CN115895948A - Lactobacillus paracasei capable of efficiently inhibiting growth of mold and culture method and application thereof - Google Patents
Lactobacillus paracasei capable of efficiently inhibiting growth of mold and culture method and application thereof Download PDFInfo
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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Abstract
The application relates to the field of microorganisms, and particularly discloses lactobacillus paracasei for efficiently inhibiting the growth of mold and a culture method and application thereof. A Lactobacillus paracasei AB0401 with high-efficiency inhibition of mold growth has a preservation number of CGMCC 25070, and the preservation date is 6 months and 13 days in 2022 years; the culture method comprises the following steps: s1 isolation, S2 inoculation, S3 primary activation and S4 monoclonal selection. The lactobacillus paracasei AB0401 can be used in fermented foods and microbial preparations, and has the advantage of efficiently inhibiting the growth of mold.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to lactobacillus paracasei for efficiently inhibiting the growth of mold, a culture method and application thereof.
Background
When the food is placed daily, the food is easily polluted and invaded by microorganisms to be rotten and deteriorated, the nutritive value and the sanitary quality of the food are reduced, and the health of a human body is possibly damaged. Molds are a common group of microorganisms causing food spoilage, some of which can convert food into toxic substances and some of which can produce mycotoxins in food. Both the mold and mycotoxin have great harm to human health, and may cause chronic poisoning, carcinogenesis, teratogenesis and the like.
The lactobacillus can inhibit the propagation of putrefying bacteria and pathogenic bacteria, produce organic acids such as lactic acid, acetic acid and the like, produce substances such as organic acid cell-like protein and the like and relevant metabolites influencing the generation of bacterial toxicants, and researches show that the food can prolong the shelf life through proper lactobacillus fermentation, and has the advantages of better flavor, higher safety and the like.
The lactobacillus paracasei belongs to the genus lactobacillus in the taxonomy of bacteria, and metabolites such as organic acid, bacteriocin and phenyllactic acid of the lactobacillus paracasei can effectively inhibit the growth of putrefying bacteria, so that the lactobacillus paracasei has great development and application values in the food industry. However, there is still a large room for improvement in the mold inhibitory effect of lactobacillus paracasei in food products, and therefore, the inventors have studied how to improve the mold inhibitory activity of lactobacillus paracasei.
Disclosure of Invention
In order to improve the bacteriostatic effect of lactobacillus paracasei in food, the application provides lactobacillus paracasei for efficiently inhibiting the growth of mold and a culture method and application thereof.
In a first aspect, the application provides a lactobacillus paracasei for efficiently inhibiting the growth of mold, which adopts the following technical scheme:
lactobacillus paracasei (L.) having high inhibitory activity against the growth of moldLactobacillus paracasei ) AB0401, preservation number CGMCC 25070, preservation date 2022, 6 months and 13 days.
By adopting the technical scheme, the lactobacillus paracasei AB0401 can efficiently inhibit the growth of the mildew.
Preferably, the 16S rDNA of the lactobacillus paracasei AB0401 is shown as SEQ ID NO. 1.
By adopting the technical scheme, the strain can be confirmed to be lactobacillus paracasei.
In a second aspect, the present application provides a method for culturing lactobacillus paracasei capable of efficiently inhibiting mold growth, which adopts the following technical scheme:
a method for culturing lactobacillus paracasei capable of efficiently inhibiting the growth of mold comprises the following steps:
s1, separation: screening the fermented dairy product, and separating to obtain lactobacillus paracasei;
s2, inoculation: inoculating lactobacillus paracasei separated from the S1 on a culture medium plate to obtain a strain;
s3, primary activation: culturing the strain obtained by inoculating the S2 at 37 ℃ for 20-28h under an anaerobic condition to obtain a primary activated strain;
s4, selecting single clone: selecting appropriate 2-3 single colonies from the preliminary activated strains obtained by the preliminary activation of S3, transferring the single colonies to a enrichment medium, and culturing for 46-50h under the anaerobic condition at 37 ℃ to obtain activated lactobacillus paracasei. By adopting the technical scheme, a large amount of activated lactobacillus paracasei AB0401 which can effectively inhibit the growth of the mold can be obtained.
