CN106434465B - Method for realizing high-density fermentation of lactic acid bacteria by adopting amorphous solid attachments - Google Patents

Method for realizing high-density fermentation of lactic acid bacteria by adopting amorphous solid attachments Download PDF

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CN106434465B
CN106434465B CN201610892909.1A CN201610892909A CN106434465B CN 106434465 B CN106434465 B CN 106434465B CN 201610892909 A CN201610892909 A CN 201610892909A CN 106434465 B CN106434465 B CN 106434465B
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fermentation
lactobacillus
lactic acid
acid bacteria
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CN106434465A (en
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杜新永
郭兴峰
王雷
李燕
孙小凡
曾庆华
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Liaocheng University
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Abstract

The invention discloses a technology for realizing high-density fermentation of lactic acid bacteria by adopting an indefinite solid attachment, which comprises the following steps: (1) selecting a good lactobacillus strain which is vigorous in growth and can reach a logarithmic growth phase within 4-8h after inoculation for activation, and inoculating the activated lactobacillus strain into a lactobacillus culture medium; (2) performing fermentation culture at 37 deg.C, and harvesting thallus after culture. The fermentation technology provided by the invention does not need material supplement, and can complete the fermentation process at one time; a general fermentation tank can be adopted, and a special fermentation tank does not need to be invested; the invention can obtain the lactobacillus thallus, and all production processes taking the lactobacillus biomass as the fermentation purpose can be applied; after fermentation, the total number of bacterial colonies can reach 1010‑12cfu/mL; the obtained thalli has high temperature resistance and strong stress resistance, and the survival rate of the thalli in simulated gastric acid and cholate environments is improved by 2-3 times.

