CN113652359B - Lactic acid bacteria freeze-dried powder, preparation method and freeze-dried protective agent thereof - Google Patents
Lactic acid bacteria freeze-dried powder, preparation method and freeze-dried protective agent thereof Download PDFInfo
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- CN113652359B CN113652359B CN202110756881.XA CN202110756881A CN113652359B CN 113652359 B CN113652359 B CN 113652359B CN 202110756881 A CN202110756881 A CN 202110756881A CN 113652359 B CN113652359 B CN 113652359B
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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
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- 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
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the field of microbial preparations, and particularly relates to lactobacillus freeze-dried powder, a preparation method and a freeze-dried protective agent thereof. The invention synergistically improves the viable count, the survival rate and the preservation time limit of the lactobacillus freeze-dried powder through stress resistance induction and the freeze-drying protective agent, has universality for different lactobacillus, can improve the survival rate of the lactobacillus in the freeze-drying process to more than 90 percent, improves the stability of the lactobacillus in the shelf life by more than 20 percent compared with that before optimization, prolongs the preservation time limit of the lactobacillus, and provides technical support for the application of the lactobacillus.
Description
Technical Field
The invention belongs to the field of microbial preparations, and particularly relates to lactobacillus freeze-dried powder, a preparation method and a freeze-dried protective agent thereof.
Background
Probiotics are a class of active microorganisms beneficial to a host by colonizing the human body and altering the flora composition of a part of the host. By regulating the immune function of host mucous membrane and system or regulating the balance of flora in intestinal tract, the effect of promoting nutrient absorption and maintaining intestinal health is achieved, so that single microorganism or mixed microorganism with definite composition beneficial to health is produced. The beneficial bacteria or fungi in human body or animal body mainly include yeast, probiotic spore bacteria, clostridium butyricum, lactobacillus, actinomycetes, etc.
Lactic acid bacteria (lactic acid bacteria, LAB), a generic term for a class of bacteria that can utilize fermentable carbohydrates to produce large amounts of lactic acid. A large number of researches show that lactic acid bacteria can regulate normal flora of gastrointestinal tract of an organism, maintain microecological balance, improve food digestibility and biological value, reduce serum cholesterol, control endotoxin, inhibit growth and reproduction of putrefying bacteria in the intestinal tract and generation of putrefying products, produce nutrient substances, stimulate tissue development, thereby having effects on nutritional status, physiological functions, cell infection, drug effect, toxic reaction, immune reaction, tumorigenesis, aging process, sudden emergency reaction and the like of the organism. Thus, the physiological function of lactic acid bacteria is closely related to the life activities of the living body, so that if lactic acid bacteria stop growing, it is difficult to keep the human or animal body healthy. Also, lactic acid bacteria are widely used in many industries such as the light industry, food, medicine and feed industry.
The lactobacillus has high activity in the application process, so the lactobacillus is often prepared into high-concentration freeze-dried lactobacillus products in industry. On one hand, the fermented beverage can be used as a direct-vat starter for producing dairy products and fermented foods, and on the other hand, the fermented beverage can be directly prepared into solid beverage for direct eating. The most central problem in the application process of lactic acid bacteria is to ensure the number of viable bacteria in the shelf life and reduce the attenuation rate of the lactic acid bacteria. However, the research on the existing lactobacillus freeze-dried powder shows that the attenuation rate is lower when the lactobacillus freeze-dried powder is placed in a low-temperature environment with the temperature less than 10 ℃, the two-year survival rate can be more than 80 percent, but the attenuation of the lactobacillus is severe when the lactobacillus freeze-dried powder is stored in a normal-temperature environment with the temperature of 25 ℃ or a higher-temperature environment with the temperature of 37 ℃, and the 2-year survival rate is generally less than 50 percent, so that the efficacy of a lactobacillus product is greatly reduced.
