CN111647510A - Bifidobacterium infantis freeze-dried powder, preparation method and composite protective agent used by same - Google Patents
Bifidobacterium infantis freeze-dried powder, preparation method and composite protective agent used by same Download PDFInfo
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- CN111647510A CN111647510A CN202010561524.3A CN202010561524A CN111647510A CN 111647510 A CN111647510 A CN 111647510A CN 202010561524 A CN202010561524 A CN 202010561524A CN 111647510 A CN111647510 A CN 111647510A
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- 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
Abstract
The invention relates to the related technical field of food processing, and particularly provides bifidobacterium infantis freeze-dried powder, a preparation method and a compound protective agent used by the same. The first aspect provides a freeze-dried powder composite protective agent for bifidobacteria, which comprises 10-30 wt% of skimmed milk powder, 0.1-5.0 wt% of amino acid and salt thereof, 3-25 wt% of disaccharide or/and polysaccharide, 1-5 wt% of small molecular polyol and the balance of phosphate buffer solution, wherein the pH value of the composite protective agent is 6.5-8.5; by adopting the combined action of the specific composite protective agent and the auxiliary agent, the freeze-drying survival rate of the bifidobacterium infantis and the viable count of the bacterial powder can be obviously improved, and the problems of low survival rate, few viable counts, short storage period and the like of the bifidobacterium infantis in the freeze-drying process are solved.
Description
Technical Field
The invention relates to the related technical field of food processing, and particularly provides bifidobacterium infantis freeze-dried powder, a preparation method and a compound protective agent used by the same.
Background
In recent years, probiotics are widely applied to the fields of functional dairy products, health-care foods, microecologics, medical treatment and the like, specifically to active microorganisms which have functional health benefits on hosts and have the advantages of regulating the human body immunity function, improving the microecological balance of intestinal flora, reducing blood fat and cholesterol, promoting digestion and absorption of nutrient substances and the like. The common probiotics include lactobacillus, bifidobacterium, clostridium butyricum, yeast and the like. The bifidobacteria, as the most important physiological bacteria in the intestinal tract of the body, are closely related to the growth and metabolism of the human body and play an important role in the aspects of human health and diseases. Specifically, the bifidobacterium has the physiological functions of inhibiting the growth of pathogenic bacteria in the intestinal tract, preventing diarrhea or constipation, providing vitamins, improving the immunity of the organism and the like. Currently, more than 30 species of bifidobacteria have been found, including Bifidobacterium infantis (Bifidobacterium infantis), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium bifidum (Bifidobacterium bifidum), and the like.
The bifidobacterium belongs to gram-positive bacteria, is obligate anaerobic, has variable forms, is mostly in V-shaped, Y-shaped, bent and other forms, does not form spores, has poor acid resistance and is difficult to keep activity in the processes of production, storage and transportation. Research shows that the number of live bacteria in the liquid bifidobacterium preparation is reduced quickly in the storage process, and the liquid bifidobacterium preparation can be only stored for 5-7 days under the condition of refrigeration at 4 ℃, so that the preparation of the high-activity bifidobacterium powder is very important, and the processing, storage and transportation of later-stage products are facilitated.
The vacuum freeze drying technology is one of the most effective methods for preparing and storing the bifidobacterium, and can well maintain the biological activity of the thalli. In the process of preparing the bacterial powder, water is not directly sublimated in a liquid state under vacuum and low temperature conditions, the bacterial strain is subjected to the action of two drastic factors of freezing and drying, if the bacterial strain is directly frozen and dried under the condition of not adding a protective agent, the permeability and the fluidity of a microbial cell membrane can be changed, the dynamic balance in cells can be damaged, enzyme protein molecules are passivated, and the like, so that the bacterial cell can be killed, and researches show that if the bacterial strain is directly frozen and dried, the survival rate of the bacterial cell is only about 10-30%. In the freeze drying process, factors influencing the freeze drying survival rate of microorganisms and the viable count of bacterial powder are complex, and the factors mainly comprise the physiological characteristics of strains, culture medium components, culture conditions, a harvest period, pre-freezing conditions, initial bacterial suspension concentration, a freeze drying protective agent, the pH value of the freeze drying protective agent, the freezing rate, residual moisture, rehydration conditions, sub-lethal treatment and the like. The freeze-drying protective agent is a key factor which is most complex and difficult to select, and at present, methods for improving the freeze-drying survival rate of the bifidobacterium infantis are mostly focused on the aspect, however, the more the addition amount of the protective agent is, the lower the viable count of the bacterial powder per unit mass is, and some research results on the freeze-dried bacterial powder of the bifidobacterium indicate that the density of the high-density thallus prepared by fermenting the bifidobacterium is basically exponentially reduced after freeze-drying, so that the freeze-dried powder capable of well maintaining the viable count of the bacterial powder is necessary to be prepared.
