other lactic acid bacteria in that the number of viable bacteria of the bacterium is higher, a large amount of acid can be produced, the PH value in water is stable and not increased, and the produced acidic substances can degrade heavy metals; since this bacterium is an anaerobic bacterium (facultative aerobe), it produces a characteristic lactobacillin, a biological preservative, during the propagation process. In the middle and later period of cultivation, as the excrement and the residual feed of animals are increased, the animals sink to the bottom of the pond and are rotten, a lot of germs are bred, a large amount of ammonia nitrogen and nitrite are generated, and the phenomenon that the bottom is killed by theft is serious. If the lactobacillus plantarum is used for a long time, the rot of bottom excrement and residual feed can be well inhibited, the increase of ammonia nitrogen and nitrite is reduced, the consumption of chemical degradation elements is greatly reduced, and the breeding cost is reduced.
The lactobacillus plantarum has a plurality of health-care effects, such as ① having a certain immunoregulation effect, ② having an inhibition effect on pathogenic bacteria, ③ reducing serum cholesterol content and preventing cardiovascular diseases, ④ maintaining the balance of flora in intestinal tracts, ⑤ promoting nutrient absorption, ⑥ relieving lactose intolerance, ⑦ inhibiting the formation of tumor cells and the like.
The invention aims to provide a lactobacillus plantarum proliferation agent which is effective in stimulating the growth of lactobacillus plantarum, particularly in cow's milk.
Disclosure of Invention
The invention aims to provide a lactobacillus plantarum proliferation agent which is effective in stimulating the growth of lactobacillus plantarum, particularly in cow's milk.
Lactobacillus plantarum LP45 is a strain known in the art and known to have various health benefits, and Lactobacillus plantarum LP45 used in the present invention was purchased from Hibei Yiran Biotech, Inc.
In the research and practice process of the invention group, the fermentation supernatant of the lactobacillus plantarum LP45 is found to have weak capacity of stimulating the proliferation of the lactobacillus plantarum, the fermentation process is further optimized and induced, and the effect of highly efficiently stimulating the proliferation of the lactobacillus plantarum can be achieved.
The technical problem to be solved by the invention can be realized by the following technical scheme.
A lactobacillus plantarum proliferation agent which is:
and (3) lactobacillus plantarum LP45 supernatant.
Preferably, the fermentation supernatant is obtained by fermentation in a medium containing betaine.
As a further preferred method for preparing the fermentation supernatant, the method comprises:
(1) activating strains:
a loop of lactobacillus plantarum LP45 was inoculated into LB solid medium for streaking and aerobically cultured at 37 ℃ for 24 hours. Selecting single colony with good growth in the plate, inoculating the single colony in LB liquid culture medium for activation, and finally adjusting the concentration to 108cfu/ml Lactobacillus plantarum LP45 seed fluid.
(2) Inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 3% by volume for fermentation, wherein the culture temperature is 25-30 ℃, the rotation speed of a shaking table is 150-200 rpm/min, and culturing for 48-62h to obtain a pure culture;
the formula of the fermentation medium is as follows:
1L of distilled water +20g of glucose +3g of betaine +1g of trehalose +15g of peptone +5g of sodium chloride +0.5g of beef extract +20g of agar, pH 7.0.
(3) And (3) centrifuging the pure culture obtained in the step (2) for 15min at the rotation speed of 1200rpm and the temperature of 4 ℃, and performing sterile filtration on the supernatant to obtain the culture, wherein the pore diameter of a filtration membrane is 0.22 mu m.
The method for applying the lactobacillus plantarum proliferation agent to promoting lactobacillus plantarum growth comprises the following steps:
(1) preparing lactobacillus plantarum seed liquid;
(2) dissolving a lactobacillus plantarum proliferation agent in water to prepare a solution with the concentration of 10%;
(3) inoculating the lactobacillus plantarum seed solution prepared in the step (1) into sterilized raw milk in a ratio of 2% (v/v), adding the lactobacillus plantarum proliferation agent obtained in the step (2) with the volume of 5% of the raw milk, uniformly mixing, and standing for culture.
Preferably, the culture is carried out at 35 ℃ for 12 hours.
