CN113337435A - Probiotic composition and application thereof in food - Google Patents
Probiotic composition and application thereof in food Download PDFInfo
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- CN113337435A CN113337435A CN202110638090.7A CN202110638090A CN113337435A CN 113337435 A CN113337435 A CN 113337435A CN 202110638090 A CN202110638090 A CN 202110638090A CN 113337435 A CN113337435 A CN 113337435A
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- freeze
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- deionized water
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- 235000018291 probiotics Nutrition 0.000 title claims abstract description 51
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- 230000000529 probiotic effect Effects 0.000 title claims abstract description 40
- 235000013305 food Nutrition 0.000 title claims description 16
- 239000000843 powder Substances 0.000 claims abstract description 95
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 75
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 75
- 241001468157 Lactobacillus johnsonii Species 0.000 claims abstract description 35
- 235000013406 prebiotics Nutrition 0.000 claims abstract description 8
- 238000000855 fermentation Methods 0.000 claims description 78
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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
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/151—Johnsonii
Abstract
The invention discloses a probiotic composition. The probiotic composition is composed of bacillus natto freeze-dried powder, lactobacillus johnsonii freeze-dried powder and viscidin akmansia freeze-dried powder, prebiotics, other auxiliary materials or additives are not required to be additionally added in the prepared probiotic composition, and the effective viable count of the bacillus natto freeze-dried powder, the lactobacillus johnsonii freeze-dried powder and the viscidin akmansia freeze-dried powder can be up to 2.0 multiplied by 1011And simultaneously has good effects of reducing blood sugar and blood fat and preventing constipation.
Description
Technical Field
The invention relates to the technical field of food, in particular to a probiotic composition and application thereof in food.
Background
The probiotics is a microbial preparation or product which contains enough viable bacteria and has definite composition, and can change the composition of flora at a certain part of a host through colonization, thereby generating the microbes which are beneficial to the health of the host. It is currently generally accepted that as probiotics, the following conditions must be met: (1) the bacterial strain is derived from the normal flora of a target host, has no pathogenicity, does not produce toxin, and does not carry transferable drug-resistant factors; (2) can produce various beneficial active substances or antibacterial substances, and has promoting effects on animal health growth and immunity improvement; (3) can resist gastric acid and bile salt in small intestine, maintain its bioactivity, and can be attached and fixed in animal intestinal tract; (4) it is suitable for production process, and can maintain activity and genetic stability during processing and storage.
As is known, intestinal microorganisms have great influence on human health, probiotics can control the reproduction of toxic microorganisms in the intestinal tract, and when the probiotics grow, move and reproduce in the digestive tract, the probiotics can be converted to form or directly produce various nutrients such as short chain fatty acids, amino acids, vitamins and the like. In addition, the probiotics can also increase the activity of natural killer cells, activate macrophages, enhance the level of immunoglobulin, particularly IgA, improve lactose intolerance, promote gastrointestinal motility, eliminate carcinogenic factors, reduce cholesterol and the like.
Chinese patent (application No. 201811633592.5) discloses a probiotic composition and its use, food and pharmaceutical products, the probiotic composition being characterized in that it comprises: 1-5 parts by weight of bifidobacterium breve; 0.4-2 parts by weight of lactobacillus rhamnosus; and 0.5-2 parts by weight of bifidobacterium lactis, and further comprises: inulin, fructo-oligosaccharides, erythritol, maltodextrin and silicon dioxide. Mainly solves the technical problems of relieving inflammation in vivo and promoting fat metabolism so as to achieve the effects of losing weight and lowering lipid. However, the invention has high viable count, and the shelf life of the viable count is greatly limited, thereby limiting the efficacy in other aspects.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a probiotic composition and application thereof in food.
In order to solve the technical problems, the invention adopts the technical scheme that:
a probiotic composition comprises Bacillus natto lyophilized powder, Lactobacillus johnsonii lyophilized powder, and Ackermansia muciniphila lyophilized powder.
Preferably, the probiotic composition consists of the following components in percentage by mass: 20-60 wt% of bacillus natto freeze-dried powder, 20-40 wt% of lactobacillus johnsonii freeze-dried powder and 20-40 wt% of akkermansia muciniphila freeze-dried powder; more preferably, the probiotic composition consists of the following components in percentage by mass: 50 wt% of bacillus natto freeze-dried powder, 25 wt% of lactobacillus johnsonii freeze-dried powder and 25 wt% of akmansia muciniphila freeze-dried powder.
B, bacillus natto: the bacillus natto has the effects of reducing and stabilizing blood sugar, can be applied to the preparation of medicines or preparations for reducing and stabilizing blood sugar, and has obvious effects of promoting the in vivo reduction of blood sugar and stabilizing blood sugar in intestinal expression; meanwhile, the bacillus natto has the functions of obviously losing weight, reducing the level of serum triglyceride, reducing the weight of white fat and reducing the diameter of fat cells; in addition, the bacillus natto enters the intestinal tract of an animal and survives for a long time, so that the immunity of the animal is enhanced for the growth and development of the animal, the number of dominant bacteria in the intestinal tract can be adjusted, the nutrient substances in the intestinal tract are changed, and the constipation is prevented.
Lactobacillus johnsonii: the lactobacillus johnsonii has the effects of improving obesity, reducing body fat and cholesterol, stabilizing intestinal bacteria phase, maintaining intestinal ecology with more bacteria and less bacteria, and further stimulating a mucosa to strengthen an immune system. The lactobacillus johnsonii can stimulate receptors on dendritic cells on the intestinal wall and combine, activate and activate transfer proteins in cells to move into nuclei to release a large amount of cytokines, promote secretion increase of leucocyte and interferon gamma, and inhibit immunoglobulin IgE by stimulating intestinal tract regulation immune response, so that an allergy phenomenon of excessive immune response is improved; meanwhile, the composition has good gastric acid and bile salt tolerance and cell adhesion, can colonize in the intestinal tract of a human body for more than 60 days, and also has good inhibition effect on pathogenic bacteria such as enterococcus faecalis, Escherichia coli, salmonella typhimurium and the like, thereby achieving the effects of cleaning and protecting the intestinal tract.
Akkermansia muciniphila: the akkermansia muciniphila has the function of improving the intestinal barrier. When the intestinal barrier is damaged, the intestinal permeability is increased, and pathogenic microorganisms, endotoxin and the like in the intestinal tract can enter blood or tissues through the intestinal barrier system, so that the body is in a disease state. Therefore, maintaining the intestinal barrier function normally plays a crucial role in the health status of the body. In addition, numerous studies have shown that the occurrence, development and outcome of many diseases (e.g., obesity, diabetes, pancreatitis, fatty liver, inflammatory bowel disease, cancer, etc.) are clinically closely related to intestinal barrier dysfunction. Therefore, intervention treatment for intestinal barrier dysfunction has a very positive effect on blocking disease progression and accelerating disease recovery.