Disclosure of Invention
At present, the effect of a large amount of researches on the surface protective agent has strain specificity, and the types and the contents of protective agents required by different strains are different.
In order to achieve the technical effects, the first aspect of the invention provides a bifidobacterium lyophilized powder composite protective agent, which comprises, by weight, 10-30% of skimmed milk powder, 0.1-5.0% of amino acid and salt thereof, 3-25% of disaccharide or/and polysaccharide, 1-5% of small molecular polyol and the balance of phosphate buffer, wherein the composite protective agent has a pH of 6.5-8.5; preferably, the composite protective agent comprises 16-22% of skimmed milk powder, 0.2-3.0% of amino acid and salt thereof, 5-20% of disaccharide or/and polysaccharide, 2.5-3.6% of small molecular polyol and the balance of phosphate buffer solution, and the pH value of the composite protective agent is 7.0-7.5; further preferably, the composite protective agent comprises 18-21% of skimmed milk powder, 1.2-2.0% of amino acid and salt thereof, 12-17% of disaccharide or/and polysaccharide, 2.8-3.2% of small molecular polyol and the balance of phosphate buffer solution, and the pH value of the composite protective agent is 7.0.
The defatted milk powder is prepared by removing fat from fresh milk and drying, can be rented as a skeleton in the freeze-drying process of biological products except that the fat can be reduced to about 1 percent, prevents low-molecular substances from carbonizing and oxidizing, protects active substances from being influenced by high temperature, enables the freeze-dried finished products to be in a porous and loose sponge shape, but also needs to control the weight ratio of a composite protective agent in the defatted milk powder, is easy to cause an allergic phenomenon in the use process when the content of the composite protective agent exceeds 30 weight percent, and reduces the viable count of bacteria powder in the obtained infant bifidobacterium freeze-dried powder when the content of the composite protective agent is less than 10 weight percent. The manufacturer of the skimmed milk powder is not particularly limited, and in one embodiment, the skimmed milk powder is purchased from nzmp New Zenland.
The protective agent of the invention contains a high molecular substance of skimmed milk powder, and also comprises saccharides, such as disaccharides and/or polysaccharides, and low molecular substances of amino acids, which are beneficial for promoting microorganisms to keep stable living state and relieving water molecules.
However, in practical experiments, it can be found that different protective agents are suitable for different microorganisms, different protective agents have different mechanisms of action on different cells, and different degrees of protection on somatic cells, and similarly, not all amino acids and salts thereof are suitable for freeze-drying bifidobacterium infantis, possibly due to their biological properties; preferably, the amino acid and the salt thereof are sodium glutamate, and the applicant finds that when the sodium glutamate is used as a component in the composite protective agent, the survival rate of bifidobacterium infantis in the obtained freeze-dried powder can be effectively improved, the viable count of the bacterial powder is improved, and the problem of wall adhesion in the freeze-drying process is reduced.
In some embodiments, the disaccharide is selected from any one or a combination of more of trehalose, sucrose, maltose, lactose; preferably, the polysaccharide is selected from any one or more of starch, cellulose, inulin, pectin, pollen polysaccharide, stigma Maydis polysaccharide, fructus Jujubae polysaccharide, fructus fici polysaccharide, tea polysaccharide, Aloe polysaccharide, folium Mori polysaccharide, longan polysaccharide, tea polysaccharide, Aloe polysaccharide, folium Mori polysaccharide, and longan polysaccharide; however, in the experimental process, it is found that not all the sugar micromolecule substances can improve the survival rate of the bifidobacterium infantis and the viable count of the bacterial powder, which is also an expression of the bacterial specificity of the effect of the protective agent.
