CN113652359A - Lactobacillus freeze-dried powder, preparation method and freeze-drying protective agent thereof - Google Patents
Lactobacillus freeze-dried powder, preparation method and freeze-drying protective agent thereof Download PDFInfo
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- CN113652359A CN113652359A CN202110756881.XA CN202110756881A CN113652359A CN 113652359 A CN113652359 A CN 113652359A CN 202110756881 A CN202110756881 A CN 202110756881A CN 113652359 A CN113652359 A CN 113652359A
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- lactic acid
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims 1
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- 229920002774 Maltodextrin Polymers 0.000 claims 1
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Classifications
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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
- C12N1/04—Preserving or maintaining viable microorganisms
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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
- C12N1/20—Bacteria; Culture media therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
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- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of microbial preparations, and particularly relates to lactic acid bacteria freeze-dried powder, a preparation method and a freeze-drying protective agent thereof. The stress resistance induction and the freeze-drying protective agent are used for synergistically improving the viable count, the survival rate and the storage time limit of the lactobacillus freeze-dried powder, the freeze-drying protective agent has universality for different lactobacillus, the survival rate of the lactobacillus in the freeze-drying process can be improved to more than 90%, the stability of the lactobacillus in the shelf life is improved by more than 20% compared with that before optimization, the storage time limit of the lactobacillus is prolonged, and a technical support is provided for the application of the lactobacillus.
Description
Technical Field
The invention belongs to the field of microbial preparations, and particularly relates to lactic acid bacteria freeze-dried powder, a preparation method and a freeze-drying protective agent thereof.
Background
Probiotics are active microorganisms which are beneficial to a host and change the composition of flora at a certain part of the host by colonizing in a human body. The health of the intestinal tract is kept by promoting the absorption of nutrients by regulating the immune function of the host mucous membrane and the system or by regulating the balance of flora in the intestinal tract, so that single microorganisms or mixed microorganisms with definite compositions which are beneficial to the health are generated. The beneficial bacteria or fungi in human body or animal body mainly include yeast, probiotic bacillus, clostridium butyricum, lactobacillus, actinomycetes, etc.
Lactic Acid Bacteria (LAB) are a general term for a group of bacteria that can utilize fermentable carbohydrates to produce large amounts of lactic acid. A large number of researches show that the lactobacillus can regulate normal flora of gastrointestinal tracts of organisms, keep microecological balance, improve the digestibility and the biovalue of food, reduce serum cholesterol, control endotoxin, inhibit growth and propagation of putrefying bacteria and generation of putrefying products in intestinal tracts, produce nutrient substances and stimulate tissue development, thereby having effects on the nutritional state, physiological functions, cell infection, drug effect, toxic reaction, immune reaction, tumorigenesis, aging process, sudden emergency reaction and the like of the organisms. Therefore, the physiological functions of lactic acid bacteria are closely related to the vital activities of the body, and it can be said that if the growth of lactic acid bacteria stops, it is difficult for the human body or animal body to live healthily. As such, lactic acid bacteria are widely used in many industries such as the light industry, food industry, pharmaceutical industry, and feed industry.
The lactobacillus should maintain high activity in the application process, so the lactobacillus is often industrially prepared into a high-concentration freeze-dried lactobacillus product. On one hand, the product can be used as a direct vat set starter for producing dairy products and fermented foods, and on the other hand, the product can be directly prepared into solid beverage for direct eating. The most central problem in the application process of the lactic acid bacteria is to ensure the viable count of the lactic acid bacteria in the shelf life and reduce the decay rate of the lactic acid bacteria. However, the research on the existing lactobacillus freeze-dried powder shows that the attenuation rate is low and the two-year survival rate can be more than 80% when the lactobacillus freeze-dried powder is placed in a low-temperature environment of less than 10 ℃, but the lactobacillus is severely attenuated when the lactobacillus freeze-dried powder is stored in a normal-temperature environment of 25 ℃ or a high-temperature environment of 37 ℃, the 2-year survival rate is generally less than 50%, and the effect of the lactobacillus product is greatly reduced.