The preparation method of the bacillus natto freeze-dried powder comprises the following steps:
s1, strain activation: marking out the bacillus natto on a solid culture medium A, and culturing for 18-30h at 32-40 ℃ to obtain activated bacillus natto; the preparation method of the solid culture medium A comprises the following steps: uniformly mixing 1-6 wt% of peptone, 1-4 wt% of glucose, 0.2-1 wt% of NaCl, 1-3 wt% of agar powder and the balance of deionized water, and sterilizing at the temperature of 110-;
s2, expanding culture: inoculating activated bacillus natto into a liquid culture medium A, wherein the inoculation amount is 1-5 wt%, and performing shake culture at 34-42 ℃ and 200-; the preparation method of the liquid culture medium A comprises the following steps: 6-14 wt% of skim milk powder, 1-4 wt% of organic inulin, 0.1-2 wt% of potassium dihydrogen phosphate, 0.0-1 wt% of magnesium sulfate and the balance of deionized water are uniformly mixed, 1-3mol/L of sodium carbonate is adopted to adjust the pH of the solution to be neutral, and the solution is sterilized for 10-30min at the temperature of 110-;
s3, fermentation: inoculating the seed liquid A into a fermentation medium A according to the inoculation amount of 2-8 wt%, and fermenting at 30-38 ℃ for 20-30h to obtain a fermentation product A; the preparation method of the fermentation medium A comprises the following steps: uniformly mixing 1-5 wt% of pea polypeptide, 1-4 wt% of lactose, 0.5-2 wt% of peptone, 0.1-1 wt% of dipotassium hydrogen phosphate, 0.05-1 wt% of manganese sulfate, 0.01-0.5 wt% of sodium selenite and the balance of deionized water, adjusting the pH of the solution to be neutral by adopting 1-3mol/L of sodium carbonate, and sterilizing at the temperature of 110-;
s4, collecting and emulsifying bacterial sludge: centrifuging the fermentation product A at 4000-7000rpm, discarding the supernatant, collecting bacterial sludge A, adding the bacterial sludge A into the freeze-drying protective agent A according to the mass ratio of 1 (1-4), and emulsifying at 100-300rpm for 15-40min to obtain an emulsion A; the lyoprotectant A is prepared as follows: uniformly mixing 2-8 wt% of glycerol, 10-30 wt% of skim milk powder, 2-6 wt% of ascorbic acid, 0.5-3 wt% of sodium glutamate, 0.5-2 wt% of prebiotics and the balance of deionized water;
s5, freeze-drying and crushing: freezing the emulsion A at-20-60 deg.C for 2-5h, drying at-40-60 deg.C under vacuum degree of 0.1-0.5mbar for 10-30h,pulverizing, and sieving with 10-40 mesh sieve to obtain Bacillus natto lyophilized powder. Aiming at the defects of low viable count and difficult high-density fermentation of the common fermented bacillus natto, the lactobacillus johnsonii fermented by adopting the special fermentation medium and the fermentation method thereof has obviously improved thallus density compared with the common fermentation, and the viable count is not less than 2 multiplied by 1010cfu/g, which is difficult to achieve and overcome by the prior art, the viable count is greatly improved, and the high-density fermentation of the high viable bacillus natto is realized;
meanwhile, the bergamot extract is added as a nutrient of the bacillus natto, and active substances such as linalyl acetate, limonene, linalool, aldehydes, ketones, esters and the like which are main components of the bergamot extract can effectively promote the growth and the reproduction of the bacillus natto, and can inhibit the growth of mixed bacteria and improve the survival rate of the bacillus natto; in addition, the pea polypeptide is added into the fermentation medium, belongs to the active ingredient of the probiotic preparation of the bioactive peptide, and can play a role of a freeze-drying protective agent, so that the damage of freeze drying on the probiotic is prevented, the live bacteria survival rate of the bacillus natto is improved, the production period of the prepared bacillus natto is shortened, the production cost is reduced, and the production efficiency is improved.
Preferably, the preparation method of the bacillus natto freeze-dried powder comprises the following steps:
s1, strain activation: marking out the bacillus natto on a solid culture medium A, and culturing for 18-30h at 32-40 ℃ to obtain activated bacillus natto; the preparation method of the solid culture medium A comprises the following steps: uniformly mixing 1-6 wt% of peptone, 1-4 wt% of glucose, 0.2-1 wt% of NaCl, 1-3 wt% of agar powder and the balance of deionized water, and sterilizing at the temperature of 110-;
s2, expanding culture: inoculating activated bacillus natto into a liquid culture medium A, wherein the inoculation amount is 1-5 wt%, and performing shake culture at 34-42 ℃ and 200-; the preparation method of the liquid culture medium A comprises the following steps: 6-14 wt% of skim milk powder, 1-4 wt% of organic inulin, 0.1-2 wt% of potassium dihydrogen phosphate, 0.0-1 wt% of magnesium sulfate and the balance of deionized water are uniformly mixed, 1-3mol/L of sodium carbonate is adopted to adjust the pH of the solution to be neutral, and the solution is sterilized for 10-30min at the temperature of 110-;
s3, fermentation: inoculating the seed liquid A into a fermentation medium A according to the inoculation amount of 2-8 wt%, and fermenting at 30-38 ℃ for 20-30h to obtain a fermentation product A; the preparation method of the fermentation medium A comprises the following steps: 1-5 wt% of pea polypeptide, 1-4 wt% of lactose, 0.5-2 wt% of peptone, 0.1-1 wt% of dipotassium hydrogen phosphate, 0.05-1 wt% of manganese sulfate, 0.01-0.5 wt% of sodium selenite, 0.01-0.8 wt% of nutrient and the balance of deionized water are uniformly mixed, 1-3mol/L of sodium carbonate is adopted to adjust the pH of the solution to be neutral, and the solution is sterilized for 10-30min at the temperature of 110-;
s4, collecting and emulsifying bacterial sludge: centrifuging the fermentation product A at 4000-7000rpm, discarding the supernatant, collecting bacterial sludge A, adding the bacterial sludge A into the freeze-drying protective agent A according to the mass ratio of 1 (1-4), and emulsifying at 100-300rpm for 15-40min to obtain an emulsion A; the lyoprotectant A is prepared as follows: uniformly mixing 2-8 wt% of glycerol, 10-30 wt% of skim milk powder, 2-6 wt% of ascorbic acid, 0.5-3 wt% of sodium glutamate, 0.5-2 wt% of prebiotics and the balance of deionized water;
s5, freeze-drying and crushing: freezing the emulsion A at-20-60 ℃ for 2-5h, drying at-40-60 ℃ under vacuum degree of 0.1-0.5mbar for 10-30h, taking out, pulverizing, and sieving with 10-40 mesh sieve to obtain Bacillus natto lyophilized powder.
The nutritional agent in the S3 is a bergamot extract, and the preparation method of the bergamot extract comprises the following steps:
t1, cleaning, drying and crushing bergamot, sieving with a 30-70-mesh sieve, taking 50-100 parts by weight of bergamot powder, adding 800-1200 parts by weight of aqueous solution for mixing, adding 0.01-1 wt% of pectinase, adjusting the pH value to 3-5 by using 1-3mol/L hydrochloric acid, performing enzymolysis at constant temperature of 40-50 ℃ for 20-30min, continuously stirring at room temperature and 200-400rpm for 30-60min, and filtering to obtain filter residue A and filtrate A;
t2, taking all filter residues A of T1, adding 800-1200 parts by weight of 30-60 wt% ethanol aqueous solution for mixing, placing the mixture at 40-60 ℃, ultrasonic power of 300-500W and ultrasonic frequency of 30-60kHz for ultrasonic 0.5-2h, filtering to obtain filtrate B, combining the filtrate A and the filtrate B, and reducing the concentration to 1/10-1/20 of the original volume at 80 ℃ to obtain concentrated solution;
t3, vacuum drying the concentrated solution of T2 at 70-90 deg.C to constant weight to obtain fructus Citri Sarcodactylis extract.
The prebiotics in the S4 are any one of isomaltooligosaccharide, fructo-oligosaccharide, malto-oligosaccharide, raffinose and isomerized lactose.