Preferably, the saccharide substance comprises trehalose and inulin, and further preferably, the weight ratio of trehalose to inulin is 1: (0.35-0.95); further preferably, the weight ratio of trehalose to inulin is 1: (0.6 to 0.83); the applicant finds that when trehalose and inulin act together with sodium glutamate in a synergistic manner, particularly when the total weight ratio of disaccharide and polysaccharide is 5-22%, and the weight ratio of trehalose to inulin is 1: (0.6-0.95), the survival rate of the bifidobacterium infantis in the obtained freeze-dried powder and the viable count of the bacterial powder can be improved, the non-reducing sugar consisting of two glucoses, the inulin and other components in the system act together to form a unique protective film on the surface of a cell membrane, so that the biomolecular structure is effectively protected from being damaged, the life process and the biological characteristics of a living body are maintained, a hydrogen bond is formed with protein, the requirement of forming the hydrogen bond with a charged group on the surface of the protein is met, the activity of bacterial cells is stabilized, the survival rate of the bifidobacterium infantis in the obtained freeze-dried powder and the viable count of the bacterial powder are improved, when the content of trehalose is more, the osmotic pressure in the system is changed, the oxidation-reduction potential of the system is improved, the consumption of oxygen in the system is reduced, and.
In addition, applicants have also found that the combination protectant system also requires the inclusion of small molecule polyols such as propylene glycol, glycerol, butylene glycol, mannitol, xylitol, and the like, and as such, not all small molecule polyols are suitable, preferably glycerol; the applicant unexpectedly finds that when the glycerin is used for synergistic action with the sodium glutamate, the trehalose and the inulin, the occupation phenomenon of the obtained freeze-dried powder can be effectively avoided, the structure is fluffy and is not agglomerated, the survival rate of the bifidobacterium infantis in the obtained freeze-dried powder and the viable count of the bacterial powder can be further improved, probably due to the synergistic action of the glycerin, the sodium glutamate, the trehalose and the inulin, the protective effect can be exerted in the freeze-drying process to replace water molecules to form hydrogen bonds with phosphate groups in bacterial cell membrane phospholipid or with polar groups of bacterial protein, the structural integrity and the functional integrity of the cell membrane and the protein can be protected, the separation of crystal water can be effectively avoided in the freeze-drying stage, the adhesion of the freeze-dried powder on the wall surface can be reduced, and the problems of freeze-dried powder agglomeration and agglomeration caused by the.
Preferably, in some embodiments, the preparation method of the composite protective agent comprises:
(1) dissolving skimmed milk powder and micromolecular polyalcohol in phosphate buffer solution, and sterilizing at high temperature to obtain solution A;
(2) dissolving disaccharide or/and polysaccharide in phosphate buffer solution, and sterilizing at high temperature to obtain solution B;
(3) dissolving amino acid and its salt in phosphate buffer solution, filtering with filter membrane for sterilization to obtain solution C;
(4) mixing solution A, solution B and solution C under aseptic condition.
Further preferably, in some embodiments, the preparation method of the composite protective agent comprises:
(1) dividing phosphate buffer into three parts according to the volume ratio of 1:1:1, dissolving skimmed milk powder and micromolecular polyol in one part of phosphate buffer, and sterilizing at 105-108 ℃ for 10-20 min to obtain solution A;
(2) dissolving disaccharide or/and polysaccharide in another part of phosphate buffer solution, and sterilizing at 110-112 ℃ for 10-20 min to obtain solution B;
(3) dissolving amino acid and its salt in the rest phosphate buffer solution, and filtering with 0.22 μm filter membrane for sterilization to obtain solution C;
(4) mixing solution A, solution B and solution C under aseptic condition.
The composite protective agent is prepared by sterilizing different components in different modes or at different temperatures in the preparation process, the obtained composite protective agent can be dissolved fully and uniformly, and protein denaturation is easy to occur when the skim milk powder is sterilized at a higher temperature to form precipitates; in addition, the skim milk powder and the carbohydrate are respectively dissolved in the phosphate buffer solution and then mixed, so that the uniform system is formed by full dissolution, and the situation that the dissolution degree is influenced by the formation of larger cohesion and agglomeration due to the direct mixing of the skim milk powder and the carbohydrate is avoided.