By adopting the technical scheme, a large amount of activated lactobacillus paracasei AB0401 can be obtained by culture.
Preferably, the fermented milk product in the S1 separation is the traditional yak milk fermented yogurt in the autonomous state of the Tibetan region of Alba, sichuan of China.
By adopting the technical scheme, the strain is derived from traditional fermented food, has food properties and has stronger antibacterial activity.
Preferably, the culture medium used in the S2 inoculation is MRS medium.
By adopting the technical scheme, the MRS culture medium is a lactic acid bacteria culture medium, so that other mixed bacteria can be eliminated, the strain screening is facilitated, and the selection of the target strain with good growth vigor and good activity is facilitated.
Preferably, the S2 inoculation is carried out by using a plate streaking method, and the streaking times are 2-3 times.
By adopting the technical scheme, the characteristics of the bacterial colony can be conveniently observed, and the bacterial strain can be purified and preliminarily activated.
Preferably, the enrichment medium used in the S4 singleton is MRS broth.
By adopting the technical scheme, the MRS broth is commonly used for selective enrichment culture of food lactobacillus, and further activation and enrichment of lactobacillus paracasei can be realized.
In a third aspect, the application provides an application of lactobacillus paracasei for efficiently inhibiting the growth of mold, and the following technical scheme is adopted:
the application of the lactobacillus paracasei AB0401 for efficiently inhibiting the growth of mold is disclosed, and the lactobacillus paracasei is applied to fermented food.
By adopting the technical scheme, the antibacterial effect of the fermented food is enhanced, and the addition amount of the preservative in the fermented food can be reduced.
In a fourth aspect, the application provides an application of lactobacillus paracasei for efficiently inhibiting the growth of mold, and the following technical scheme is adopted:
the application of lactobacillus paracasei AB0401 for efficiently inhibiting the growth of mold is applied to microbial preparations.
By adopting the technical scheme, the microbial preparation with high-efficiency bacteriostasis can be provided.
In summary, the present application has the following beneficial effects:
1. the lactobacillus paracasei AB0401 is screened from the traditional yak milk fermented yogurt and has food attribute and stronger antibacterial activity;
2. the cultured lactobacillus paracasei AB0401 can efficiently inhibit the growth of mould;
3. the lactobacillus paracasei AB0401 can be applied to fermented food, so that the fermented food can efficiently inhibit the growth of mold, thereby reducing the addition amount of preservatives;
4. the lactobacillus paracasei AB0401 can be applied to a microbial preparation and provides a high-efficiency bacteriostatic microbial preparation.
Drawings
FIG. 1 is a graph showing the results of bacteriostasis experiments of different lactobacilli on Aspergillus niger.
FIG. 2 is a graph showing the results of experiments on Aspergillus flavus inhibition by different lactobacilli.
FIG. 3 is a graph showing the results of experiments on the inhibition of Rhizopus oryzae by different lactobacilli.
FIG. 4 is a graph showing the results of a two-layer plate bacteriostatic experiment on Lactobacillus paracasei AB0401.
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples.
Lactobacillus paracasei (see examples)Lactobacillus paracasei ) AB0401, deposited in China general microbiological culture Collection center on 13 th 6 th 2022 month, with the preservation number of CGMCC 25070. The microbial research institute of the national academy of sciences No. 3, xilu No. 1, beijing, chaoyang, address.
In the present application, MRS medium was purchased from Beijing road bridge technology, inc.; MRS broth, purchased from Beijing road bridge technology corporation; PDA medium, purchased from Beijing road bridge technology GmbH; aspergillus nigerAspergillus niger) Aspergillus flavus (A) andAspergillus flavus) Rhizopus oryzae (A. Oryzae) ((A. Oryzae))Rhizopus oryzae) Rhizopus stolonifer (Rhizopus nigricans) ((A))Rhizopus stolonifer) Penicillium expansum (Penicillium expansum) Are purchased from China general microbiological culture Collection center (CGMCC).