The preparation method of the lactobacillus johnsonii freeze-dried powder comprises the following steps:
w1, strain activation: taking Lactobacillus johnsonii to streak on a solid culture medium B, and statically culturing for 30-48h at the constant temperature of 30-45 ℃ to obtain activated Lactobacillus johnsonii; the solid medium B was prepared as follows: uniformly mixing 1-5 wt% of peptone, 1-4 wt% of glucose, 0.1-1 wt% of magnesium sulfate, 1-5 wt% of agar powder and the balance of deionized water, and sterilizing at the temperature of 110-;
w2, expanding culture: inoculating activated Lactobacillus johnsonii in a liquid culture medium B, wherein the inoculation amount is 1-5 wt%, and culturing in a shaking table at 32-40 ℃ for 18-36h to obtain a seed solution B; the preparation method of the liquid culture medium B comprises the following steps: the beef extract is 10-20 wt%, the peptone is 10-30 wt%, the glucose is 20-40 wt%, the sodium acetate is 2-8 wt%, the citric acid diamine is 0.5-2 wt%, the dipotassium phosphate is 0.1-1 wt%, the Tween-800.1-1 wt%, the magnesium sulfate is 0.1-0.5 wt%, and the balance is deionized water, and the beef extract is uniformly mixed and sterilized at the temperature of 110-;
w3, fermentation: inoculating the seed liquid B into a fermentation medium B according to the inoculation amount of 2-6 wt%, and performing static fermentation at 30-45 ℃ for 8-18h to obtain a fermentation product B; the preparation method of the fermentation medium B comprises the following steps: the beef extract is 10-20 wt%, the peptone is 10-30 wt%, the glucose is 20-40 wt%, the sodium acetate is 2-8 wt%, the citric acid diamine is 0.5-2 wt%, the dipotassium phosphate is 0.1-1 wt%, the Tween-800.1-1 wt%, the magnesium sulfate is 0.1-0.5 wt%, and the balance is deionized water, and the beef extract is uniformly mixed and sterilized at the temperature of 110-;
w4, bacterial sludge collection and emulsification: centrifuging the fermentation product B at 10000-15000rpm for 10-30min, removing the supernatant, collecting bacterial sludge B, adding the bacterial sludge B into the freeze-drying protective agent B according to the mass ratio of 1 (1-4), and emulsifying at 150-200rpm for 10-30min to obtain an emulsion B; the preparation method of the freeze-drying protective agent B comprises the following steps: uniformly mixing 10-30 wt% of skimmed milk powder, 2-8 wt% of trehalose, 3-8 wt% of glycerol, 1-4 wt% of maltodextrin and the balance of deionized water;
w5, lyophilization and pulverization: freezing the emulsion B at-40 to-50 ℃ for 2-8h, drying at-50 to-70 ℃ under vacuum at 0.1 to 0.5mbar for 40-60h, taking out, pulverizing, and sieving with 10-40 mesh sieve to obtain Lactobacillus johnsonii lyophilized powder.
The preparation method of the akkermansia muciniphila freeze-dried powder comprises the following steps:
x1, strain activation: taking Ackermanella muciniphila to streak on a solid culture medium C, and culturing for 20-28h at the constant temperature of 35-40 ℃ to obtain activated Ackermanella muciniphila; the solid medium C was prepared as follows: uniformly mixing 1-5 wt% of casein peptone, 0.5-2 wt% of glucose, 0.5-2 wt% of fucose, 0.1-1 wt% of threonine, 1-5 wt% of agar powder and the balance of deionized water, adjusting the pH to 6.0-7.0 by using 1-3mol/L hydrochloric acid solution, and sterilizing at the temperature of 110-;
x2, expanding culture: inoculating Ackermanella muciniphila in a liquid culture medium C with the inoculation amount of 1-4 wt%, and culturing in a shaker at 35-40 deg.C for 20-28h to obtain a seed solution C; the preparation method of the liquid culture medium C comprises the following steps: uniformly mixing 10-30 wt% of beef extract, 1-5 wt% of casein peptone, 0.5-2 wt% of glucose, 0.5-2 wt% of fucose, 0.1-0.5 wt% of threonine, 0.1-0.5 wt% of magnesium sulfate and the balance of deionized water, adjusting the pH to 6.0-7.0 by using 1-3mol/L hydrochloric acid solution, and sterilizing at the temperature of 110-;
x3, fermentation: inoculating the seed liquid C into a fermentation culture medium C according to the inoculation amount of 2-8 wt%, and performing static fermentation at 35-40 ℃ for 12-24h to obtain a fermentation product C; the preparation method of the fermentation medium C comprises the following steps: uniformly mixing 10-30 wt% of beef extract, 1-5 wt% of casein peptone, 0.5-2 wt% of glucose, 0.5-2 wt% of fucose, 0.1-0.5 wt% of threonine, 0.1-0.5 wt% of magnesium sulfate and the balance of deionized water, adjusting the pH to 6.0-7.0 by using 1-3mol/L hydrochloric acid solution, and sterilizing at the temperature of 110-;
x4, bacterial sludge collection and emulsification: placing the fermentation product B obtained in the step X3 at 6000-10000rpm for centrifugation for 10-30min, removing the supernatant, collecting bacterial sludge C, adding the bacterial sludge C into the freeze-drying protective agent C according to the mass ratio of 1 (1-4), and emulsifying at 100-300rpm for 10-30min to obtain an emulsion C; the preparation method of the freeze-drying protective agent C comprises the following steps: uniformly mixing 10-30 wt% of skimmed milk powder, 2-8 wt% of glycerol, 3-10 wt% of oligomeric maltose and the balance of deionized water;
x5, lyophilization and pulverization: freezing the emulsion C at-40 to-50 ℃ for 2-8h, drying at-50 to-70 ℃ under a vacuum degree of 0.1 to 0.5mbar for 40-60h, taking out of the bin, crushing, and sieving with a 10-40 mesh sieve to obtain the adhesive protein akkermansia freeze-dried powder.
The probiotic composition is applied to food.
The invention has the beneficial effects that:
1. according to the probiotic composition and the application thereof in food, through the optimal selection and reasonable matching of the raw materials, the composite probiotics comprise the bacillus natto freeze-dried powder, the lactobacillus johnsonii freeze-dried powder and the akkermansia muciniphila freeze-dried powder, the growth and the propagation of beneficial bacteria in intestinal tracts and the intestinal tract health are facilitated, and the three components synergistically play a role in protecting the intestinal tracts, improving the intestinal tract microbial balance, regulating the intestinal tract function and improving the immunity of organisms.
2. The bergamot extract is used as a nutrient in the fermentation process of the bacillus natto, and the main components of the bergamot extract are active substances such as linalyl acetate, limonene, linalool, aldehydes, ketones, esters and the like, so that the growth and the propagation of the bacillus natto can be effectively promoted, the growth of mixed bacteria can be inhibited, and the survival rate of the bacillus natto can be improved.
3. The probiotic composition prepared by the invention does not need to be additionally added with prebiotics, other auxiliary materials or additives, the effective viable count of the bacillus natto, the lactobacillus johnsonii and the akkermansia muciniphila can reach as high as 200 hundred million/g, the survival rate of the viable count after being placed for a long time is still very high, and the probiotic composition also has good effects of reducing blood sugar and blood fat and preventing constipation.
Detailed Description
The above summary of the present invention is described in further detail below with reference to specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples.
Introduction of some raw materials in this application:
the Lactobacillus johnsonii in the examples is deposited in China center for type culture Collection with a deposition number of CCTCC NO: m20191013.
In the examples, Bacillus natto, Bacillus subtilis, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation numbers are: CGMCC No. 5769.
Examples Akkermansia muciniphila, purchased from the china industrial microbial cultures collection management center, strain collection No.: CICC 24917.
In the examples, skim milk powder was purchased from Zhengzhou Baister food additives Co., Ltd, food grade.
In the examples, organic inulin was purchased from food science and technology limited, mingjie, in the following amounts: 99 percent and is in food grade.
In the embodiment, the pea polypeptide is purchased from Gansu Probiotics and auspicious biotechnology limited, the total protein is more than or equal to 90 percent, the peptide content is more than or equal to 80 percent, the molecular weight is less than 1000, and the pea polypeptide is in food grade.
In the embodiment, the bergamot is purchased from the professional cooperation society for planting Xianggang Chinese medicinal materials in the heat exchanger in the Bozhou city.
In the examples, fucose was purchased from Zhengzhou encaste food additives Co., Ltd, food grade.
In the examples, pectinase was purchased from Huizhou Yuanle Biotech, Inc., and the enzyme activity: 10 ten thousand U/g and food grade.