The invention provides a bifidobacterium infantis freeze-dried powder, and the compound protective agent is the bifidobacterium infantis freeze-dried powder compound protective agent.
The third aspect of the invention provides a preparation method of the bifidobacterium infantis freeze-dried powder, which is obtained by mixing wet bacterial sludge and a composite protective agent and freeze-drying.
Preferably, in some embodiments, the weight ratio of the wet bacterial sludge to the composite protective agent is (0.2-3.5): 1; further preferably, the weight ratio of the wet bacterial sludge to the composite protective agent is (0.5-3): 1; or further preferably, the weight ratio of the wet bacterial sludge to the composite protective agent is (1-2): 1; or further preferably, the weight ratio of the wet bacterial sludge to the composite protective agent is 1: 1.
in some embodiments, the wet bacterial sludge is prepared by the steps of bacterial activation, primary bacterial culture, secondary bacterial culture, fermentation culture and bacterial collection.
Specifically, the preparation process of the wet bacterial sludge comprises the following steps:
(1) activating strains: dissolving Bifidobacterium infantis strain stored at low temperature in 20-30mL of sterile physiological saline, shaking, mixing, inoculating into TPY liquid culture medium according to inoculum size of 2-6% by volume, anaerobically culturing at 35-38 deg.C for 8-15h, and continuously activating for 2 generations in TPY liquid culture medium according to the same inoculum size and culture conditions to obtain 3 rd generation liquid activated culture;
(2) primary strain culture: inoculating the 3 rd generation liquid activated culture in a first-stage strain liquid culture medium with the inoculation amount of 2% -6%, and terminating the culture after anaerobic culture at 35-38 ℃ for 8-15 h;
(3) and (3) secondary strain culture: inoculating the first-stage strain liquid culture in a second-stage strain liquid culture medium with the inoculation amount of 2% -6%, and terminating the culture after anaerobic culture at 35-38 ℃ for 8-15 h;
(4) fermentation culture: inoculating the second-level strain liquid culture in a liquid fermentation culture medium with the inoculation amount of 2% -6%, carrying out anaerobic culture at 35-38 ℃, supplementing a sodium hydroxide solution to stabilize the pH value of a fermentation liquor of the second-level strain liquid culture to 6-6.5, and terminating the culture after culturing for 12-20 h;
(5) and (3) collecting thalli: centrifuging the fermentation broth at 4 deg.C and 5000rpm for 20min, and removing the supernatant to obtain wet bacterial sludge.
The Bifidobacterium infantis strain is purchased from Shanghai Reineckia organisms.
The TPY liquid culture medium is not particularly limited, and can be purchased from the market, and the purchasing manufacturer is not particularly limited, and in one embodiment, the TPY liquid culture medium is purchased from Qingdao Haibo biotechnology limited.
The culture medium for the first-stage and second-stage strains is not particularly limited, and may be a strain culture medium component well known to those skilled in the art; the fermentation medium is also not particularly limited in the present invention and may be a fermentation medium component well known to those skilled in the art.
In some embodiments, the primary strain culture medium and the secondary strain culture medium are the same, and the composition and weight ratio of the primary strain culture medium and the secondary strain culture medium are as follows: 12-18 g of casein peptone, 3-8 g of soybean peptone, 3-8 g of yeast extract powder, 7-13 g of beef extract, 7-13 g of glucose, 3-8 g of lactose, 0.5-1.5 g of sodium acetate, 1-5 g of citric acid diamine, 0.5-4.5 g of dipotassium hydrogen phosphate, 1-3 g of magnesium sulfate, 0.1-0.8 g of L-cysteine and 1000mL of distilled water.
In some embodiments, the fermentation medium comprises, by weight: 12-18 g of casein peptone, 12-18 g of soybean peptone, 3-8 g of yeast extract powder, 12-18 g of digestive serum powder, 12-18 g of beef extract, 12-18 g of glucose, 3-8 g of lactose, 0.5-2.5 g of sodium acetate, 1-5 g of diamine citrate, 1-3 g of dipotassium hydrogen phosphate, 0.5-2.5 g of magnesium sulfate, 0.5-1.5 g of sorbitol, 0.1-0.6 g of glutamic acid, 0.1-0.45 g of L-cysteine and 1000mL of distilled water.