For preservation of microbial preparations, patent CN111647510A provides a bifidobacterium infantis freeze-dried powder, a preparation method and a compound protective agent used by the bifidobacterium infantis freeze-dried powder, wherein the compound 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 micromolecular polyalcohol and the balance of phosphate buffer solution, the pH of the compound protective agent is 6.5-8.5, and the bifidobacterium infantis freeze-dried powder can remarkably improve the freeze-dried survival rate and the viable count of bacterial powder and prolong the shelf life of the bacterial powder; patent CN103041383B provides a heat-resistant freeze-drying protective agent for live vaccine, freeze-dried live vaccine and 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 1-9% of common Lu Nike, and the shelf life of the live vaccine is prolonged at 37 ℃ and 20 ℃. This indicates that the choice of a suitable lyoprotectant for the microorganism can significantly extend the shelf life of the microorganism.
Therefore, it is needed to provide a lactic acid bacteria freeze-drying protective agent and freeze-drying powder thereof, which further improve the freeze-drying survival rate and the preservation time limit.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides lactic acid bacteria freeze-dried powder, a preparation method and a freeze-dried protective agent thereof. The stress resistance induction and freeze-drying protective agent proportion of the invention improves the survival rate and the storage life of the lactic acid bacteria, and can obviously improve the viable count of the lactic acid bacteria.
In order to achieve the above object, the present invention provides the following technical solutions:
in one aspect, the invention provides a lactobacillus freeze-drying protective agent, which comprises small molecular polyalcohol and/or saccharide with the content of 100-400g/L, free amino acid or salt substance thereof with the content of 5-20g/L, vitamin C or salt substance thereof with the content of 5-30g/L, high molecular protective agent with the content of 10-100g/L and buffer salt with the content of 5-30 g/L.
Specifically, the pH of the lyoprotectant is 5.0-6.5.
Specifically, the content ratio of the small molecular polyalcohol and/or saccharide to the free amino acid or the salt substance thereof is 5-80:1.
More specifically, the content ratio of the small molecular polyalcohol and/or saccharide to the free amino acid or the salt substance thereof is 10-30:1.
Preferably, the content ratio of the small molecular polyalcohol and/or saccharide to the free amino acid or the salt substance thereof is 20:1.
Specifically, the small molecule polyalcohol 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, acacia, konjac gum and sodium carboxymethyl cellulose.
Specifically, the buffer salt comprises phosphate.
More specifically, the buffer salts are potassium dihydrogen phosphate/sodium and dipotassium hydrogen phosphate/sodium, and the pH of the mixed solution is controlled to be 5.0-6.5 by adding the buffer salts, so that the damage of hydrogen ions to thalli along with the drying and concentration is prevented.
Specifically, the lyoprotectant can reduce the damage of ice crystal formation to thalli in the process of lyophilization and the damage of dehydration and drying to thalli and enzyme activity.
In another aspect, the present invention provides a method for culturing lactic acid bacteria to improve survival rate by stress resistance induction, the method comprising the steps of: when residual sugar is left for 0.5-1%, the temperature is reduced, the temperature of the fermentation liquid is reduced to 10-25 ℃, and simultaneously alkali control is stopped, so that the pH of the culture medium is reduced to 4-5 along with the consumption of the residual sugar, and the whole process lasts for 3-5 hours.
In still another aspect, the present invention provides a method for preparing lactic acid bacteria lyophilized powder, comprising the steps of:
(1) Culturing and fermenting lactobacillus, and then performing the stress resistance induction culture method;
(2) Centrifuging the lactobacillus solution 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 lactobacillus freeze-dried powder.
Specifically, the step (1) of culturing and fermenting the lactic acid bacteria is to inoculate the lactic acid bacteria into a culture medium for culturing and fermenting. The culture medium used for the culture and fermentation of lactic acid bacteria and the culture medium for the culture and fermentation of lactic acid bacteria in the present invention is not particularly limited, and may be a fermentation step and a culture medium composition of a lactic acid bacteria culture medium which are well known to those skilled in the art.
Specifically, in the step (3), the mass ratio of the bacterial mud to the freeze-drying protective agent 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 1h, and keep for 3-4h; the primary drying is to control the temperature of the laminate to rise to-25 to-30 ℃ for 2 hours, and keep the laminate to the primary drying end point; and the secondary drying is to control the temperature of the laminate to be raised to 22-30 ℃ for 1h, and keep the laminate to reach the secondary drying end point.