Isomaltooligosaccharides in the examples were obtained from seian university harvest biotechnology limited, type: IMO-500, Cat number: 45000, content of active substance: 99 percent and is in food grade.
Example 1
A probiotic composition is bacillus natto freeze-dried powder.
The preparation method of the bacillus natto freeze-dried powder comprises the following steps:
s1, strain activation: marking out the bacillus natto on a solid culture medium A, and culturing for 24 hours at 37 ℃ to obtain activated bacillus natto; the preparation method of the solid culture medium A comprises the following steps: uniformly mixing 3 wt% of peptone, 2 wt% of glucose, 0.5 wt% of NaCl, 2 wt% of agar powder and the balance of deionized water, and sterilizing at 120 ℃ for 20 min;
s2, expanding culture: inoculating activated bacillus natto into a liquid culture medium A, wherein the inoculation amount is 2 wt%, and performing shaking culture at 38 ℃ and 300rpm for 12 hours to obtain a seed solution A; the preparation method of the liquid culture medium A comprises the following steps: uniformly mixing 10 wt% of skim milk powder, 2 wt% of organic inulin, 1 wt% of potassium dihydrogen phosphate, 0.2 wt% of magnesium sulfate and the balance of deionized water, adjusting the pH of the solution to be neutral by adopting 2mol/L sodium carbonate, and sterilizing at 120 ℃ for 20 min;
s3, fermentation: inoculating the seed liquid A into a fermentation medium A according to the inoculation amount of 5 wt%, and fermenting at 35 ℃ for 24h to obtain a fermentation product A; the preparation method of the fermentation medium A comprises the following steps: uniformly mixing 2 wt% of pea polypeptide, 2 wt% of lactose, 1 wt% of peptone, 0.2 wt% of dipotassium hydrogen phosphate, 0.1 wt% of manganese sulfate, 0.05 wt% of sodium selenite, 0.1 wt% of a nutrient and the balance of deionized water, adjusting the pH of the solution to be neutral by adopting 2mol/L sodium carbonate, and sterilizing at 120 ℃ for 20 min;
s4, collecting and emulsifying bacterial sludge: centrifuging the fermentation product A at 5000rpm for 15min, removing supernatant, collecting bacterial sludge A, adding the bacterial sludge A into the freeze-drying protective agent A according to the mass ratio of 1:2, and emulsifying at 200rpm for 30min to obtain an emulsion A; the lyoprotectant A is prepared as follows: uniformly mixing 5 wt% of glycerol, 20 wt% of skim milk powder, 4 wt% of ascorbic acid, 1.5 wt% of sodium glutamate, 1 wt% of isomaltooligosaccharide and the balance of deionized water;
s5, freeze-drying and crushing: freezing the emulsion A at-40 deg.C for 3h, drying at-50 deg.C under vacuum degree of 0.2mbar for 20h, taking out, pulverizing, and sieving with 20 mesh sieve to obtain Bacillus natto lyophilized powder.
The nutritional agent is a bergamot extract, and the preparation method of the bergamot extract comprises the following steps:
t1, cleaning, drying and crushing bergamot, sieving with a 50-mesh sieve, taking 80 parts by weight of bergamot powder, adding 1000 parts by weight of deionized water solution for mixing, adding 0.1 wt% of pectinase, adjusting pH to 4 by using 2mol/L hydrochloric acid, carrying out enzymolysis at a constant temperature of 45 ℃ for 25min, continuously stirring at room temperature and 300rpm for 40min, and filtering to obtain filter residue A and filtrate A;
t2, taking all filter residues A of T1, adding 1000 parts by weight of 40 wt% ethanol aqueous solution, mixing, putting into ultrasonic waves with the ultrasonic power of 400W and the ultrasonic frequency of 45kHz at 50 ℃ for 1h, filtering to obtain filtrate B, combining the filtrate A and the filtrate B, and reducing and concentrating the filtrate A and the filtrate B to 1/15 of the original volume at 80 ℃ to obtain concentrated solution;
t3, vacuum drying the concentrated solution of T2 at 80 deg.C to constant weight to obtain fructus Citri Sarcodactylis extract.
Example 2
A probiotic composition consisting of the following ingredients: 50 wt% of bacillus natto freeze-dried powder and 50 wt% of lactobacillus johnsonii freeze-dried powder;
the preparation method of the bacillus natto freeze-dried powder is the same as that of the example 1.
The preparation method of the lactobacillus johnsonii freeze-dried powder comprises the following steps:
w1, strain activation: taking Lactobacillus johnsonii to streak on a solid culture medium B, and statically culturing for 36h at the constant temperature of 38 ℃ to obtain activated Lactobacillus johnsonii; the solid medium B was prepared as follows: uniformly mixing 3 wt% of peptone, 2 wt% of glucose, 0.5 wt% of magnesium sulfate, 2 wt% of agar powder and the balance of deionized water, and sterilizing at 120 ℃ for 20 min;
w2, expanding culture: inoculating activated lactobacillus johnsonii into a liquid culture medium B, wherein the inoculation amount is 2 wt%, and culturing in a shaking table at 36 ℃ for 24 hours to obtain a seed solution B; the preparation method of the liquid culture medium B comprises the following steps: uniformly mixing 15 wt% of beef extract, 15 wt% of peptone, 30 wt% of glucose, 5 wt% of sodium acetate, 1 wt% of diamine citrate, 0.5 wt% of dipotassium phosphate, 0.2 wt% of tween-800.5, 0.2 wt% of magnesium sulfate and the balance of deionized water, and sterilizing at 120 ℃ for 20 min;
w3, fermentation: inoculating the seed liquid B into a fermentation medium B according to the inoculation amount of 4 wt%, and performing static fermentation for 10h at 36 ℃ to obtain a fermentation product B; the preparation method of the fermentation medium B comprises the following steps: 15 wt% of beef extract, 15 wt% of peptone, 30 wt% of glucose, 5 wt% of sodium acetate, 1 wt% of diamine citrate, 0.5 wt% of dipotassium phosphate, 0.2 wt% of tween-800.5 and 0.2 wt% of magnesium sulfate, and sterilizing at 120 ℃ for 20 min;
w4, bacterial sludge collection and emulsification: centrifuging the fermentation product B at 12000rpm for 15min, removing supernatant, collecting bacterial sludge B, adding the bacterial sludge B into the freeze-drying protective agent B according to the mass ratio of 1:2, and emulsifying at 180rpm for 15min to obtain an emulsion B; the preparation method of the freeze-drying protective agent B comprises the following steps: uniformly mixing 20 wt% of skimmed milk powder, 5 wt% of trehalose, 5 wt% of glycerol, 2 wt% of maltodextrin and the balance of deionized water;
w5, lyophilization and pulverization: freezing the emulsion B at-45 deg.C for 3h, drying at-60 deg.C under vacuum degree of 0.2mbar for 48h, taking out, pulverizing, and sieving with 20 mesh sieve to obtain Lactobacillus johnsonii lyophilized powder.
Example 3
A probiotic composition consisting of the following ingredients: 50 wt% of bacillus natto freeze-dried powder and 50 wt% of akkermansia muciniphila freeze-dried powder;
the preparation method of the bacillus natto freeze-dried powder is the same as that of the example 1.