Preferably, the preparation method of the bifidobacterium infantis freeze-dried powder comprises the steps of mixing wet bacterial sludge and a composite protective agent, balancing for 20 min-60 min at the temperature of 30-45 ℃, pre-freezing for 3-10 h at the temperature of-20-60 ℃, and then carrying out freeze drying to obtain the bifidobacterium infantis freeze-dried powder; further preferably, the preparation method of the bifidobacterium infantis freeze-dried powder comprises the steps of mixing wet bacterial sludge and a composite protective agent, balancing for 20 min-60 min at the temperature of 30-45 ℃, pre-freezing for 3-10 h at the temperature of-30-50 ℃, and then freeze-drying for 16-22 h at the pressure of 3-8Pa to obtain the bifidobacterium infantis freeze-dried powder.
Preferably, in some embodiments, the fermentation medium further comprises an auxiliary agent, the auxiliary agent comprises a second component amino acid, a second component polyol and distilled water, and the relative molecular weight of the second component polyol is 100-400 g/mol; further preferably, the auxiliary agent comprises 0.01-0.5% of second component polyol, 0.01-0.3% of second component amino acid and the balance of distilled water in percentage by weight; further preferably, the auxiliary agent comprises 0.02-0.3% of second component polyol, 0.02-0.15% of second component amino acid and the balance of distilled water; further preferably, the auxiliary agent comprises 0.06-0.2% of the second component polyol, 0.06-0.11% of the second component amino acid and the balance of distilled water.
In some embodiments, the second component amino acid may be selected from any one or more combinations of glycine, alanine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, glutamine, methionine, serine, threonine, cysteine, proline, histidine, arginine; preferably glutamic acid.
In some embodiments, the second component polyol may be selected from any one or combination of sorbitol, xylitol, maltitol, glycerin, mannitol; sorbitol is preferred.
The applicant finds that, unexpectedly, when sorbitol and glutamic acid are prepared into a sterile solution with a certain concentration and added into a thallus fermentation culture medium, the survival rate of bifidobacterium infantis in the obtained freeze-dried powder and the viable count of the powder are improved, probably because the addition agent is added into the fermentation culture medium, the addition agent is better adsorbed on the surfaces of thallus cells under the condition of not influencing thallus fermentation, and is better adsorbed on the surfaces of the thallus cells under the synergistic action with a composite protective agent in the freeze-drying process, the change of a protein space structure is hindered, the acting force between the thallus and water molecules is reduced, meanwhile, the rapid metabolic death caused by excessive water can be effectively avoided, and the phenomenon that the cells are damaged or sublethal is avoided.
When the auxiliary agent is used, the auxiliary agent is filtered and sterilized by a 0.22-micron filter membrane, and then is added into a thallus fermentation culture medium in a sterile room, so that the auxiliary agent is obtained.
Compared with the prior art, the invention has the following beneficial effects:
(1) the composite protective agent suitable for the bifidobacterium infantis is formed by adopting specific carbohydrate, amino acid and salt substances thereof, the survival rate of the bifidobacterium infantis in the obtained freeze-dried powder and the viable count of the bacterial powder can be effectively improved, and the problems of wall adhesion, deep color, caking and the like in the freeze-drying process can be avoided;
(2) according to the application, the specific auxiliary agent and the compound protective agent are adopted in the preparation process of the freeze-dried powder, so that the survival rate of the bifidobacterium infantis freeze-dried powder and the viable count of the bacterial powder can be improved, and the survival rate can be highThe content of the active bacteria is more than 85 percent, and the number of the active bacteria of the bacterial powder can be as high as 1.5 × 1011cfu/g or more.
Detailed Description
The present invention will be specifically described below by way of examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention. In addition, the starting materials used are all commercially available, if not otherwise stated, of the kind or type known to those skilled in the art.
Skim milk powder was purchased from nzmp NewZenland in the following examples; TPY liquid medium was purchased from Qingdao Haibo Biotech limited; bifidobacterium infantis strains were purchased from Shanghai Reineckia.