For the preservation of microbial preparations, patent CN111647510A provides a bifidobacterium infantis lyophilized powder, a preparation method and a composite protective agent used by the same, wherein the composite protective agent comprises 10-30% of skimmed milk powder, 0.1-5.0% of amino acid and salts thereof, 3-25% of disaccharide or/and polysaccharide, 1-5% of small molecular polyol and the balance of phosphate buffer solution, and the pH of the composite protective agent is 6.5-8.5, the bifidobacterium infantis lyophilized powder can significantly improve the lyophilized survival rate of strains and the viable count of the strains powder, and prolong the storage life thereof; patent CN103041383B provides a heat-resistant freeze-drying protective agent for live vaccines, live vaccine freeze-dried powder and a preparation method thereof, wherein the heat-resistant freeze-drying protective agent comprises 46-90% of oligosaccharide, 1-9% of amino acid, 1-9% of gelatin, 1-9% of casein hydrolysate, 1-9% of polyvinylpyrrolidone, 1-9% of glycerol and 681-9% of Pluronic, and the storage life of the live vaccines for viruses is prolonged at 37 ℃ and 20 ℃. This indicates that the selection of a suitable cryoprotectant for the microorganisms significantly extends the shelf life of the microorganisms.
Therefore, it is highly desirable to provide a lactic acid bacteria freeze-drying protective agent and freeze-dried powder thereof, which further improve the freeze-drying survival rate and the storage time limit.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a lactic acid bacteria freeze-dried powder, a preparation method and a freeze-drying protective agent thereof. The survival rate and the storage life of the lactic acid bacteria are improved by the stress resistance induction and freeze-drying protective agent proportion, and the viable count of the lactic acid bacteria can be obviously improved.
In order to achieve the above object, the present invention provides the following technical solutions:
in one aspect, the invention provides a lactic acid bacteria freeze-drying protective agent, which comprises 100-400g/L of micromolecular polyalcohol and/or saccharide, 5-20g/L of free amino acid or salt thereof, 5-30g/L of vitamin C or salt thereof, 10-100g/L of polymer protective agent and 5-30g/L of buffer salt.
Specifically, the pH value of the lyoprotectant is 5.0-6.5.
Specifically, the content ratio of the small molecular polyol and/or the saccharide to the free amino acid or the salt substance thereof is 5-80: 1.
More specifically, the content ratio of the small molecular polyol and/or the saccharide to the free amino acid or the salt substance thereof is 10-30: 1.
Preferably, the content ratio of the small-molecule polyol and/or the saccharide to the free amino acid or the salt substance thereof is 20: 1.
Specifically, the small molecule polyol and/or saccharide comprises one or more of glycerol, mannitol, trehalose, sucrose, glucose, lactose, sucrose and maltodextrin.
Specifically, the free amino acid comprises one or more of glutamic acid, proline, leucine, isoleucine, valine, alanine, phenylalanine, aspartic acid, methionine, glycine, lysine, threonine, arginine and tyrosine.
Specifically, the polymer protective agent comprises one or more of milk protein, Arabic gum, konjac glucomannan and sodium carboxymethylcellulose.
Specifically, the buffer salt comprises phosphate.
More specifically, the buffer salts are potassium/sodium dihydrogen phosphate and dipotassium hydrogen phosphate/sodium, and the pH of the mixed solution is controlled to 5.0-6.5 by adding the buffer salts, so that the damage of hydrogen ions to the bacterial cells along with the drying and concentration is prevented.
Specifically, the freeze-drying protective agent can reduce the damage of ice crystal formation to thalli and the damage of dehydration and drying to thalli and enzyme activity in the freeze-drying process.
In another aspect, the present invention provides a culture method for enhancing survival rate of lactic acid bacteria by inducing stress tolerance, wherein the culture method comprises the following steps: when 0.5-1% of residual sugar is left in the fermentation, cooling to 10-25 deg.C, and stopping controlling alkali to reduce pH of the culture medium to 4-5 with residual sugar consumption, wherein the whole process lasts for 3-5 h.