The preparation method of the akkermansia muciniphila freeze-dried powder comprises the following steps:
x1, strain activation: taking Ackermanella muciniphila to streak on a solid culture medium C, and culturing for 24h at the constant temperature of 37 ℃ to obtain activated Ackermanella muciniphila; the solid medium C was prepared as follows: uniformly mixing 3 wt% of casein peptone, 1 wt% of glucose, 1 wt% of fucose, 0.2 wt% of threonine, 3 wt% of agar powder and the balance of deionized water, adjusting the pH to 6.5 by using 2mol/L hydrochloric acid, and sterilizing at 120 ℃ for 20 min;
x2, expanding culture: inoculating the activated akkermansia muciniphila in a liquid culture medium C, wherein the inoculation amount is 2 wt%, and culturing in a shaker at 37 ℃ for 24h to obtain a seed solution C; the preparation method of the liquid culture medium C comprises the following steps: uniformly mixing 15 wt% of beef extract, 3 wt% of casein peptone, 1 wt% of glucose, 1 wt% of fucose, 0.2 wt% of threonine, 0.2 wt% of magnesium sulfate and the balance of deionized water, adjusting the pH to 6.5 by using 2mol/L hydrochloric acid, and sterilizing at 120 ℃ for 20 min;
x3, fermentation: inoculating the seed solution C into a fermentation medium C according to the inoculation amount of 4 wt%, and performing static fermentation at 37 ℃ for 18h to obtain a fermentation product C; the preparation method of the fermentation medium C comprises the following steps: uniformly mixing 15 wt% of beef extract, 3 wt% of casein peptone, 1 wt% of glucose, 1 wt% of fucose, 0.2 wt% of threonine, 0.2 wt% of magnesium sulfate and the balance of deionized water, adjusting the pH to 6.5 by using 2mol/L hydrochloric acid, and sterilizing at 120 ℃ for 20 min;
x4, bacterial sludge collection and emulsification: placing the fermentation product B obtained in the step X3 at 8000rpm, centrifuging for 20min, removing supernatant, collecting bacterial sludge C, adding the bacterial sludge C into the freeze-drying protective agent C according to the mass ratio of 1:2, and emulsifying at 200rpm for 15min to obtain emulsion C; the preparation method of the freeze-drying protective agent C comprises the following steps: uniformly mixing 20 wt% of skimmed milk powder, 5 wt% of glycerol, 5 wt% of oligomeric maltose and the balance of deionized water;
x5, lyophilization and pulverization: freezing the emulsion C at-45 deg.C for 3h, drying at-60 deg.C under vacuum degree of 0.2mbar for 48h, taking out, pulverizing, and sieving with 20 mesh sieve to obtain the lyophilized powder of Acermanniella muciniphila.
Example 4
A probiotic composition consisting of the following ingredients: 50 wt% of bacillus natto freeze-dried powder, 25 wt% of lactobacillus johnsonii freeze-dried powder and 25 wt% of akkermansia muciniphila freeze-dried powder.
The preparation method of the bacillus natto freeze-dried powder is the same as that of the example 1.
The preparation method of the lactobacillus johnsonii freeze-dried powder is the same as that of the example 2.
The preparation method of the akkermansia muciniphila freeze-dried powder is the same as that in example 3.
The viable count of the bacillus natto freeze-dried powder is 3.7 multiplied by 1011cfu/g, viable count in Lactobacillus johnsonii freeze-dried powder 8.3 x 1011cfu/g, viable count in Ackermanella mucronata freeze-dried powder 2.0 x 1011cfu/g。
Example 5
Essentially the same as example 1, except that: the preparation method of the bacillus natto freeze-dried powder comprises the following steps:
s1, strain activation: marking out the bacillus natto on a solid culture medium A, and culturing for 24 hours at 37 ℃ to obtain activated bacillus natto; the preparation method of the solid culture medium A comprises the following steps: uniformly mixing 3 wt% of peptone, 2 wt% of glucose, 0.5 wt% of NaCl, 2 wt% of agar powder and the balance of deionized water, and sterilizing at 120 ℃ for 20 min;
s2, expanding culture: inoculating activated bacillus natto into a liquid culture medium A, wherein the inoculation amount is 2 wt%, and performing shaking culture at 38 ℃ and 300rpm for 12 hours to obtain a seed solution A; the preparation method of the liquid culture medium A comprises the following steps: uniformly mixing 10 wt% of skim milk powder, 2 wt% of organic inulin, 1 wt% of potassium dihydrogen phosphate, 0.2 wt% of magnesium sulfate and the balance of deionized water, adjusting the pH of the solution to be neutral by adopting 2mol/L sodium carbonate, and sterilizing at 120 ℃ for 20 min;
s3, fermentation: inoculating the seed liquid A into a fermentation medium A according to the inoculation amount of 5 wt%, and fermenting at 35 ℃ for 24h to obtain a fermentation product A; the preparation method of the fermentation medium A comprises the following steps: uniformly mixing 2 wt% of pea polypeptide, 2 wt% of lactose, 1 wt% of peptone, 0.2 wt% of dipotassium hydrogen phosphate, 0.1 wt% of manganese sulfate, 0.05 wt% of sodium selenite and the balance of deionized water, adjusting the pH of the solution to be neutral by adopting 2mol/L sodium carbonate, and sterilizing at 120 ℃ for 20 min;
s4, collecting and emulsifying bacterial sludge: centrifuging the fermentation product A at 5000rpm for 15min, removing supernatant, collecting bacterial sludge A, adding the bacterial sludge A into the freeze-drying protective agent A according to the mass ratio of 1:2, and emulsifying at 200rpm for 30min to obtain an emulsion A; the lyoprotectant A is prepared as follows: uniformly mixing 5 wt% of glycerol, 20 wt% of skim milk powder, 4 wt% of ascorbic acid, 1.5 wt% of sodium glutamate, 1 wt% of isomaltooligosaccharide and the balance of deionized water;
s5, freeze-drying and crushing: freezing the emulsion A at-40 deg.C for 3h, drying at-50 deg.C under vacuum degree of 0.2mbar for 20h, taking out, pulverizing, and sieving with 20 mesh sieve to obtain Bacillus natto lyophilized powder.
Example 6
Essentially the same as example 1, except that: the preparation method of the bacillus natto freeze-dried powder comprises the following steps:
s1, strain activation: marking out the bacillus natto on a solid culture medium A, and culturing for 24 hours at 37 ℃ to obtain activated bacillus natto; the preparation method of the solid culture medium A comprises the following steps: uniformly mixing 3 wt% of peptone, 2 wt% of glucose, 0.5 wt% of NaCl, 2 wt% of agar powder and the balance of deionized water, and sterilizing at 120 ℃ for 20 min;
s2, expanding culture: inoculating activated bacillus natto into a liquid culture medium A, wherein the inoculation amount is 2 wt%, and performing shaking culture at 38 ℃ and 300rpm for 12 hours to obtain a seed solution A; the preparation method of the liquid culture medium A comprises the following steps: uniformly mixing 10 wt% of skim milk powder, 2 wt% of organic inulin, 1 wt% of potassium dihydrogen phosphate, 0.2 wt% of magnesium sulfate and the balance of deionized water, adjusting the pH of the solution to be neutral by adopting 2mol/L sodium carbonate, and sterilizing at 120 ℃ for 20 min;
s3, fermentation: inoculating the seed liquid A into a fermentation medium A according to the inoculation amount of 5 wt%, and fermenting at 35 ℃ for 24h to obtain a fermentation product A; the preparation method of the fermentation medium A comprises the following steps: uniformly mixing 2 wt% of lactose, 1 wt% of peptone, 0.2 wt% of dipotassium phosphate, 0.1 wt% of manganese sulfate, 0.05 wt% of sodium selenite, 0.1 wt% of nutrient and the balance of deionized water, adjusting the pH of the solution to be neutral by adopting 2mol/L sodium carbonate, and sterilizing at 120 ℃ for 20 min;
s4, collecting and emulsifying bacterial sludge: centrifuging the fermentation product A at 5000rpm for 15min, removing supernatant, collecting bacterial sludge A, adding the bacterial sludge A into the freeze-drying protective agent A according to the mass ratio of 1:2, and emulsifying at 200rpm for 30min to obtain an emulsion A; the lyoprotectant A is prepared as follows: uniformly mixing 5 wt% of glycerol, 20 wt% of skim milk powder, 4 wt% of ascorbic acid, 1.5 wt% of sodium glutamate, 1 wt% of isomaltooligosaccharide and the balance of deionized water;
s5, freeze-drying and crushing: freezing the emulsion A at-40 deg.C for 3h, drying at-50 deg.C under vacuum degree of 0.2mbar for 20h, taking out, pulverizing, and sieving with 20 mesh sieve to obtain Bacillus natto lyophilized powder.