Examples
EXAMPLE 1 isolation of Lactobacillus paracasei AB0401
S1, separation: collecting traditional yak milk fermented yogurt in the region of autonomous state of the Tibetan Alba, sichuan China, separating out single colony, identifying lactobacillus by a microscope, further determining 16S rDNA, determining lactobacillus paracasei, preserving and numbering AB0401, and obtaining lactobacillus paracasei;
s2, inoculation: inoculating lactobacillus paracasei separated from S1 on an MRS culture medium plate by using a plate streaking method, and streaking for 3 times to obtain a strain;
s3, primary activation: culturing the strain obtained by inoculating the S2 for 24 hours under the anaerobic condition at 37 ℃ to obtain a primary activated strain;
s4, selecting single clone: appropriate 3 single colonies of the primary activated species obtained from the S3 primary activation were picked and transferred to MRS broth, and cultured at 37 ℃ for 48h to obtain 100mL of activated Lactobacillus paracasei.
EXAMPLE 2 identification of Lactobacillus paracasei AB0401
The results of the gene sequence test of 16S rDNA of Lactobacillus paracasei AB0401 are as follows:
CCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAAGGTGGGACAAATGATTAGGGTGAAGTCGTAACAAG
according to the gene sequence result of 16S rDNA, the strain can be determined to be lactobacillus paracasei.
Example 3 Lactobacillus paracasei AB0401 and other Lactobacillus 24-well plate bacteriostasis experiments
Preparation of a mould spore suspension: inoculating mould on a PDA (personal digital assistant) plate by using an inoculating needle, culturing for 4 days at 25 ℃, washing mould spores on the mould plate by using normal saline, filtering by using absorbent cotton to obtain spore suspension, sucking 200 mu L of the spore suspension into a 96-well plate, measuring the absorbance by using an enzyme-linked immunosorbent assay (OD 600) instrument, diluting to the OD value of 0.15, and placing in a refrigerator for later use. The mould is Aspergillus niger, aspergillus flavus and Rhizopus oryzae.
Preparing fermentation liquor: sucking activated lactobacillus bacterial liquid by using a sterile suction head, inoculating the lactobacillus bacterial liquid into MRS broth in a proportion of 1vol%, and culturing the MRS broth in a constant-temperature incubator at 37 ℃ for 48 hours to obtain fermentation liquid. Wherein the lactobacillus is selected from traditional yak milk fermented yogurt collected from the region of autonomous state of the Tibetan Alba of Sichuan China.
24-well plate bacteriostasis experiment: respectively sucking 1mL of mould spore suspension, adding into a 24-pore plate containing 1mL of lactobacillus fermentation liquor and MRS liquid, culturing at 25 ℃ for 48-72h, and observing the size of a bacteriostatic zone, wherein the results are shown in figures 1-3 and table 1.
TABLE 1 results of inhibition zone of Lactobacillus paracasei AB0401 and other lactobacillus fermentation liquid
As can be seen from Table 1, streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus have certain inhibition effects on Aspergillus niger and Aspergillus flavus, but have almost no inhibition effect on Rhizopus oryzae; and the lactobacillus paracasei AB0401 has better inhibition effect on aspergillus niger, aspergillus flavus and rhizopus oryzae.
Example 4 bacteriostatic test on 96-well plate of Lactobacillus paracasei AB0401
Preparation of a mould spore suspension: inoculating mould on a PDA (personal digital assistant) plate by using an inoculating needle, culturing for 4 days at 25 ℃, washing mould spores on the mould plate by using normal saline, filtering by using absorbent cotton to obtain spore suspension, sucking 200 mu L of the spore suspension into a 96-well plate, measuring absorbance by using an enzyme-labeling instrument OD600, diluting to an OD value of 0.15, and placing in a refrigerator for later use. The mould is Aspergillus niger, aspergillus flavus, rhizopus oryzae, rhizopus stolonifer and Penicillium expansum.
Preparing fermentation liquor: sucking activated Lactobacillus paracasei AB0401 by using a sterile sucker, inoculating the activated Lactobacillus paracasei AB0401 into MRS broth in a proportion of 1vol%, and culturing the MRS broth in a constant-temperature incubator at 37 ℃ for 48 hours to obtain fermentation liquid.