In another aspect, the invention provides a method for preparing a lactic acid bacteria freeze-dried powder, which comprises the following steps:
(1) culturing lactobacillus, fermenting, and inducing stress resistance;
(2) centrifuging the lactobacillus liquid obtained by culturing in the step (1), and collecting bacterial sludge;
(3) and (3) mixing the bacterial sludge obtained by centrifugation in the step (2) with the freeze-drying protective agent to obtain a mixed solution, and freeze-drying the mixed solution to obtain the lactic acid bacteria freeze-dried powder.
Specifically, the lactobacillus is inoculated into a culture medium for culture and fermentation in the step (1). The medium used in the present invention for culturing and fermenting lactic acid bacteria and culturing and fermenting lactic acid bacteria is not particularly limited, and may be a fermentation step of a lactic acid bacteria medium and a medium component well known to those skilled in the art.
Specifically, the mass ratio of the bacterial sludge to the freeze-drying protective agent in the step (3) is 1: 1-2.
Specifically, the freeze drying in the step (3) is completed in a freeze dryer, and comprises prefreezing, primary drying and secondary drying; the pre-freezing is to control the temperature of the laminate to be reduced to-45 to-50 ℃ within 1 hour and keep the temperature for 3 to 4 hours; the primary drying is to control the temperature of the laminate to be increased to-25 to-30 ℃ for 2h, and keep the temperature to the primary drying end point; and the secondary drying is to control the temperature of the laminate to 22-30 ℃ within 1 hour and keep the laminate until the end point of the secondary drying.
Specifically, the initial viable count of the lactobacillus freeze-dried powder is 1 multiplied by 1011-1×1012CFU/g。
Specifically, the lactic acid bacteria include, but are not limited to, Streptococcus thermophilus (Streptococcus thermophilus), Lactococcus lactis (Lactobacillus lactis), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus casei (Lactobacillus casei), Lactobacillus paracasei (Lactobacillus paracasei), Bifidobacterium (Bifidobacterium species), Lactobacillus acidophilus (Lactobacillus acidophilus), and Lactobacillus rhamnosus (Lactobacillus rhamnophilus) and Lactobacillus bulgaricus (Lactobacillus bulgaricus).
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention improves the viable count, the survival rate and the preservation time limit of the lactobacillus freeze-dried powder by the two aspects of the stress resistance induction and the freeze-drying protective agent.
(2) The freeze-drying protective agent and the freeze-drying powder preparation method have universality on different lactic acid bacteria, the survival rate of the lactic acid bacteria in the freeze-drying process can be improved to more than 90%, the stability of the lactic acid bacteria in the shelf life is improved by more than 20% compared with that before optimization, the storage time limit of the lactic acid bacteria is prolonged, and technical support is provided for the application of the lactic acid bacteria.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
The examples, where no specific techniques or conditions are indicated, are carried out according to the techniques or conditions described in the literature of the art (for example, see J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, scientific Press, ed. by Huang Pe, et al) or according to the instructions of the product.
Description of the source of the strain: the lactic acid bacteria involved in the experiment were from Jiangsu microbial health, Inc., Danisc or Hansen, all of which are commercially available.
EXAMPLE 1 preparation of lyophilized powder of Bifidobacterium lactis
1. Reagent
(1) Seed culture medium: MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 0.5g/L of sodium acetate, 10g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05g/L, Tween-801 mL/L and cysteine hydrochloride 0.5 g/L.
(2) Fermentation medium: MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 40g/L of glucose, 0.5g/L of sodium acetate, 10g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05g/L, Tween-801 mL/L and cysteine hydrochloride 0.5 g/L.
(3) Freeze-drying protective agent: 150g/L trehalose and 50g/L maltodextrin, wherein the free amino acids or salts comprise 2g/L glutamic acid, leucine, isoleucine, valine and alanine respectively, 10g/L vitamin C salt, 20g/L milk protein, 10g/L Arabic gum, 5g/L dipotassium hydrogen phosphate and 5g/L potassium dihydrogen phosphate.