Example 1
The embodiment 1 of the invention provides a composite protective agent for bifidobacterium infantis freeze-dried powder, which comprises 20% of skimmed milk powder, 1.6% of sodium glutamate, 16% of disaccharide and polysaccharide, 2% of small molecular polyol and the balance of phosphate buffer solution in percentage by weight; the small molecular polyol is glycerol;
the disaccharide is trehalose, the polysaccharide is inulin, and the weight ratio of the trehalose to the inulin is 1: 0.82;
the preparation method of the composite protective agent comprises the following steps:
(1) dividing phosphate buffer into three parts according to the volume ratio of 1:1:1, dissolving skimmed milk powder and micromolecular polyalcohol in one part of phosphate buffer, and sterilizing at 107 deg.C for 15min to obtain solution A;
(2) dissolving disaccharide and polysaccharide in another phosphoric acid buffer solution, and sterilizing at 110 deg.C for 15min to obtain solution B;
(3) dissolving amino acid and its salt in the rest phosphate buffer solution, and filtering with 0.22 μm filter membrane for sterilization to obtain solution C;
(4) mixing solution A, solution B and solution C under aseptic condition.
Example 2
The embodiment 2 of the invention provides a composite protective agent for bifidobacterium infantis freeze-dried powder, which comprises 25 percent of skimmed milk powder, 0.5 percent of sodium glutamate, 8 percent of disaccharide and polysaccharide, 1.5 percent of micromolecular polyhydric alcohol and the balance of phosphate buffer solution in percentage by weight; the small molecular polyol is glycerol;
the disaccharide is trehalose, the polysaccharide is inulin, and the weight ratio of the trehalose to the inulin is 1: 0.6;
the preparation method of the composite protective agent is the same as that of the example 1.
Example 3
The embodiment 3 of the invention provides a composite protective agent for bifidobacterium infantis freeze-dried powder, which comprises 13 percent of skimmed milk powder, 2 percent of sodium glutamate, 10.5 percent of disaccharide and polysaccharide, 4 percent of micromolecular polyol and the balance of phosphate buffer solution according to the weight percentage of the composite protective agent; the small molecular polyol is glycerol;
the disaccharide is trehalose, the polysaccharide is inulin, and the weight ratio of the trehalose to the inulin is 1: 0.35;
the preparation method of the composite protective agent is the same as that of the example 1.
Example 4
Embodiment 4 of the present invention provides a composite protective agent for bifidobacterium infantis lyophilized powder, which is the same as embodiment 3 in specific embodiment except that glycerol is replaced by mannitol.
Example 5
Embodiment 5 of the present invention provides a composite protective agent for bifidobacterium infantis lyophilized powder, which is the same as embodiment 3 in specific embodiment except that sorbitol is replaced with glycerol.
Example 6
Embodiment 6 of the present invention provides a composite protective agent for bifidobacterium infantis lyophilized powder, which is implemented in the same manner as embodiment 3 except that lactose is substituted for inulin.
Example 7
Embodiment 7 of the present invention provides a composite protective agent for bifidobacterium infantis lyophilized powder, which is implemented in the same manner as embodiment 3 except that inulin is replaced by pectin.
Example 8
Embodiment 8 of the present invention provides a composite protective agent for bifidobacterium infantis lyophilized powder, which is the same as embodiment 3 in specific implementation manner, except that sodium glutamate is replaced by Vc-Na.
Example 9
Embodiment 9 of the present invention provides a composite protective agent for bifidobacterium infantis lyophilized powder, which is different from embodiment 3 in that sodium glutamate is replaced by lysine.
Example 10
Embodiment 10 of the present invention provides a composite protective agent for bifidobacterium infantis lyophilized powder, which is prepared in the same manner as in embodiment 3, except that the total content of disaccharide and polysaccharide is replaced by 35 wt%.