Description

Method for realizing high-density fermentation of lactic acid bacteria by adopting amorphous solid attachments
Technical Field
The invention relates to the field of food microbial fermentation engineering, in particular to a method for realizing high-density fermentation of lactic acid bacteria by adopting an amorphous solid attachment.
Background
Lactic acid bacteria are a generic term for a group of gram-positive bacteria that utilize fermentable carbohydrates to produce large amounts of lactic acid. Lactic acid bacteria are a group of microorganisms beneficial to the human body and widely distributed in the natural world. The lactobacillus fermentation can produce a large amount of metabolites such as organic acids, alcohols and various amino acids, and has physiological effects of inhibiting putrefaction, improving digestibility, preventing cancer, etc. The application history of lactic acid bacteria is very long, and the lactic acid bacteria are widely applied to the fermented dairy industry, the food processing industry, the feed preparation, the medicine and health and the prevention and treatment of livestock diseases nowadays.
With the rapid development of the fermented dairy product industry in China, the efficient concentrated yoghurt starter culture is actively researched and vigorously developed, and the method has important significance for promoting the industrialization process of the lactobacillus starter culture in China and promoting the development of the fermented dairy product industry in China. The key point of the preparation of the lactic acid bacteria hyperconcentration leaven is to realize the high-activity and high-density culture of the lactic acid bacteria, so that the research of the high-density culture engineering technology of the lactic acid bacteria has important practical significance and broad prospect.
The high-density culture of lactic acid bacteria can be used for producing leavening agents, probiotic preparations, lactic acid, bacteriocins, and other products, but acidification and metabolic products of fermentation liquor of the lactic acid bacteria in the culture process can inhibit the growth of the lactic acid bacteria, so that the density of cultured thalli and the continuity of biomass accumulation are seriously inhibited.
In the process of culturing the thalli, the product inhibition coefficient influences the growth rate and the product conversion rate. While controlling the pH change only partially slows this inhibition. In the prior art, in order to realize high-density fermentation of lactic acid bacteria, (1) material supplement and a membrane filtration device are adopted to dilute or remove metabolic waste and relieve growth inhibition; or (2) fixing cells by adopting immobilization or microencapsulation to prevent the metabolites from influencing the growth of the lactic acid bacteria. In addition, a biomembrane induction method (3) can be adopted to enable the lactobacillus to form a biomembrane through exogenous stimulation, so that high-density culture is realized; finally, the scholars observe the cell growth cycle through the mathematical model (4) and calculate the variation trend of the cell concentration, the biosynthesis activity, the substrate consumption rate and the like, and finally obtain the optimal target product production model or high-density culture.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for realizing high-density fermentation of lactic acid bacteria by adopting an amorphous solid attachment, and the amorphous solid suspension is adopted to induce the lactic acid bacteria to generate a biological membrane, so that high-density culture of the lactic acid bacteria is realized.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for realizing high-density fermentation of lactic acid bacteria by adopting an amorphous solid attachment comprises the following steps:
(1) selecting a good lactobacillus strain which is vigorous in growth and can reach a logarithmic growth phase within 4-8h after inoculation for activation, and inoculating the activated lactobacillus strain into a lactobacillus culture medium;
(2) performing fermentation culture at 37 deg.C, and harvesting thallus after culture.
In the method for realizing high-density fermentation of lactic acid bacteria by using the amorphous solid attachment, the preferable scheme is that the lactic acid bacteria strain in the step (1) is one of lactobacillus salivarius, lactobacillus fermentum and pediococcus acidilactici.
In the method for realizing high-density fermentation of lactic acid bacteria by using the amorphous solid attachments, the preferable scheme is that the inoculation amount of the lactic acid bacteria strain in the step (1) is 0.5 ~ 1%.
In the method for realizing high-density fermentation of lactic acid bacteria by using the amorphous solid attachment, the preferable scheme is that the lactic acid bacteria culture medium in the step (1) is a PY culture medium.
In the method for realizing high-density fermentation of lactic acid bacteria by using the amorphous solid attachment, the preferred scheme is that the PY culture medium is composed of the following raw materials in parts by weight: 50g of unhydrolyzed macromolecular protein, 5g of peptone, 5g of tryptone, 10g of yeast powder, 20g of anhydrous sodium acetate and KH2PO46g，MgSO4∙7H2O 0.6g，MnSO4∙4H20.2g of O and 1000g of water.
In the method for realizing high-density fermentation of lactic acid bacteria by using the amorphous solid attachment, the pH value of the PY culture medium is preferably 6.8.
In the method for realizing high-density fermentation of lactic acid bacteria by using the amorphous solid attachments, the preferable scheme is that the fermentation time in the step (2) is 36-48 h.
Compared with the prior art, the invention has the beneficial effects that:
(1) the fermentation method provided by the invention does not need material supplement and can complete the fermentation process at one time; a general fermentation tank can be adopted, and a special fermentation tank does not need to be invested;
(2) the invention can obtain the lactobacillus thallus, and all production processes taking the lactobacillus biomass as the fermentation purpose can be applied, while the immobilized (biomembrane) high-density culture method can not obtain the lactobacillus thallus and only takes the lactobacillus metabolite as the target product;
(3) according to the fermentation method provided by the invention, after the fermentation is finished, the total number of bacterial colonies can reach 1010-12cfu/mL; the obtained starter is wrapped by the curdlin, so that most of thallus embedding is realized;
(4) the thalli obtained by the fermentation method has high temperature resistance and strong stress resistance, and the survival rate of the thalli in simulated gastric acid and bile salt environments is improved by 2-3 times.
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FIGS. 1-3 are graphs showing a comparison of the total number of colonies obtained by the fermentation process of the present invention and a control.
FIGS. 4-6 are graphs showing the comparison of the high temperature resistance of the fermentation products of the present invention and the control.
FIGS. 7-9 are graphs showing the gastric acid tolerance of the fermentation products of the present invention and the control group compared with each other.