2. The experimental method comprises the following steps:
inoculating a bifidobacterium lactis storage glycerol tube into a shake flask seed culture medium according to the inoculation amount of 2%, and culturing at the constant temperature of 37 ℃ for 16h to obtain a seed solution; inoculating the seed solution into a fermentation tank according to the inoculation amount of 2% (v/v) for culture, supplementing sodium hydroxide to control the pH value in the fermentation process to be 5.5, carrying out constant-temperature culture at 37 ℃ until 0.5% of residual sugar remains, stopping supplementing sodium hydroxide, continuously reducing the pH value of a culture medium due to continuous acid production of bifidobacterium lactis, simultaneously opening cold water to reduce the temperature of the fermentation tank to 10 ℃, keeping the whole low-pH and low-temperature environment for 4 hours, and centrifuging to obtain bacterial sludge; uniformly mixing the bacterial sludge and a protective agent 1:1(w/w), adjusting the pH value of the re-suspension to 5.5, and carrying out freeze drying. The method comprises pre-freezing, primary drying and secondary drying, wherein the pre-freezing is to cool the control laminate to-45 ℃ within 1h and keep the temperature for 4h, the primary drying is to heat the control laminate to-25 ℃ within 1.3h and keep the temperature for 30h, and the secondary drying is to heat the control laminate to 25 ℃ within 1h and keep the temperature for 20h, so that the freeze-dried powder of the bifidobacterium lactis is obtained. Detecting the number of living bacteria and calculating the survival rate. And respectively storing the prepared freeze-dried powder at 25 ℃ for 6 months, and after storing the freeze-dried powder at 37 ℃ for 6 months, detecting the number of viable bacteria and calculating the survival rate.
Example 2
Compared with example 1, the difference between this example 2 and example 1 is that, when bifidobacterium lactis is subjected to fermentation culture and residual sugar is 0.5%, the operation of cooling and reducing the pH is not carried out, and the bacterial sludge is obtained by direct centrifugation, and the rest of the operation steps are the same as those of example 1.
Example 3
Compared with example 1, the difference of this example 3 is that the lyoprotectant comprises the following components: trehalose 50g/L and maltodextrin 50g/L, free amino acids or salts including glutamic acid, leucine, isoleucine, valine, alanine each 4g/L, vitamin C salt 30g/L, milk protein 40g/L, disodium hydrogen phosphate 15g/L, potassium dihydrogen phosphate 15g/L, the remaining procedure was identical to example 1.
Example 4
Compared with example 1, the difference of this example 4 is that the lyoprotectant comprises the following components: trehalose 150g/L and maltodextrin 250g/L, free amino acids or salts including glutamic acid, leucine, isoleucine, valine, alanine each 1g/L, vitamin C salt 5g/L, gum arabic 10g/L, dipotassium hydrogen phosphate 2.5g/L, sodium dihydrogen phosphate 2.5g/L, the rest of the procedure was identical to example 1.
Example 5
Compared with example 1, the difference of this example 5 is that the lyoprotectant comprises the following components: trehalose 25g/L and maltodextrin 25g/L, free amino acids or salts including glutamic acid, leucine, isoleucine, valine, alanine each 0.5g/L, vitamin C salt 4g/L, milk protein 2.5g/L, acacia gum 1g/L, dipotassium hydrogen phosphate 2g/L, sodium dihydrogen phosphate 1g/L, the remaining procedures were consistent with example 1.
Example 6
Compared with example 1, the difference of this example 6 is that the lyoprotectant comprises the following components: trehalose 100g/L and maltodextrin 100g/L, free amino acids or salts including glutamic acid, leucine, isoleucine, valine, alanine each 15g/L, vitamin C salt 40g/L, milk protein 20g/L, gum arabic 20g/L, dipotassium hydrogen phosphate 10g/L, potassium dihydrogen phosphate 10g/L, the rest of the procedure was identical to example 1.