The nutritional agent is a bergamot extract, and the preparation method of the bergamot extract comprises the following steps:
t1, cleaning, drying and crushing bergamot, sieving with a 50-mesh sieve, taking 80 parts by weight of bergamot powder, adding 1000 parts by weight of deionized water solution for mixing, adding 0.1 wt% of pectinase, adjusting pH to 4 by using 2mol/L hydrochloric acid, carrying out enzymolysis at a constant temperature of 45 ℃ for 25min, continuously stirring at room temperature and 300rpm for 40min, and filtering to obtain filter residue A and filtrate A;
t2, taking all filter residues A of T1, adding 1000 parts by weight of 40 wt% ethanol aqueous solution, mixing, putting into ultrasonic waves with the ultrasonic power of 400W and the ultrasonic frequency of 45kHz at 50 ℃ for 1h, filtering to obtain filtrate B, combining the filtrate A and the filtrate B, and reducing and concentrating the filtrate A and the filtrate B to 1/15 of the original volume at 80 ℃ to obtain concentrated solution;
t3, vacuum drying the concentrated solution of T2 at 80 deg.C to constant weight to obtain fructus Citri Sarcodactylis extract.
Test example 1
And (3) measuring the viable count:
and (3) measuring the viable count before freeze-drying: the Bacillus natto bacterial sludge, the Lactobacillus johnsonii bacterial sludge and the Ackermansia muciniphila bacterial sludge collected in the above examples 1 and 5-6 are taken and put into 10ml of 0.85% (m/v) physiological saline to be shaken evenly, and the viable count is measured by a gradient dilution method and an agar culture medium.
Fully shaking the cultured bacteria liquid on a clean bench, and continuously diluting all samples by 10 times with the dilution degree of 10-1、10-2、10-3、10-4、10-5、10-6And 10-7. Selecting appropriate dilution, uniformly inoculating 1mL of the dilution into a modified MRS agar culture medium which is sterilized, cooled to about 45 ℃, shaking uniformly, culturing at 37 ℃ for 48h, selecting the dilution with about 100 colony counts, selecting 2 dilutions for each sample, performing 3 repeated averaging on each dilution, and calculating the unit viable count (cfu/g).
And (3) measuring the viable count after freeze-drying: taking 1g of Bacillus natto freeze-dried powder, Lactobacillus johnsonii freeze-dried powder and Ackermanella mucronata freeze-dried powderAdding 10mL of 0.85% (m/v) physiological saline into a centrifuge tube for restoration, fully oscillating until the freeze-dried powder is completely dissolved, and respectively diluting with 10 degree of dilution-1、10-2、10-3、10-4、10-5、10-6And 10-7. Selecting appropriate dilution, uniformly inoculating 1mL of the diluted solution into a modified MRS agar culture medium which is sterilized and then cooled to about 45 ℃, shaking uniformly, and culturing at 37 ℃ for 48h, wherein the dilution with about 100 colonies is selected.
TABLE 1 detection results of viable count before lyophilization
| Bacillus natto (cfu/g) | |
| Example 1 | 9.8×1011 |
| Example 5 | 4.1×1010 |
| Example 6 | 6.8×1010 |
TABLE 2 detection results of viable count after lyophilization
| Natto bacillus rodFungus (cfu/g) | |
| Example 1 | 3.7×1011 |
| Example 5 | 1.4×1010 |
| Example 6 | 2.8×1010 |
From the results, the content of the viable count can be effectively improved by adding the bergamot extract serving as a nutrient in the preparation process of the bacillus natto, the content of the viable count can also be improved to a certain extent by adding the pea polypeptide, and the viable count is also effectively improved after freeze-drying. The reason is that the main components of the bergamot extract, such as linalyl acetate, limonene, linalool, aldehydes, ketones, esters and other active substances, can effectively promote the growth and the propagation of the bacillus natto, and can inhibit the growth of mixed bacteria and improve the survival rate of the bacillus natto; in addition, the pea polypeptide belongs to the effective component of the probiotic preparation of the bioactive peptide, and simultaneously can play the role of a freeze-drying protective agent, so that the damage of freeze-drying to probiotics is prevented, the survival rate of viable bacteria of bacillus natto is improved, the defatted milk powder in the freeze-dried powder can prevent the cell damage by stabilizing the composition of cell membranes, the defatted milk powder also contains protein and can provide a protective layer for the cells, in addition, a porous structure is generated in the freeze-drying process, so that the dehydration is easier, the survival rate of thalli can be improved, the excessive moisture of the product is removed by freeze-drying, and the stability of the finished product is improved; meanwhile, the pea polypeptide and the probiotics are freeze-dried together, so that the activity of the probiotics is kept, the effective viable count of the finished product is higher, and the activity of the pea polypeptide can be improved. Meanwhile, the probiotic composition prepared by the invention has a production period, so that the production cost is reduced and the production efficiency is improved.
Test example 2
Rat testing: SPF-class Wistar rats weighing 150 + -20 g were selected and randomly and evenly divided into experimental groups (examples 1-6) of 20 rats each with half of males and females. The rats are numbered before the test, the probiotic composition in the examples 1-6 is prepared into a probiotic composition solution with the concentration of 100mg/kg by warm boiled water at 37 ℃, the probiotic composition solution in the examples 1-6 is perfused for 3 times a day by an experimental group of rats with the dosage of 0.5mL each time, warm boiled water at 37 ℃ is perfused for 3 times a day by a blank group of rats, the rats in the experimental group and the blank group freely eat and drink water and are continuously fed for 30 days, and the performance, behavior, toxicity performance and death condition of the rats are observed every day.
1. After feeding for 30 days, fasting was performed for 10 hours, and blood was collected from abdominal aorta of rats, serum was separated, and Total Cholesterol (TCH), Triglyceride (TG) and blood sugar (GLU) contents of rats were measured using an enzymatic kit.
TABLE 3 measurement results of Total Cholesterol (TCH), Triglyceride (TG) and blood sugar (GLU)
2. Mouse peripheral blood immune cells and peritoneal macrophages: separately collecting eyeballs of mice in blank group and test group, collecting total peripheral blood (with mouse hemorrhagic shock as standard), and collecting with Tris-NH4After the CI solution hypotonically destroys the red blood cells, immune cells are counted, and then the mice after blood collection are dissected on a clean workbench, and abdominal cavity macrophages are taken for counting.
TABLE 4 blood immunocyte and peritoneal macrophage assay results
| Peripheral blood immune cells (1X 10)6/mL) | Peritoneal macrophage (1X 10)6/mL) | |
| Example 1 | 2.31 | 4.02 |
| Example 2 | 2.51 | 4.23 |
| Example 3 | 2.58 | 4.28 |
| Example 4 | 2.73 | 4.81 |
| Example 5 | 2.04 | 3.84 |
| Example 6 | 2.01 | 3.78 |
| Blank group | 1.68 | 2.94 |
From the results, the probiotic composition prepared by the invention has good effects of reducing blood sugar and blood fat, and also has good immune effect.
Test example 3
And (3) constipation prevention test: 210 constipation patients are randomly selected as subjects, the age, sex, illness condition and other data of the patients have no significant difference and are comparable, the age of the patients ranges from 20 to 70 years, wherein 50% of men and 50% of women are randomly divided into 7 groups, each group is divided into 30 cases, the subjects take the probiotic compositions of examples 1 to 6 of the invention respectively 30 minutes before dinner every day, one group does not take any product of the same type as a control group, 1.5g of the composition is taken every time, and the observation is carried out after 20 days of taking. The therapeutic effect of the probiotic composition of the present invention was evaluated on three scales of "significant effect", "effective" and "ineffective", and the condition of constipation patients was recorded separately.