FIGS. 10-12 are graphs showing the bile salt tolerance of the fermentation products of the present invention and the control group in comparison.
Detailed Description
The technical solution of the present invention is described in detail below with reference to examples and experimental examples, but the scope of protection is not limited thereto. The raw materials used in the invention can be purchased from the market.
Example 1 a method for achieving high density fermentation of lactic acid bacteria using an amorphous solid attachment, comprising the steps of:
(1) selecting excellent lactobacillus salivarius strains which are vigorous in growth and can reach the logarithmic growth phase within 4-8h after inoculation for activation, inoculating the activated lactobacillus salivarius strains into a PY culture medium with the pH of 6.8, wherein the inoculation amount of the lactobacillus salivarius strains is 0.5 ~ 1%, and the PY culture medium is composed of the following raw materials in parts by weight of 50g of unhydrolyzed macromolecular protein, 5g of peptone, 5g of tryptone, 10g of yeast powder, 20g of anhydrous sodium acetate and 20g of KH2PO46g，MgSO4∙7H2O0.6g，MnSO4∙4H20.2g of O and 1000g of water;
(2) fermenting and culturing at 37 deg.C for 36-48h, and harvesting thallus after culture.
Example 2 a method for achieving high density fermentation of lactic acid bacteria using an amorphous solid attachment, comprising the steps of:
(1) selecting a good lactobacillus fermentum strain which is vigorous in growth and can reach a logarithmic growth phase within 4-8h after inoculation for activation, inoculating the activated lactobacillus fermentum strain into a PY culture medium with the pH of 6.8, wherein the inoculation amount of the lactobacillus fermentum strain is 0.5 ~ 1%, and the PY culture medium is composed of the following raw materials in parts by weight of 50g of unhydrolyzed macromolecular protein, 5g of peptone, 5g of tryptone, 10g of yeast powder, 20g of anhydrous sodium acetate, and 20g of KH2PO46g，MgSO4∙7H2O0.6g，MnSO4∙4H20.2g of O and 1000g of water;
(2) fermenting and culturing at 37 deg.C for 36-48h, and harvesting thallus after culture.
Example 3 a method for achieving high density fermentation of lactic acid bacteria using an amorphous solid attachment, comprising the steps of:
(1) selecting excellent pediococcus acidilactici strain which is vigorous in growth and can reach a logarithmic growth phase within 4-8h after inoculation for activation, inoculating the activated pediococcus acidilactici strain into a PY culture medium with the pH of 6.8, wherein the inoculation amount of the pediococcus acidilactici strain is 0.5 ~ 1%, and the PY culture medium is composed of the following raw materials in parts by weight of 50g of unhydrolyzed macromolecular protein, 5g of peptone, 5g of tryptone, 10g of yeast powder, 20g of anhydrous sodium acetate and 20g of KH sodium acetate2PO46g，MgSO4∙7H2O0.6g，MnSO4∙4H20.2g of O and 1000g of water;
(2) fermenting and culturing at 37 deg.C for 36-48h, and harvesting thallus after culture.
Experimental examples experimental studies of a method of implementing high-density fermentation of lactic acid bacteria using an amorphous solid attachment of the present invention:
1. test object
Experimental groups: the fermentation method of the culture medium added with the unhydrolyzed protein is used for respectively culturing the lactobacillus salivarius, the lactobacillus fermentum and the pediococcus acidilactici at high density.
Control group: lactobacillus salivarius, Lactobacillus fermentum and Pediococcus acidilactici were cultured at high density by fermentation with a basal medium without addition of unhydrolyzed protein.
2. Test method
2.1 measurement of Total colony count
The total number of bacterial colonies is determined by plate counting method, after 1mL sample is diluted with 10 times of sterile water in gradient, 200 mul is sucked and poured into a culture dish, then MRS agar culture medium with temperature not more than 70 ℃ is poured, after culturing for 48h at 37 ℃, the bacterial colonies visible to naked eyes are counted, the number of the bacterial colonies is required to be 30 ~ 300 effective dilution gradient, the number of the bacterial colonies in the original sample is calculated according to the dilution gradient, and as can be seen from the graph 1-3, the total number of the bacterial colonies is 10 h after the culture of the basic culture medium (control group) is finished (24 h)8-9cfu/mL, whereas a total number of 10 colonies could be obtained at the end of fermentation in the method of the invention (experimental group)10-12cfu/mL, corresponding to 100 ~ 1000 fold of control.
2.2 high temperature resistance test
The method comprises the steps of respectively taking lactic acid bacteria liquid of a control group and an experimental group which are cultured for 12 hours under an aseptic condition, transferring 1mL of the lactic acid bacteria liquid into an aseptic centrifuge tube, carrying out water bath at 50 ℃, 60 ℃ and 70 ℃ for 30min, then rapidly cooling ice water to normal temperature, then measuring the total number of bacterial colonies after the initial water bath and the high-temperature water bath, and judging the high-temperature resistance of the lactic acid bacteria of different treatment groups according to the survival rate of the bacteria, wherein as shown in the figure 4-6, when the lactic acid bacteria are cultured by the method, the high-temperature resistance of the bacteria is remarkably enhanced, particularly, the lactic acid bacteria are treated at 60 ℃ for 30min, all the lactic acid bacteria of the control group die, the experimental groups have the survival rates of different degrees, but when the lactic acid bacteria are treated at 50 ℃, the survival rate of the bacteria of the control group is 73 8586.
2.3 simulated gastric juice and bile salt preparation and experimental method
Simulated gastric fluid reference formulation JJ Ahire et al (2011): peptone 8.3, glucose 3.5, NaCL2.05, KH2PO40.6， CaCl20.11, KCL 0.37, bile salt0.05, lysozyme 0.1, pepsin 13.3 (unit: mg/mL), the final pH was adjusted to 2.50 with 1mol/L HCl. It is diluted 10 times when used.
When the survival rate of the bacteria in the simulated gastric juice is measured, 10000 g of lactobacillus bacteria liquid is taken for centrifugation for 5 min, after supernatant fluid is removed, the lactobacillus bacteria liquid is changed into the equivalent simulated gastric juice (diluted by 10 times), and after the lactobacillus bacteria liquid is cultured for 30-180 min at 37 ℃, the total number of bacterial colonies is measured. Fig. 7-9 show that the lactic acid bacteria in the experimental group and the control group have certain tolerance to simulated gastric acid, and the survival rate of the bacteria is rapidly reduced along with the prolonging of the treatment time. The survival rate of the experimental group is significantly higher than that of the control group (P < 0.05).
The bile salt is prepared by preparing corresponding bile salt solution according to the mass fraction of 0.5-2%. FIGS. 10-12 show that the tested lactic acid bacteria all show strong tolerance to bile salts. The survival rate of the thallus is reduced along with the increase of the concentration of the bile salts. Overall, the survival rate of the bacteria in the experimental group is significantly higher than that in the control group (P < 0.05).
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims (5)