Example 7
Compared with example 1, the difference of this example 7 is that the lyoprotectant comprises the following components: 250g/L trehalose and 150g/L maltodextrin, 0.5g/L each of free amino acids or salts including glutamic acid, leucine, isoleucine, valine and alanine, 10g/L vitamin C salt, 20g/L milk protein, 10g/L acacia gum, 5g/L dipotassium hydrogen phosphate and 5g/L potassium dihydrogen phosphate, and the rest of the procedure was the same as in example 1.
EXAMPLE 8 preparation of lyophilized powder of Streptococcus thermophilus
1. Reagent
(1) Seed culture medium: MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 0.5g/L of sodium acetate, 10g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05g/L and Tween-801 mL/L.
(2) Fermentation medium: MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 40g/L of glucose, 0.5g/L of sodium acetate, 10g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05g/L and Tween-801 mL/L.
(3) Freeze-drying protective agent: 20g/L of glycerin, 80g/L of sucrose and 60g/L of maltodextrin, wherein free amino acids or salts comprise 2g/L of glutamic acid, valine, alanine and aspartic acid respectively, 20g/L of vitamin C salt, 100g/L of milk powder, 15g/L of dipotassium hydrogen phosphate and 15g/L of potassium dihydrogen phosphate.
2. The experimental method comprises the following steps:
inoculating a streptococcus thermophilus preservation glycerol tube into a shake flask seed culture medium according to the inoculation amount of 2%, and culturing at the constant temperature of 37 ℃ for 16h to obtain a seed solution; inoculating the seed solution into a fermentation tank according to the inoculation amount of 2% (v/v) for culture, supplementing ammonia water to control the pH value in the fermentation process to be 5.5, carrying out constant-temperature culture at 37 ℃ until 1% of residual sugar remains, stopping supplementing ammonia water, continuously reducing the pH value of a culture medium due to continuous acid production of streptococcus thermophilus, simultaneously opening cold water to reduce the temperature of the fermentation tank to 15 ℃, keeping the whole low-pH and low-temperature environment for 3 hours, and centrifuging to obtain bacterial sludge; uniformly mixing the bacterial sludge and a protective agent 1:2(w/w), adjusting the pH value of the re-suspension to 5.8, and carrying out freeze drying. The method comprises prefreezing, primary drying and secondary drying, wherein the prefreezing is that the temperature of a control laminate is reduced to minus 50 ℃ within 1h, and is kept for 3h, the primary drying is that the temperature of the control laminate is increased to minus 25 ℃ within 2h, and is kept for 30h, and the secondary drying is that the temperature of the control laminate is increased to 30 ℃ within 1h, and is kept to a secondary drying end point, so that the streptococcus thermophilus freeze-dried powder is obtained. Detecting the number of living bacteria and calculating the survival rate. And respectively storing the prepared freeze-dried powder at 25 ℃ for 6 months, and after storing the freeze-dried powder at 37 ℃ for 6 months, detecting the number of viable bacteria and calculating the survival rate.
Example 9
The difference between this example 9 and example 8 is that the streptococcus thermophilus is cultured by fermentation, when 1% of residual sugar is left, the operation of cooling and lowering pH is not carried out, the direct centrifugation is carried out to obtain bacterial sludge, and the rest of the operation steps are the same as example 8.
EXAMPLE 10 preparation of lyophilized powder of Lactobacillus acidophilus
1. Reagent
(1) Seed culture medium: MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 0.5g/L of sodium acetate, 10g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05g/L and Tween-801 mL/L.
(2) Fermentation medium MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 40g/L of glucose, 0.5g/L of sodium acetate, 10g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05g/L and Tween-801 mL/L.
(3) Freeze-drying protective agent: 20g/L of sucrose, 20g/L of galactose and 180g/L of maltodextrin, wherein free amino acids or salts comprise 2g/L of glutamic acid, valine, proline and arginine respectively, 5g/L of vitamin C salt, 2g/L of Arabic gum, 10g/L of dipotassium hydrogen phosphate and 10g/L of potassium dihydrogen phosphate.