The invention has the advantages that:
(1) the preparation method of the proliferation agent provided by the invention is simple, the cost is low, and the used bacterial strain is probiotics and can improve other health-care probiotic effects;
(2) the proliferation agent prepared by the invention has the growth promoting effect on the lactobacillus plantarum in milk, and the preferred embodiment has the capability of highly efficiently and remarkably promoting the lactobacillus plantarum to be enhanced, so that the application potential of the lactobacillus plantarum in the practical product development of the application space is greatly improved.
Detailed Description
The following examples of the present invention are described in detail, and are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention.
Specific examples of the present invention are described below.
Example 1
The following experimental groups were set up and 5 replicates of each group were averaged.
Group 1:
and (3) lactobacillus plantarum LP45 supernatant.
Preferably, the fermentation supernatant is obtained by fermentation in a medium containing betaine.
As a further preferred method for preparing the fermentation supernatant, the method comprises:
(1) activating strains:
a loop of lactobacillus plantarum LP45 was inoculated into LB solid medium for streaking and aerobically cultured at 37 ℃ for 24 hours. Selecting single colony with good growth in the plate, inoculating the single colony in LB liquid culture medium for activation, and finally adjusting the concentration to 108cfu/ml Lactobacillus plantarum LP45 seed fluid.
(2) Inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 3% by volume for fermentation, wherein the culture temperature is 30 ℃, the rotating speed of a shaking table is 200 rpm/min, and culturing for 48-62h to obtain a pure culture;
the formula of the fermentation medium is as follows:
1L of distilled water +20g of glucose +3g of betaine +1g of trehalose +15g of peptone +5g of sodium chloride +0.5g of beef extract +20g of agar, pH 7.0.
(3) And (3) centrifuging the pure culture obtained in the step (2) for 15min at the rotation speed of 1200rpm and the temperature of 4 ℃, and performing sterile filtration on the supernatant to obtain the culture, wherein the pore diameter of a filtration membrane is 0.22 mu m.
Group 2, medium without betaine:
and (3) lactobacillus plantarum LP45 supernatant.
Preferably, the fermentation supernatant is obtained by fermentation in a medium containing betaine.
As a further preferred method for preparing the fermentation supernatant, the method comprises:
(1) activating strains:
a loop of lactobacillus plantarum LP45 was inoculated into LB solid medium for streaking and aerobically cultured at 37 ℃ for 24 hours. Selecting single colony with good growth in the plate, inoculating the single colony in LB liquid culture medium for activation, and finally adjusting the concentration to 108cfu/ml Lactobacillus plantarum LP45 seed fluid.
(2) Inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 3% by volume for fermentation, wherein the culture temperature is 30 ℃, the rotating speed of a shaking table is 200 rpm/min, and culturing for 48-62h to obtain a pure culture;
the formula of the fermentation medium is as follows:
1L of distilled water +20g of glucose +1g of trehalose +15g of peptone +5g of sodium chloride +0.5g of beef extract +20g of agar, pH 7.0.
(3) And (3) centrifuging the pure culture obtained in the step (2) for 15min at the rotation speed of 1200rpm and the temperature of 4 ℃, and performing sterile filtration on the supernatant to obtain the culture, wherein the pore diameter of a filtration membrane is 0.22 mu m.
Group 3, LB broth with 1% betaine.
Example 2
The method for measuring the growth promoting effect of the lactobacillus plantarum ST-III on milk specifically comprises the following steps:
(1) preparation of lactobacillus plantarum ST-III seed liquid: inoculating a loop of pure lactobacillus to MRS solid culture medium, streaking, and carrying out anaerobic culture at 37 ℃ for 24 h. Selecting single colony with better growth in the plate, inoculating the single colony in MRS liquid culture medium for activation, and performing anaerobic culture to 108cfu/ml lactobacillus plantarum seed fluid. (ii) a
(2) Dissolving the proliferation agent of groups 1-3 in water to prepare a solution with a concentration of 10%;
(3) inoculating the lactobacillus plantarum seed solution prepared in the step (1) into sterilized raw milk in a ratio of 2% (v/v), adding the lactobacillus plantarum proliferation agent obtained in the step (2) with the volume of 5% of the raw milk, uniformly mixing, and standing for culture.