Preparing lactobacillus fermentation supernatant: centrifuging the fermentation liquor at 12000 rpm at 4 deg.C for 10min, sucking the supernatant with sterile syringe, filtering with 0.22 μm filter membrane to obtain sample to be detected, packaging with 10mL clean sterile small tube, and storing in 4 deg.C refrigerator.
ph6.5 fermentation supernatant: adding a small amount of 2mol/L NaOH solution into the subpackaged fermentation supernatant, measuring the pH value by using a pH meter, repeating for many times until the pH value of the supernatant is 6.5-7.0, and storing in a refrigerator at 4 ℃.
Bacteriostatic experiments on 96-well plates: respectively sucking 100 μ L of mold spore suspension, adding into 96-well plate containing 100 μ L of lactobacillus fermentation supernatant, pH6.5 fermentation supernatant and MRS liquid, culturing at 25 deg.C for 48 hr, measuring absorbance at OD600 position of enzyme labeling instrument, and recording the measurement results of lactobacillus fermentation supernatant and pH6.5 fermentation supernatant as OD LAB And the measurement result of the MRS-containing liquid is recorded as OD control . Bacteriostatic rate% =100- (OD) LAB ×100/OD control ) The calculation results are shown in Table 2.
TABLE 2 results of the bacteriostasis rate of Lactobacillus paracasei AB0401 fermentation liquid
As can be seen from Table 2, the lactic acid bacteria fermentation supernatant has a good inhibition effect on 5 kinds of moulds, and the inhibition rate is about 90%;
the lactobacillus paracasei can be metabolized to generate organic acid with broad-spectrum antibacterial effect, and under the condition of pH6.5, the organic acid basically loses efficacy, but the fermentation supernatant with pH6.5 still has certain antibacterial effect, which shows that non-acid antibacterial substances exist in the fermentation liquid of the lactobacillus paracasei, so that the lactobacillus paracasei AB0401 can effectively inhibit the growth of mold.
Example 5 double-layer plate bacteriostasis experiment of Lactobacillus paracasei AB0401
Preparing a lactobacillus bottom-layer flat plate: inoculating two bacterial liquid strips of lactobacillus paracasei AB0401 on an MRS plate by using an inoculating loop, airing, and then placing in an incubator at 37 ℃ for culturing for 48h.
Preparation of a mould spore suspension: inoculating mould on a PDA (personal digital assistant) plate by using an inoculating needle, culturing for 4 days at 25 ℃, washing mould spores on the mould plate by using normal saline, filtering by using absorbent cotton to obtain spore suspension, sucking 200 mu L of the spore suspension into a 96-well plate, measuring the absorbance by using an enzyme-linked immunosorbent assay (OD 600) instrument, diluting to the OD value of 0.15, and placing in a refrigerator for later use. The mould is Aspergillus niger, aspergillus flavus, rhizopus oryzae, and Rhizopus stolonifer.
Preparation of a mould spore PDA culture medium: the prepared mold spore suspension is sucked and added into the PDA culture medium which is not solidified after being cooled to below 50 ℃ in a proportion of 0.1 vol%.
Double-layer flat plate bacteriostasis experiment: pouring the PDA culture medium containing the mould spore suspension on the upper layer of the cultured lactobacillus bottom flat plate, solidifying, culturing at 25 ℃ for 48h, observing, photographing and recording. The results are shown in FIG. 4.
As can be seen from FIG. 4, lactobacillus paracasei AB0401 has inhibitory effects on four molds, among which the inhibitory effects on Aspergillus niger and Aspergillus flavus are the best and the inhibitory effect on Rhizopus oryzae is the worst.
Comparative example 1
Preparation of a mould spore suspension: inoculating mould on a PDA (personal digital assistant) plate by using an inoculating needle, culturing for 4 days at 25 ℃, washing mould spores on the mould plate by using normal saline, filtering by using absorbent cotton to obtain spore suspension, sucking 200 mu L of the spore suspension into a 96-well plate, measuring absorbance by using an enzyme-labeling instrument OD600, diluting to an OD value of 0.15, and placing in a refrigerator for later use. The mould is Aspergillus niger, aspergillus flavus and Rhizopus oryzae.