2. The experimental method comprises the following steps:
inoculating a shake flask seed culture medium into a lactobacillus acidophilus preservation glycerol tube according to the inoculation amount of 2%, and culturing at the constant temperature of 37 ℃ for 16h to obtain a seed solution; inoculating the seed liquid into a fermentation tank according to the inoculation amount of 2% (v/v) for culturing, supplementing ammonia water to control the pH value in the fermentation process to be 5.5, culturing at the constant temperature of 37 ℃ until 1% of residual sugar remains, stopping supplementing ammonia water, continuously reducing the pH value of a culture medium due to continuous acid production of lactobacillus acidophilus, simultaneously opening cold water to reduce the temperature of the fermentation tank to 15 ℃, keeping the whole low-pH and low-temperature environment for 5 hours, and centrifuging to obtain bacterial sludge; uniformly mixing the bacterial sludge and a protective agent 1:1(w/w), adjusting the pH value of the re-suspension to 6.0, and carrying out freeze drying. The method comprises prefreezing, primary drying and secondary drying, wherein the prefreezing is to reduce the temperature of a control laminate to-50 ℃ within 1h and keep the temperature for 3h, the primary drying is to raise the temperature of the control laminate to-25 ℃ within 2h and keep the temperature for 30h, the secondary drying is to raise the temperature of the control laminate to 30 ℃ and keep the temperature to a secondary drying end point, and the lactobacillus acidophilus freeze-dried powder is obtained. Detecting the number of living bacteria and calculating the survival rate. And respectively storing the prepared freeze-dried powder at 25 ℃ for 6 months, and after storing the freeze-dried powder at 37 ℃ for 6 months, detecting the number of viable bacteria and calculating the survival rate.
Example 11
Compared with example 10, the difference between this example 11 and example 10 is that when lactobacillus acidophilus is subjected to fermentation culture and 1% of residual sugar is left, the operation of cooling and reducing the pH is not performed, and the direct centrifugation is performed to obtain bacterial sludge, and the rest of the operation steps are the same as those of example 10.
Experimental example 1 survival rate detection
1. Detecting the viable count of the freeze-dried bacterial powder: weighing 1g of freeze-dried bacterial powder, adding the freeze-dried bacterial powder into 9mL of physiological saline containing glass beads, oscillating for 30min, taking 1mL of bacterial liquid to 9mL of physiological saline for gradient dilution, taking 100 mu L of diluent, coating the diluent on an MRS solid culture medium, coating 3 gradients, enabling 3 gradients to be parallel, putting a coated culture dish into a 37 ℃ constant temperature incubator for culturing for 48h, taking out and counting.
2. Detecting the number of viable bacteria in the bacterial sludge emulsion:
weighing 1g of bacterial sludge emulsion, adding the bacterial sludge emulsion into 9mL of physiological saline containing glass beads, oscillating for 30min, taking 1mL of bacterial liquid to 9mL of physiological saline for gradient dilution, taking 100 mu L of diluent, coating the diluent on an MRS solid culture medium, coating 3 gradients, wherein 3 gradients are parallel, putting a coated culture dish into a 37 ℃ constant temperature incubator, culturing for 48h, taking out and counting, and multiplying by the weight of bacterial sludge to obtain the total number of viable bacteria before freeze-drying; after freeze-drying, counting the bacterial powder according to the step 1.
3. Measuring the content of mud and water:
10g of bacterial sludge is taken, dried in an oven at 105 ℃ to constant weight, and the water content is calculated.
Freeze-drying survival rate is the number of viable bacteria per gram of bacterial powder/the number of viable bacteria per gram of bacterial sludge/the moisture content of the bacterial sludge after freeze-drying;
the survival rate of the shelf life is the number of live bacteria per gram of the fungus powder/the number of live bacteria per gram of the fungus powder after freeze-drying;
shelf life viable count: placing the freeze-dried bacterial powder in incubators at 25 ℃ and 37 ℃ for 6 months respectively to detect the number of viable bacteria.
The technical method comprises the following steps: GB 4789.35-2016 national standard for food safety, food microbiology test, and lactobacillus test.
The specific test results are shown in table 1 below.