Preferably, the culture is performed at 35 ℃ for 12 hours, and the culture is observed at 0.6 hour and 12 hours, respectively. Observing the curd condition of the culture medium in the culture process; ST-III colonies in milk medium were counted in log CFU/mL using MRS plate pour method.
The results are shown in table 1 below:
TABLE 1
Group 1 Group 2 Group 3
0h colony count (log CFU/mL) 7.16 7.16 7.16
6h colony count (log CFU/mL) 7.32 8.89 6.61
12h colony count (log CFU/mL) 7.69 9.05 4.30
Curd condition at 0h Non-curd Non-curd Non-curd
Curd condition of 6h Curd Non-curd Non-curd
Curd condition of 12h Curd Curd Non-curd
Therefore, the fermentation supernatant can obviously promote the proliferation of the lactobacillus plantarum in the milk, and in the preparation of the supernatant, the stimulation and induction of the betaine are very important, and the effect of the supernatant obtained by the culture of the culture medium without the betaine is very weak.
It is to be understood that the foregoing is only a preferred embodiment of the invention and that modifications, variations and changes may be made in the invention without departing from the spirit or scope of the invention as defined in the appended claims.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims (6)

1. A lactobacillus plantarum proliferation agent which is:
and (3) lactobacillus plantarum LP45 supernatant.
2. A proliferation agent as claimed in claim 1, which is characterized in that:
the fermentation supernatant is obtained by fermentation in a culture medium containing betaine.
3. The propagation agent of claim 2, wherein the fermentation supernatant is prepared by:
(1) activating strains:
inoculating a loop of lactobacillus plantarum LP45 into LB solid culture medium for streaking, and carrying out aerobic culture at 37 ℃ for 24 h; selecting single colony with good growth in the plate, inoculating the single colony in LB liquid culture medium for activation, and finally adjusting the concentration to 108cfu/ml lactobacillus plantarum LP45 seed fluid;
(2) inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 3% by volume for fermentation, wherein the culture temperature is 25-30 ℃, the rotation speed of a shaking table is 150-200 rpm/min, and culturing for 48-62h to obtain a pure culture;
(3) and (3) centrifuging the pure culture obtained in the step (2) for 15min at the rotation speed of 1200rpm and the temperature of 4 ℃, and performing sterile filtration on the supernatant to obtain the culture, wherein the pore diameter of a filtration membrane is 0.22 mu m.
4. The proliferation agent of claim 3, wherein the fermentation medium is formulated as:
1L of distilled water +20g of glucose +3g of betaine +1g of trehalose +15g of peptone +5g of sodium chloride +0.5g of beef extract +20g of agar, pH 7.0.
5. Use of a Lactobacillus plantarum proliferation agent according to claims 1-4 in a method for promoting Lactobacillus plantarum growth.
6. The method of claim 5, comprising the steps of:
(1) preparing lactobacillus plantarum seed liquid;
(2) dissolving a lactobacillus plantarum proliferation agent in water to prepare a solution with the concentration of 10%;
(3) inoculating the lactobacillus plantarum seed solution prepared in the step (1) into sterilized raw milk in a ratio of 2% (v/v), adding the lactobacillus plantarum proliferation agent obtained in the step (2) with the volume of 5% of the raw milk, uniformly mixing, and standing for culture.
CN201911418565.0A 2019-12-31 2019-12-31 Lactobacillus plantarum proliferation agent Withdrawn CN110923186A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115895963A (en) * 2022-11-29 2023-04-04 内蒙古九禾农业科技发展有限公司 Preparation method of rhizoma atractylodis stem and leaf extract and application of rhizoma atractylodis stem and leaf extract in promoting proliferation of lactobacillus plantarum

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115895963A (en) * 2022-11-29 2023-04-04 内蒙古九禾农业科技发展有限公司 Preparation method of rhizoma atractylodis stem and leaf extract and application of rhizoma atractylodis stem and leaf extract in promoting proliferation of lactobacillus plantarum
CN115895963B (en) * 2022-11-29 2024-05-14 内蒙古九禾农业科技发展有限公司 Preparation method of rhizoma atractylodis stem and leaf extract and application of rhizoma atractylodis stem and leaf extract in promoting proliferation of lactobacillus plantarum

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