Specifically, the initial viable count of the lactobacillus freeze-dried powder is 1 multiplied by 10 11 -1×10 12 CFU/g。
Specifically, the lactic acid bacteria include, but are not limited to, streptococcus thermophilus (Streptococcus thermophilus), lactococcus lactis (Lactococcus lactis), lactobacillus fermentum (Lactobacillus fermentum), lactobacillus plantarum (Lactobacillus plantarum), lactobacillus brevis (Lactobacillus breris), lactobacillus casei (Lactobacillus casei), lactobacillus paracasei (Lactobacillus paracasei), bifidobacterium (Bifidobacterium species), lactobacillus acidophilus (Lactobacillus acidophilus) and lactobacillus rhamnosus (Lactobacillus rhamnosus) and lactobacillus bulgaricus (Lactobacillus bulgaricus).
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention synergistically improves the viable count, the survival rate and the preservation time limit of the lactobacillus freeze-dried powder through stress resistance induction and the freeze-drying protective agent.
(2) The freeze-drying protective agent and the preparation method of the freeze-drying powder have universality for different lactic acid bacteria, can improve the survival rate of the lactic acid bacteria in the freeze-drying process to more than 90%, improve the stability of the lactic acid bacteria in the shelf life by more than 20% compared with that before optimization, prolong the preservation time limit of the lactic acid bacteria, and provide technical support for the application of the lactic acid bacteria.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
The examples are not to be construed as a specific technique or condition, and are carried out according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sam Brooks et al, J. Mi. Cloning Experimental guidelines, third edition, scientific Press, et al, huang Peitang et al) or according to the specifications of the product.
Strain source description: the lactic acid bacteria involved in the experiments were from Jiangsu Microsoft Biometrics, denmark or Hansen, and the strains were commercially available.
EXAMPLE 1 preparation of lyophilized powder of Bifidobacterium lactis
1. Reagent(s)
(1) Seed culture medium: MRS liquid medium: 10g/L peptone, 10g/L beef extract, 20g/L glucose, 0.5g/L sodium acetate, 10g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05g/L, tween-80 1mL/L, cysteine hydrochloride 0.5g/L.
(2) Fermentation medium: MRS liquid medium: 10g/L peptone, 10g/L beef extract, 40g/L glucose, 0.5g/L sodium acetate, 10g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05g/L, tween-80 1mL/L, cysteine hydrochloride 0.5g/L.
(3) Lyoprotectant: trehalose 150g/L and maltodextrin 50g/L, wherein the free amino acids or salts comprise glutamic acid, leucine, isoleucine, valine and alanine 2g/L each, vitamin C salt 10g/L, milk protein 20g/L, acacia 10g/L, dipotassium hydrogen phosphate 5g/L and potassium dihydrogen phosphate 5g/L.
2. The experimental method comprises the following steps:
inoculating shake flask seed culture medium into a bifidobacterium lactis preservation glycerol tube according to an inoculum size of 2%, and culturing at a constant temperature of 37 ℃ for 16 hours to obtain seed liquid; inoculating the seed solution into a fermentation tank according to an inoculum size of 2% (v/v), culturing, supplementing sodium hydroxide to control the pH of the fermentation process to be 5.5, culturing at a constant temperature of 37 ℃ until residual sugar is 0.5%, stopping supplementing sodium hydroxide, continuously reducing the pH 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 ℃, maintaining the whole low-pH and low-temperature environment for 4 hours, and centrifuging to obtain bacterial sludge; uniformly mixing the bacterial mud with a protective agent in a ratio of 1:1 (w/w), regulating the pH of the heavy suspension to 5.5, and freeze-drying. The method comprises the steps of prefreezing, primary drying and secondary drying, wherein prefreezing is to control the temperature of the laminate to be reduced to-45 ℃ in 1h, keeping for 4h, primary drying is to control the temperature of the laminate to be increased to-25 ℃ in 1.3h, keeping for 30h, and secondary drying is to control the temperature of the laminate to be increased to 25 ℃ in 1h, keeping for 20h, so as to obtain the bifidobacterium lactis freeze-dried powder. The number of viable bacteria was measured and the survival rate was calculated. And (3) respectively preserving the prepared freeze-dried powder at 25 ℃ for 6 months, and after preserving the freeze-dried powder at 37 ℃ for 6 months, detecting the viable count and calculating the survival rate.