Preparing fermentation liquor: sucking activated lactobacillus liquid by using a sterile suction head, inoculating the lactobacillus liquid into MRS broth in a proportion of 1vol%, and culturing in a constant-temperature incubator at 37 ℃ for 48 hours to obtain fermentation liquid. Wherein the lactobacillus paracasei ZFM54 is obtained according to the method disclosed in the Chinese patent with the publication number CN111286474A, the lactobacillus paracasei ALAC-4 is obtained according to the method disclosed in the Chinese patent with the publication number CN110229768A, and the lactobacillus paracasei TS301476 and the lactobacillus paracasei TS301489 are purchased from Ningbo Testout biotechnology Limited liability company.
24-well plate bacteriostasis experiment: respectively sucking 1mL of mould spore suspension, adding the mould spore suspension into a 24-pore plate containing 1mL of lactobacillus fermentation liquor and MRS liquid, culturing for 48-72h at 25 ℃, and observing the size of a bacteriostatic zone, wherein the results are shown in Table 3.
TABLE 3 results of inhibition zone of lactobacillus paracasei fermentation liquor
By combining the example 3 and the comparative example 1 and combining the table 1 and the table 3, different lactobacillus paracasei have certain inhibition effects on aspergillus niger, aspergillus flavus and rhizopus oryzae, and the inhibition effect on the three moulds is combined, so that the lactobacillus paracasei AB0401 has the best inhibition effect.
Application example 1
The lactobacillus paracasei AB0401 can be applied to fermented food, and the prepared fermented food has the effect of efficiently inhibiting the growth of mold.
Application example 2
The lactobacillus paracasei AB0401 can be applied to microbial preparations, and the prepared microbial preparations have the effect of efficiently inhibiting the growth of mold.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (9)
1. A Lactobacillus paracasei (Lactobacillus paracasei) AB0401 for efficiently inhibiting the growth of mold is characterized in that the preservation number is CGMCC 25070, the preservation date is 2022 years, 6 months and 13 days, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is the institute of microbiology of China academy of sciences No. 3 of North date West Lu No. 1 of the Yangtze district in Beijing.
2. The lactobacillus paracasei AB0401 with high efficiency for inhibiting mold growth according to claim 1, wherein: the 16S rDNA of the lactobacillus paracasei AB0401 is shown in SEQ ID NO 1.
3. A method for culturing Lactobacillus paracasei AB0401 which has a high inhibitory effect on the growth of mold as described in any of claims 1 to 2, comprising the steps of:
s1, separation: screening the fermented dairy product, and separating to obtain lactobacillus paracasei;
s2, inoculation: inoculating lactobacillus paracasei separated from the S1 on a culture medium flat plate to obtain a strain;
s3, primary activation: culturing the strain obtained by inoculating the S2 at 37 ℃ for 20-28h under an anaerobic condition to obtain a primary activated strain; s4, selecting monoclonal antibody: selecting appropriate 2-3 single colonies from the preliminary activated strains obtained by the preliminary activation of S3, transferring the single colonies to a enrichment medium, and culturing for 46-50h under the anaerobic condition at 37 ℃ to obtain activated lactobacillus paracasei.
4. The method for culturing Lactobacillus paracasei AB0401 with high efficiency in inhibiting mold growth according to claim 3, wherein: the fermented milk product in the S1 separation is the traditional yak milk fermented yogurt in the region of autonomous State of Alba Tibetan of Sichuan China.
5. The method for culturing Lactobacillus paracasei AB0401 with high efficiency in inhibiting mold growth according to claim 3, wherein: and the culture medium used in the S2 inoculation is an MRS culture medium.
6. The method for culturing Lactobacillus paracasei AB0401 with high efficiency in inhibiting mold growth according to claim 3, wherein: and in the S2 inoculation, a plate scribing method is used for inoculation, and the scribing times are 2-3.
7. The method for culturing Lactobacillus paracasei AB0401 with high efficiency for inhibiting mold growth according to claim 3, wherein: the enrichment culture medium used in the S4 picking monoclonal is MRS broth.
8. Use of a highly effective mould growth inhibiting lactobacillus paracasei AB0401 according to any of claims 1-2 in fermented food products.
9. Use of a Lactobacillus paracasei AB0401 with high efficiency in the inhibition of mold growth according to any of the claims 1 to 2, in a microbial preparation.
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