TABLE 5 criteria
| Grade of judgement | Criterion of judgment |
| The effect is obvious | After taking for 15 days, the patient defecates l-2 times per day, and the stool quality is soft |
| Is effective | After taking the medicine for 15 days, the patient defecates for 2 days, and the stool quality is close to normal |
| Invalidation | After taking the medicine for 15 days, the excrement condition of the patient has no obvious change |
TABLE 6 test results
| Remarkable effect (number of people) | Effective (number of people) | Invalid (number of people) | |
| Example 1 | 22 | 8 | 0 |
| Example 2 | 25 | 5 | 0 |
| Example 3 | 26 | 4 | 0 |
| Example 4 | 29 | 1 | 0 |
| Example 5 | 18 | 11 | 1 |
| Example 6 | 17 | 12 | 1 |
| Control group | 0 | 5 | 25 |
From the above results, it is clear that the probiotic composition prepared by the present invention is safe and effective, can be used for treating constipation, and achieves beneficial effects.
Claims (9)
1. The probiotic composition is characterized by consisting of bacillus natto freeze-dried powder, lactobacillus johnsonii freeze-dried powder and akkermansia muciniphila freeze-dried powder.
2. The probiotic composition according to claim 1, characterized by consisting of, in mass percent: 20-60 wt% of bacillus natto freeze-dried powder, 20-40 wt% of lactobacillus johnsonii freeze-dried powder and 20-40 wt% of akmansia muciniphila freeze-dried powder.
3. The probiotic composition according to claim 2, wherein the bacillus natto freeze-dried powder is prepared by the following method:
s1, strain activation: marking out the bacillus natto on a solid culture medium A, and culturing for 18-30h at 32-40 ℃ to obtain activated bacillus natto; the preparation method of the solid culture medium A comprises the following steps: uniformly mixing 1-6 wt% of peptone, 1-4 wt% of glucose, 0.2-1 wt% of NaCl, 1-3 wt% of agar powder and the balance of deionized water, and sterilizing at the temperature of 110-;
s2, expanding culture: inoculating activated bacillus natto into a liquid culture medium A, wherein the inoculation amount is 1-5 wt%, and performing shake culture at 34-42 ℃ and 200-; the preparation method of the liquid culture medium A comprises the following steps: 6-14 wt% of skim milk powder, 1-4 wt% of organic inulin, 0.1-2 wt% of potassium dihydrogen phosphate, 0.0-1 wt% of magnesium sulfate and the balance of deionized water are uniformly mixed, 1-3mol/L of sodium carbonate is adopted to adjust the pH of the solution to be neutral, and the solution is sterilized for 10-30min at the temperature of 110-;
s3, fermentation: inoculating the seed liquid A into a fermentation medium A according to the inoculation amount of 2-8 wt%, and fermenting at 30-38 ℃ for 20-30h to obtain a fermentation product A; the preparation method of the fermentation medium A comprises the following steps: uniformly mixing 1-5 wt% of pea polypeptide, 1-4 wt% of lactose, 0.5-2 wt% of peptone, 0.1-1 wt% of dipotassium hydrogen phosphate, 0.05-1 wt% of manganese sulfate, 0.01-0.5 wt% of sodium selenite and the balance of deionized water, adjusting the pH of the solution to be neutral by adopting 1-3mol/L of sodium carbonate, and sterilizing at the temperature of 110-;
s4, collecting and emulsifying bacterial sludge: centrifuging the fermentation product A at 4000-7000rpm, discarding the supernatant, collecting bacterial sludge A, adding the bacterial sludge A into the freeze-drying protective agent A according to the mass ratio of 1 (1-4), and emulsifying at 100-300rpm for 15-40min to obtain an emulsion A; the lyoprotectant A is prepared as follows: uniformly mixing 2-8 wt% of glycerol, 10-30 wt% of skim milk powder, 2-6 wt% of ascorbic acid, 0.5-3 wt% of sodium glutamate, 0.5-2 wt% of prebiotics and the balance of deionized water;
s5, freeze-drying and crushing: freezing the emulsion A at-20-60 ℃ for 2-5h, drying at-40-60 ℃ under vacuum degree of 0.1-0.5mbar for 10-30h, taking out, pulverizing, and sieving with 10-40 mesh sieve to obtain Bacillus natto lyophilized powder.
4. The probiotic composition according to claim 3, wherein the Bacillus natto freeze-dried powder is prepared by the following method:
s1, strain activation: marking out the bacillus natto on a solid culture medium A, and culturing for 18-30h at 32-40 ℃ to obtain activated bacillus natto; the preparation method of the solid culture medium A comprises the following steps: uniformly mixing 1-6 wt% of peptone, 1-4 wt% of glucose, 0.2-1 wt% of NaCl, 1-3 wt% of agar powder and the balance of deionized water, and sterilizing at the temperature of 110-;
s2, expanding culture: inoculating activated bacillus natto into a liquid culture medium A, wherein the inoculation amount is 1-5 wt%, and performing shake culture at 34-42 ℃ and 200-; the preparation method of the liquid culture medium A comprises the following steps: 6-14 wt% of skim milk powder, 1-4 wt% of organic inulin, 0.1-2 wt% of potassium dihydrogen phosphate, 0.0-1 wt% of magnesium sulfate and the balance of deionized water are uniformly mixed, 1-3mol/L of sodium carbonate is adopted to adjust the pH of the solution to be neutral, and the solution is sterilized for 10-30min at the temperature of 110-;
s3, fermentation: inoculating the seed liquid A into a fermentation medium A according to the inoculation amount of 2-8 wt%, and fermenting at 30-38 ℃ for 20-30h to obtain a fermentation product A; the preparation method of the fermentation medium A comprises the following steps: 1-5 wt% of pea polypeptide, 1-4 wt% of lactose, 0.5-2 wt% of peptone, 0.1-1 wt% of dipotassium hydrogen phosphate, 0.05-1 wt% of manganese sulfate, 0.01-0.5 wt% of sodium selenite, 0.01-0.8 wt% of nutrient and the balance of deionized water are uniformly mixed, 1-3mol/L of sodium carbonate is adopted to adjust the pH of the solution to be neutral, and the solution is sterilized for 10-30min at the temperature of 110-;
s4, collecting and emulsifying bacterial sludge: centrifuging the fermentation product A at 4000-7000rpm, discarding the supernatant, collecting bacterial sludge A, adding the bacterial sludge A into the freeze-drying protective agent A according to the mass ratio of 1 (1-4), and emulsifying at 100-300rpm for 15-40min to obtain an emulsion A; the lyoprotectant A is prepared as follows: uniformly mixing 2-8 wt% of glycerol, 10-30 wt% of skim milk powder, 2-6 wt% of ascorbic acid, 0.5-3 wt% of sodium glutamate, 0.5-2 wt% of prebiotics and the balance of deionized water;
s5, freeze-drying and crushing: freezing the emulsion A at-20-60 ℃ for 2-5h, drying at-40-60 ℃ under vacuum degree of 0.1-0.5mbar for 10-30h, taking out, pulverizing, and sieving with 10-40 mesh sieve to obtain Bacillus natto lyophilized powder.