1. A method for realizing high-density fermentation of lactic acid bacteria by adopting an amorphous solid attachment is characterized by comprising the following steps:
(1) selecting a good lactobacillus strain which is vigorous in growth and can reach a logarithmic growth phase within 4-8h after inoculation for activation, and inoculating the activated lactobacillus strain into a lactobacillus culture medium;
(2) performing fermentation culture at 37 deg.C, and harvesting thallus after culture;
wherein the lactobacillus culture medium is a PY culture medium;
the PY culture medium is composed of the following raw materials in parts by weight: 50g of unhydrolyzed macromolecular protein, 5g of peptone, 5g of tryptone, 10g of yeast powder, 20g of anhydrous sodium acetate and KH2PO46g，MgSO4∙7H2O 0.6g，MnSO4∙4H20.2g of O and 1000g of water.
2. The method according to claim 1, wherein the lactic acid bacteria species of step (1) is one of Lactobacillus salivarius, Lactobacillus fermentum, and Pediococcus acidilactici.
3. The method according to claim 1, wherein the lactobacillus species of step (1) is inoculated in an amount of 0.5 ~ 1%.
4. The method of claim 1, wherein the pH of the PY medium is 6.8.
5. The method according to claim 1, wherein the fermentation time in step (2) is 36-48 h.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101338283A (en) * 2007-07-06 2009-01-07 内蒙古农业大学 Lactobacillus casei and applications thereof in solid-state fermentation
CN102057046A (en) * 2008-04-09 2011-05-11 钴技术有限公司 Immobilized product tolerant microorganisms
CN103013893A (en) * 2013-01-21 2013-04-03 黑龙江八一农垦大学 Lactobacillus plantarum CCL67 and application of same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101338283A (en) * 2007-07-06 2009-01-07 内蒙古农业大学 Lactobacillus casei and applications thereof in solid-state fermentation
CN102057046A (en) * 2008-04-09 2011-05-11 钴技术有限公司 Immobilized product tolerant microorganisms
CN103013893A (en) * 2013-01-21 2013-04-03 黑龙江八一农垦大学 Lactobacillus plantarum CCL67 and application of same

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