Example 2
In example 2, the difference from example 1 is that the bifidobacterium lactis of this example was fermented and cultured to give a residual sugar content of 0.5%, and the residual sugar content was identical to that of example 1 except that the operation of lowering the temperature and lowering the pH was not performed, and the resulting bacterial sludge was directly centrifuged.
Example 3
Compared with the embodiment 1, the embodiment 3 is different in 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 4g/L each, vitamin C salt 30g/L, milk protein 40g/L, disodium hydrogen phosphate 15g/L, potassium dihydrogen phosphate 15g/L, and the remaining procedures were the same as in example 1.
Example 4
Compared with example 1, the difference of this example 4 is that the lyoprotectant in this example 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, acacia 10g/L, dipotassium hydrogen phosphate 2.5g/L, sodium dihydrogen phosphate 2.5g/L, and the remaining procedures were consistent with example 1.
Example 5
Compared with example 1, the difference of this example 5 is that the lyoprotectant in this example 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 1g/L, dipotassium hydrogen phosphate 2g/L, sodium dihydrogen phosphate 1g/L, and the remaining procedures were the same as in example 1.
Example 6
Compared with example 1, the difference of this example 6 is that the lyoprotectant in this example comprises the following components: trehalose 100g/L and maltodextrin 100g/L, free amino acids or salts including glutamic acid, leucine, isoleucine, valine, alanine 15g/L each, vitamin C salt 40g/L, milk protein 20g/L, acacia 20g/L, dipotassium hydrogen phosphate 10g/L, potassium dihydrogen phosphate 10g/L, and the remaining procedures were the same as in example 1.
Example 7
Compared with example 1, the difference of this example 7 is that the lyoprotectant in this example comprises the following components: trehalose 250g/L and maltodextrin 150g/L, free amino acids or salts including glutamic acid, leucine, isoleucine, valine, alanine each 0.5g/L, vitamin C salt 10g/L, milk protein 20g/L, acacia 10g/L, dipotassium hydrogen phosphate 5g/L, potassium dihydrogen phosphate 5g/L, and the remaining procedures were the same as in example 1.
EXAMPLE 8 preparation of lyophilized powder of Streptococcus thermophilus
1. Reagent(s)
(1) Seed culture medium: MRS liquid medium: 10g/L peptone, 10g/L beef extract, 20g/L glucose, 0.5g/L sodium acetate, 10g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05g/L, tween-80 1mL/L.
(2) Fermentation medium: MRS liquid medium: 10g/L peptone, 10g/L beef extract, 40g/L glucose, 0.5g/L sodium acetate, 10g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05g/L, tween-80 1mL/L.
(3) Lyoprotectant: 20g/L of glycerin, 80g/L of sucrose and 60g/L of maltodextrin, wherein the 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 shaking bottle seed culture medium into a streptococcus thermophilus preservation glycerol tube according to an inoculum size of 2%, and culturing at a constant temperature of 37 ℃ for 16 hours to obtain seed liquid; inoculating the seed solution into a fermentation tank according to an inoculum size of 2% (v/v), culturing, supplementing ammonia water to control the pH of the fermentation process to be 5.5, culturing at a constant temperature of 37 ℃ until residual sugar is 1%, stopping supplementing ammonia water, continuing to reduce the pH of a culture medium due to the fact that streptococcus thermophilus continues to produce acid, simultaneously opening cold water to reduce the temperature of the fermentation tank to 15 ℃, and centrifuging to obtain bacterial sludge in the whole low-pH and low-temperature environment for 3 hours; uniformly mixing the bacterial mud with a protective agent in a ratio of 1:2 (w/w), regulating the pH of the heavy suspension to 5.8, and freeze-drying. The method comprises the steps of prefreezing, primary drying and secondary drying, wherein prefreezing is to control the temperature of the laminate to be reduced to minus 50 ℃ in 1h, keeping for 3h, primary drying is to control the temperature of the laminate to be increased to minus 25 ℃ in 2h, keeping for 30h, and secondary drying is to control the temperature of the laminate to be increased to 30 ℃ in 1h, keeping to the secondary drying end point, thus obtaining the streptococcus thermophilus freeze-dried powder. The number of viable bacteria was measured and the survival rate was calculated. And (3) respectively preserving the prepared freeze-dried powder at 25 ℃ for 6 months, and after preserving the freeze-dried powder at 37 ℃ for 6 months, detecting the viable count and calculating the survival rate.