5. The probiotic composition according to claim 4, wherein the nutritional agent in S3 is a bergamot extract prepared by the following method:
t1, cleaning, drying and crushing bergamot, sieving with a 30-70-mesh sieve, taking 50-100 parts by weight of bergamot powder, adding 800-1200 parts by weight of aqueous solution for mixing, adding 0.01-1 wt% of pectinase, adjusting the pH value to 3-5 by using 1-3mol/L hydrochloric acid, performing enzymolysis at constant temperature of 40-50 ℃ for 20-30min, continuously stirring at room temperature and 200-400rpm for 30-60min, and filtering to obtain filter residue A and filtrate A;
t2, taking all filter residues A of T1, adding 800-1200 parts by weight of 30-60 wt% ethanol aqueous solution for mixing, placing the mixture at 40-60 ℃, ultrasonic power of 300-500W and ultrasonic frequency of 30-60kHz for ultrasonic 0.5-2h, filtering to obtain filtrate B, combining the filtrate A and the filtrate B, and reducing the concentration to 1/10-1/20 of the original volume at 80 ℃ to obtain concentrated solution;
t3, vacuum drying the concentrated solution of T2 at 70-90 deg.C to constant weight to obtain fructus Citri Sarcodactylis extract.
6. The probiotic composition according to claim 3 or 4, characterized in that the prebiotic in S4 is any one of isomaltooligosaccharide, fructo-oligosaccharide, malto-oligosaccharide, raffinose and isomerized lactose.
7. The probiotic composition according to claim 2, characterized in that the lactobacillus johnsonii freeze-dried powder is prepared as follows:
w1, strain activation: taking Lactobacillus johnsonii to streak on a solid culture medium B, and statically culturing for 30-48h at the constant temperature of 30-45 ℃ to obtain activated Lactobacillus johnsonii; the solid medium B was prepared as follows: uniformly mixing 1-5 wt% of peptone, 1-4 wt% of glucose, 0.1-1 wt% of magnesium sulfate, 1-5 wt% of agar powder and the balance of deionized water, and sterilizing at the temperature of 110-;
w2, expanding culture: inoculating activated Lactobacillus johnsonii in a liquid culture medium B, wherein the inoculation amount is 1-5 wt%, and culturing in a shaking table at 32-40 ℃ for 18-36h to obtain a seed solution B; the preparation method of the liquid culture medium B comprises the following steps: the beef extract is 10-20 wt%, the peptone is 10-30 wt%, the glucose is 20-40 wt%, the sodium acetate is 2-8 wt%, the citric acid diamine is 0.5-2 wt%, the dipotassium phosphate is 0.1-1 wt%, the Tween-800.1-1 wt%, the magnesium sulfate is 0.1-0.5 wt%, and the balance is deionized water, and the beef extract is uniformly mixed and sterilized at the temperature of 110-;
w3, fermentation: inoculating the seed liquid B into a fermentation medium B according to the inoculation amount of 2-6 wt%, and performing static fermentation at 30-45 ℃ for 8-18h to obtain a fermentation product B; the preparation method of the fermentation medium B comprises the following steps: the beef extract is 10-20 wt%, the peptone is 10-30 wt%, the glucose is 20-40 wt%, the sodium acetate is 2-8 wt%, the citric acid diamine is 0.5-2 wt%, the dipotassium phosphate is 0.1-1 wt%, the Tween-800.1-1 wt%, the magnesium sulfate is 0.1-0.5 wt%, and the balance is deionized water, and the beef extract is uniformly mixed and sterilized at the temperature of 110-;
w4, bacterial sludge collection and emulsification: centrifuging the fermentation product B at 10000-15000rpm for 10-30min, removing the supernatant, collecting bacterial sludge B, adding the bacterial sludge B into the freeze-drying protective agent B according to the mass ratio of 1 (1-4), and emulsifying at 150-200rpm for 10-30min to obtain an emulsion B; the preparation method of the freeze-drying protective agent B comprises the following steps: uniformly mixing 10-30 wt% of skimmed milk powder, 2-8 wt% of trehalose, 3-8 wt% of glycerol, 1-4 wt% of maltodextrin and the balance of deionized water;
w5, lyophilization and pulverization: freezing the emulsion B at-40 to-50 ℃ for 2-8h, drying at-50 to-70 ℃ under vacuum at 0.1 to 0.5mbar for 40-60h, taking out, pulverizing, and sieving with 10-40 mesh sieve to obtain Lactobacillus johnsonii lyophilized powder.
8. The probiotic composition according to claim 2, characterized in that the preparation method of the akkermansia muciniphila lyophilized powder is as follows:
x1, strain activation: taking Ackermanella muciniphila to streak on a solid culture medium C, and culturing for 20-28h at the constant temperature of 35-40 ℃ to obtain activated Ackermanella muciniphila; the solid medium C was prepared as follows: uniformly mixing 1-5 wt% of casein peptone, 0.5-2 wt% of glucose, 0.5-2 wt% of fucose, 0.1-1 wt% of threonine, 1-5 wt% of agar powder and the balance of deionized water, adjusting the pH to 6.0-7.0 by using 1-3mol/L hydrochloric acid solution, and sterilizing at the temperature of 110-;
x2, expanding culture: inoculating Ackermanella muciniphila in a liquid culture medium C with the inoculation amount of 1-4 wt%, and culturing in a shaker at 35-40 deg.C for 20-28h to obtain a seed solution C; the preparation method of the liquid culture medium C comprises the following steps: uniformly mixing 10-30 wt% of beef extract, 1-5 wt% of casein peptone, 0.5-2 wt% of glucose, 0.5-2 wt% of fucose, 0.1-0.5 wt% of threonine, 0.1-0.5 wt% of magnesium sulfate and the balance of deionized water, adjusting the pH to 6.0-7.0 by using 1-3mol/L hydrochloric acid solution, and sterilizing at the temperature of 110-;
x3, fermentation: inoculating the seed liquid C into a fermentation culture medium C according to the inoculation amount of 2-8 wt%, and performing static fermentation at 35-40 ℃ for 12-24h to obtain a fermentation product C; the preparation method of the fermentation medium C comprises the following steps: uniformly mixing 10-30 wt% of beef extract, 1-5 wt% of casein peptone, 0.5-2 wt% of glucose, 0.5-2 wt% of fucose, 0.1-0.5 wt% of threonine, 0.1-0.5 wt% of magnesium sulfate and the balance of deionized water, adjusting the pH to 6.0-7.0 by using 1-3mol/L hydrochloric acid solution, and sterilizing at the temperature of 110-;
x4, bacterial sludge collection and emulsification: placing the fermentation product B obtained in the step X3 at 6000-10000rpm for centrifugation for 10-30min, removing the supernatant, collecting bacterial sludge C, adding the bacterial sludge C into the freeze-drying protective agent C according to the mass ratio of 1 (1-4), and emulsifying at 100-300rpm for 10-30min to obtain an emulsion C; the preparation method of the freeze-drying protective agent C comprises the following steps: uniformly mixing 10-30 wt% of skimmed milk powder, 2-8 wt% of glycerol, 3-10 wt% of oligomeric maltose and the balance of deionized water;
x5, lyophilization and pulverization: freezing the emulsion C at-40 to-50 ℃ for 2-8h, drying at-50 to-70 ℃ under a vacuum degree of 0.1 to 0.5mbar for 40-60h, taking out of the bin, crushing, and sieving with a 10-40 mesh sieve to obtain the adhesive protein akkermansia freeze-dried powder.
9. Use of a probiotic composition according to any one of claims 1 to 8 in a food product.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116508993A (en) * | 2023-03-21 | 2023-08-01 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus reuteri in preparation of probiotics with metabolism regulating effect |
| CN117603884A (en) * | 2024-01-17 | 2024-02-27 | 广州同康生物科技有限公司 | Acremonium muciniphilum bacterial powder and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116508993A (en) * | 2023-03-21 | 2023-08-01 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus reuteri in preparation of probiotics with metabolism regulating effect |
| CN116508993B (en) * | 2023-03-21 | 2023-12-15 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus reuteri in preparation of probiotics with metabolism regulating effect |
| CN117603884A (en) * | 2024-01-17 | 2024-02-27 | 广州同康生物科技有限公司 | Acremonium muciniphilum bacterial powder and preparation method thereof |
| CN117603884B (en) * | 2024-01-17 | 2024-03-26 | 广州同康生物科技有限公司 | Acremonium muciniphilum bacterial powder and preparation method thereof |
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