TABLE 1 survival of lactic acid bacteria
The above are merely embodiments of the present invention, which are described in detail and with particularity, and therefore should not be construed as limiting the scope of the invention. It should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the spirit of the present invention, and these changes and modifications are within the scope of the present invention.
Claims (10)
1. A lactic acid bacteria freeze-drying protective agent is characterized in that: the freeze-drying protective agent comprises small molecular polyol and/or saccharide with the content of 100-400g/L, free amino acid or salt thereof with the content of 5-20g/L, vitamin C or salt thereof with the content of 5-30g/L, a high molecular protective agent with the content of 10-100g/L and buffer salt with the content of 5-30 g/L;
the content ratio of the micromolecule polyalcohol and/or saccharide to the free amino acid or the salt substance thereof is 5-80: 1.
2. The lyoprotectant of claim 1, wherein: the content ratio of the small molecular polyol and/or the saccharide to the free amino acid or the salt substance thereof is 10-30: 1.
3. The lyoprotectant of claim 2, wherein: the content ratio of the small molecular polyol and/or the saccharide to the free amino acid or the salt substance thereof is 20: 1.
4. The lyoprotectant of claim 3, wherein:
the micromolecular polyalcohol and/or saccharide comprises one or more of glycerol, mannitol, trehalose, sucrose, glucose, lactose, sucrose and maltodextrin;
the free amino acid comprises one or more of glutamic acid, proline, leucine, isoleucine, valine, alanine, phenylalanine, aspartic acid, methionine, glycine, lysine, threonine, arginine and tyrosine;
the macromolecular protective agent comprises one or more of milk protein, Arabic gum, konjac glucomannan and sodium carboxymethylcellulose;
the buffer salt comprises phosphate.
5. A culture method for improving the survival rate of lactic acid bacteria by stress resistance induction is characterized in that: the culture method comprises the following steps: when 0.5-1% of residual sugar is left in the fermentation, cooling to 10-25 deg.C, and stopping controlling alkali to reduce pH of the culture medium to 4-5 with residual sugar consumption, wherein the whole process lasts for 3-5 h.
6. A preparation method of lactobacillus freeze-dried powder is characterized by comprising the following steps: the preparation method comprises the following steps:
(1) culturing and fermenting lactic acid bacteria, and then carrying out the stress-resistant induction culture method according to claim 5;
(2) centrifuging the lactobacillus liquid obtained by culturing in the step (1), and collecting bacterial sludge;
(3) mixing the bacterial sludge obtained by centrifugation in the step (2) with the freeze-drying protective agent of any one of claims 1 to 4 to obtain a mixed solution, and freeze-drying the mixed solution to obtain the freeze-dried lactic acid bacteria powder.
7. The method of claim 6, wherein: the mass ratio of the bacterial sludge to the freeze-drying protective agent in the step (3) is 1: 1-2.
8. The method of claim 7, wherein: the step (3) of freeze drying is completed in a freeze dryer, and comprises prefreezing, primary drying and secondary drying; the pre-freezing is to control the temperature of the laminate to be reduced to-45 to-50 ℃ within 1 hour and keep the temperature for 3 to 4 hours; the primary drying is to control the temperature of the laminate to be increased to-25 to-30 ℃ for 2h, and keep the temperature to the primary drying end point; and the secondary drying is to control the temperature of the laminate to 22-30 ℃ within 1 hour and keep the laminate until the end point of the secondary drying.
9. The method of claim 8, wherein: the initial viable count of the lactobacillus freeze-dried powder is 1 multiplied by 1011-1×1012CFU/g。
10. The method of claim 9, wherein: the lactic acid bacteria include, but are not limited to, Streptococcus thermophilus, lactococcus lactis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei, Bifidobacterium, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus bulgaricus.
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| CN114521608A (en) * | 2022-01-27 | 2022-05-24 | 江苏雅博动物健康科技有限责任公司 | Fermentation synergistic composition of lactic acid bacteria and bacillus |
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| CN115558620A (en) * | 2022-09-30 | 2023-01-03 | 微康益生菌(苏州)股份有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
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