Example 11
Embodiment 11 of the present invention provides a bifidobacterium infantis lyophilized powder, and the method for preparing the bifidobacterium infantis lyophilized powder comprises: mixing wet bacterial sludge with compound protectant, balancing at 37 deg.C for 35min, pre-freezing at-40 deg.C for 6 hr, and freeze-drying at 3-8Pa for 21 hr to obtain the final product;
the weight ratio of the wet bacterial sludge to the composite protective agent is 1: 1; the composite protective agent is the composite protective agent obtained in example 1;
the preparation process of the wet bacterial sludge comprises the following steps:
(1) activating strains: dissolving Bifidobacterium infantis strain stored at low temperature in 20-30mL sterile physiological saline, shaking, mixing, inoculating into TPY liquid culture medium according to inoculum size of 4% volume ratio, anaerobically culturing at 37 deg.C for 9.5h, and continuously activating for 2 generations in TPY liquid culture medium according to the same inoculum size and culture conditions to obtain 3 rd generation liquid activated culture;
(2) primary strain culture: inoculating the 3 rd generation liquid activated culture in a first-stage strain liquid culture medium, wherein the inoculation amount is 4%, and terminating the culture after anaerobic culture at 37 ℃ for 12 h;
(3) and (3) secondary strain culture: inoculating the first-stage strain liquid culture in a second-stage strain liquid culture medium, wherein the inoculation amount is 4%, and terminating the culture after anaerobic culture at 37 ℃ for 12 h;
(4) fermentation culture: inoculating the second-level strain liquid culture in a liquid fermentation culture medium with the inoculum size of 4%, performing anaerobic culture at 38 ℃, supplementing sodium hydroxide solution to stabilize the pH of the fermentation liquor between 6 and 6.5, and terminating the culture after culturing for 15 h;
(5) and (3) collecting thalli: centrifuging the fermentation liquor at 4 deg.C and 5000rpm for 20min, and removing the supernatant to obtain wet bacterial sludge;
the strain culture medium used for the first-stage strain culture and the second-stage strain culture is the same, and the composition and the weight ratio of the strain culture medium are as follows: 15g of casein peptone, 5g of soybean peptone, 5g of yeast extract powder, 10g of beef extract, 10g of glucose, 5g of lactose, 1g of sodium acetate, 3g of diamine citrate, 2g of dipotassium phosphate, 1.5g of magnesium sulfate, 0.3g of L-cysteine and 1000mL of distilled water;
the fermentation medium comprises the following components in parts by weight: 15g of casein peptone, 10g of soybean peptone, 5g of yeast extract powder, 10g of digestive serum powder, 10g of beef extract, 10g of glucose, 5g of lactose, 1g of sodium acetate, 3g of diamine citrate, 2g of dipotassium hydrogen phosphate, 1.5g of magnesium sulfate, 1g of sorbitol, 0.2g of glutamic acid, 0.3g of L-cysteine and 1000mL of distilled water;
the fermentation medium also comprises an auxiliary agent, and the auxiliary agent comprises 0.12% of second component polyol, 0.08% of second component amino acid and the balance of distilled water according to the weight percentage of the auxiliary agent; the second component polyalcohol is sorbitol; the second component amino acid is glutamic acid;
the sorbitol and the glutamic acid are dissolved in 50mL of distilled water, and then are filtered and sterilized by a 0.22 mu m filter membrane, and then are added into a thallus fermentation medium in a sterile room.
Example 12
Embodiment 12 of the present invention provides a bifidobacterium infantis lyophilized powder, which is the same as in embodiment 11, except that the composite protectant is the composite protectant obtained in embodiment 2; the weight ratio of the wet bacterial sludge to the composite protective agent is 3: 1.
example 13
Embodiment 13 of the present invention provides a bifidobacterium infantis lyophilized powder, which is the same as in embodiment 11, except that the composite protectant is the composite protectant obtained in embodiment 3; the weight ratio of the wet bacterial sludge to the composite protective agent is 1: 2.5.
example 14
Embodiment 14 of the present invention provides a bifidobacterium infantis lyophilized powder, which is the same as in embodiment 11, except that the composite protectant is the composite protectant obtained in embodiment 4.
Example 15
Embodiment 15 of the present invention provides a bifidobacterium infantis lyophilized powder, which is the same as in embodiment 11 except that the composite protective agent is the composite protective agent obtained in embodiment 5.
Example 16
Embodiment 16 of the present invention provides a bifidobacterium infantis lyophilized powder, which is the same as in embodiment 11 except that the composite protectant is the composite protectant obtained in embodiment 6.