Example 9
In this example 9, compared with example 8, the difference is that the fermentation culture of Streptococcus thermophilus was performed, and when the residual sugar content was 1%, the bacterial sludge was obtained by direct centrifugation without performing the operation of lowering the temperature and lowering the pH, and the remaining operation steps were the same as in example 8.
EXAMPLE 10 preparation of lyophilized powder of Lactobacillus acidophilus
1. Reagent(s)
(1) Seed culture medium: MRS liquid medium: 10g/L peptone, 10g/L beef extract, 20g/L glucose, 0.5g/L sodium acetate, 10g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05g/L, tween-80 1mL/L.
(2) Fermentation medium MRS liquid medium: 10g/L peptone, 10g/L beef extract, 40g/L glucose, 0.5g/L sodium acetate, 10g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05g/L, tween-80 1mL/L.
(3) Lyoprotectant: sucrose 20g/L, galactose 20g/L and maltodextrin 180g/L, and the free amino acids or salts comprise glutamic acid, valine, proline and arginine 2g/L each, vitamin C salt 5g/L, acacia 2g/L, dipotassium hydrogen phosphate 10g/L and potassium dihydrogen phosphate 10g/L.
2. The experimental method comprises the following steps:
inoculating a shake flask seed culture medium into a lactobacillus acidophilus preservation glycerol tube according to an inoculum size of 2%, and culturing at a constant temperature of 37 ℃ for 16 hours to obtain seed liquid; inoculating the seed solution into a fermentation tank according to an inoculum size of 2% (v/v), culturing, supplementing ammonia water to control the pH of the fermentation process to be 5.5, culturing at a constant temperature of 37 ℃ until residual sugar is 1%, stopping supplementing ammonia water, continuing to reduce the pH of a culture medium due to the fact that lactobacillus acidophilus continues to produce acid, simultaneously opening cold water to reduce the temperature of the fermentation tank to 15 ℃, and centrifuging to obtain bacterial sludge in the whole low-pH and low-temperature environment for 5 hours; uniformly mixing the bacterial mud with a protective agent in a ratio of 1:1 (w/w), regulating the pH of the heavy suspension to 6.0, and freeze-drying. The method comprises the steps of prefreezing, primary drying and secondary drying, wherein prefreezing is to control the temperature of the laminate to be reduced to minus 50 ℃ in 1h, keeping for 3h, primary drying is to control the temperature of the laminate to be increased to minus 25 ℃ in 2h, keeping for 30h, and secondary drying is to control the temperature of the laminate to be increased to 30 ℃ in 1h, keeping to the secondary drying end point, thus obtaining the lactobacillus acidophilus freeze-dried powder. The number of viable bacteria was measured and the survival rate was calculated. And (3) respectively preserving the prepared freeze-dried powder at 25 ℃ for 6 months, and after preserving the freeze-dried powder at 37 ℃ for 6 months, detecting the viable count and calculating the survival rate.
Example 11
In this example 11, compared with example 10, the difference is that the lactobacillus acidophilus of this example was fermented and cultured to obtain residual sugar of 1%, and the residual sugar was not subjected to the operation of lowering temperature and lowering pH, and was directly centrifuged to obtain bacterial sludge, and the remaining operation steps were the same as in example 10.
Experimental example 1 survival 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 to coat in an MRS solid culture medium, coating 3 gradients, wherein 3 gradients are parallel to each other, placing the coated culture dish in a constant temperature incubator at 37 ℃ for culturing for 48h, and taking out for counting.
2. Detecting the viable count of the bacterial mud 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 to be coated in an MRS solid culture medium, coating 3 gradients, wherein 3 gradients are parallel to each other, placing a coated culture dish in a constant temperature incubator at 37 ℃ for culturing for 48h, taking out and counting, and multiplying the bacterial sludge weight to obtain the total viable count before freeze drying; after lyophilization, the bacterial powders were counted as in step 1 above.
3. And (3) measuring the content of mud water:
10g of bacterial sludge is taken, dried in an oven at 105 ℃ to constant weight, and the moisture content is calculated.
Freeze-drying survival rate = viable count per gram of bacterial powder/viable count per gram of bacterial mud/bacterial mud moisture content after freeze-drying;
shelf life survival = viable count per gram of powder per shelf life/viable count per gram of powder after lyophilization;
shelf life viable count: the lyophilized powder was placed in an incubator 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 food safety standard, food microbiology test, lactic acid bacteria test.
Specific detection results are shown in table 1 below.
TABLE 1 lactic acid bacteria survival
The foregoing is a description of embodiments of the invention, which are specific and detailed, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (7)
1. A preparation method of lactic acid bacteria freeze-dried powder is characterized by comprising the following steps: the preparation method comprises the following steps:
(1) Culturing lactobacillus to obtain seed solution, and continuously culturing the seed solution, wherein the culture method comprises the following steps: when the residual sugar is left for 0.5-1%, starting to cool, reducing the temperature of the fermentation liquor to 10-25 ℃, and stopping controlling alkali, so that the pH of the culture medium is reduced to 4-5 along with the consumption of the residual sugar, and the whole process lasts for 3-5 hours;
(2) Centrifuging the lactobacillus solution obtained by culturing in the step (1), and collecting bacterial sludge;
(3) Mixing the bacterial mud obtained by centrifugation in the step (2) with a freeze-drying protective agent to obtain a mixed solution, and freeze-drying the mixed solution to obtain lactic acid bacteria freeze-dried powder;
the freeze-drying protective agent in the step (3) comprises small molecular polyalcohol and/or saccharide with the content of 100-400g/L, free amino acid with the content of 5-20g/L, vitamin C salt with the content of 5-30g/L, high molecular protective agent with the content of 10-100g/L and phosphate with the content of 5-30 g/L;
the small molecule polyalcohol and/or saccharide comprises one or more of glycerol, trehalose, sucrose, galactose and maltodextrin;
the free amino acid is selected from glutamic acid, leucine, isoleucine, valine and alanine or glutamic acid, valine, alanine and aspartic acid or glutamic acid, valine, proline and arginine;
the polymer protective agent comprises one or more of milk protein, acacia and milk powder;
the content ratio of the small molecular polyalcohol and/or saccharide to the free amino acid or the salt substance thereof is 5-80:1.
2. The method of manufacturing according to claim 1, characterized in that: the content ratio of the small molecular polyalcohol and/or the saccharide to the free amino acid is 10-30:1.
3. The preparation method according to claim 2, characterized in that: the content ratio of the small molecular polyalcohol and/or the saccharide to the free amino acid is 20:1.
4. The method of manufacturing according to claim 1, characterized in that: the mass ratio of the bacterial mud to the freeze-drying protective agent in the step (3) is 1:1-2.
5. The method of manufacturing according to claim 1, characterized in that: 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 1h, and keep for 3-4h; the primary drying is to control the temperature of the laminate to rise to-25 to-30 ℃ for 2 hours, and keep the laminate to the primary drying end point; and the secondary drying is to control the temperature of the laminate to be raised to 22-30 ℃ for 1h, and keep the laminate to reach the secondary drying end point.
6. The method of manufacturing according to claim 5, wherein: the initial viable count of the lactobacillus freeze-dried powder is 1 multiplied by 10 11 -1×10 12 CFU/g。
7. The method of manufacturing according to claim 1, characterized in that: 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 and lactobacillus rhamnosus and lactobacillus bulgaricus.
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