Example 17
Embodiment 17 of the present invention provides a bifidobacterium infantis lyophilized powder, which is the same as in embodiment 11 except that the composite protectant is the composite protectant obtained in embodiment 7.
Example 18
Embodiment 18 of the present invention provides a bifidobacterium infantis lyophilized powder, which is the same as in embodiment 11 except that the composite protectant is the composite protectant obtained in embodiment 8.
Example 19
Embodiment 19 of the present invention provides a bifidobacterium infantis lyophilized powder, which is the same as in embodiment 11 except that the composite protectant is the composite protectant obtained in embodiment 9.
Example 20
Embodiment 20 of the present invention provides a bifidobacterium infantis lyophilized powder, which is the same as in embodiment 11 except that the composite protectant is the composite protectant obtained in embodiment 10.
Example 21
Example 21 of the present invention provides a bifidobacterium infantis lyophilized powder, which is substantially the same as example 11, except that the second component polyol is replaced with mannitol.
Example 22
Example 22 of the present invention provides a lyophilized powder of bifidobacterium infantis, which is substantially the same as in example 11, except that the amino acid of the second component is replaced with cysteine.
Example 23
Embodiment 23 of the present invention provides a bifidobacterium infantis lyophilized powder, which is prepared in the same manner as in embodiment 11, except that the content of the composite protectant is replaced with 0, and the content of the adjuvant is replaced with 0.
Example 24
Embodiment 24 of the present invention provides a composite protective agent for bifidobacterium infantis lyophilized powder, which has the same specific implementation manner as embodiment 1, except that,
the preparation method of the composite protective agent comprises the following steps:
(1) dividing phosphate buffer into two parts according to the volume ratio of 1:1, dissolving skimmed milk powder, micromolecular polyalcohol, disaccharide and polysaccharide in one part of phosphate buffer, and sterilizing at 110 deg.C for 15min to obtain solution B;
(2) dissolving amino acid and its salt in the rest phosphate buffer solution, and filtering with 0.22 μm filter membrane for sterilization to obtain solution C;
(3) mixing solution A, solution B and solution C under aseptic condition.
Performance evaluation
1. Freeze-drying survival rate determination of the bifidobacterium infantis; weighing 3g of centrifuged wet bacterial sludge, adding the weighed wet bacterial sludge into 27mL of physiological saline containing glass beads, oscillating for 30min, taking 1mL of bacterial liquid to 9mL of physiological saline for gradient dilution, taking 100ul of diluent to coat the diluent in a GAM solid culture medium, coating 3 gradients, wherein 3 gradients are parallel to each other, putting a coated culture dish into a 37 ℃ constant-temperature incubator to culture for 48h, taking out the culture dish for counting, and multiplying the count by the weight of the bacterial sludge to obtain the total number of viable bacteria before freeze-drying, wherein the total number is marked as a; after freeze-drying, counting the bacterial powder by the same method, and marking as b; the freeze-drying survival rate is recorded as c; the freeze-drying survival rate is calculated according to the formula: c is 100% of a/b;
2. and (3) determining the viable count of the bacterial powder in the bifidobacterium infantis freeze-dried powder: weighing 3g of freeze-dried bacterial powder, adding into 27mL of physiological saline containing glass beads, oscillating for 30min, taking 1mL of bacterial liquid to 9mL of physiological saline for gradient dilution, taking 100ul of diluent, coating into a GAM solid culture medium, coating 3 gradients, wherein each gradient is parallel, putting the coated culture dish into a 37 ℃ constant temperature incubator, culturing for 48h, taking out and counting.
3. And (3) detecting the freeze drying property: observing whether the obtained freeze-dried powder has wall sticking phenomenon or not, observing whether the freeze-dried powder has caking problem or not, wherein each detection embodiment corresponds to 100 samples, and the wall sticking evaluation grade and the standard are as follows: a-0-3 sticking walls appear; b, 4-10 sticky walls appear; c, 11-25 sticky walls appear; d-more than 25 sticky walls appear; the blocking evaluation rating and criteria were: a-0-3 lumps appear; b, 4-10 caking appears; c, 11-25 agglomerates appear; d-more than 25 lumps appear;
4. the composite protective agent obtained in the embodiment 1-3 is observed to be a uniform system, and has no phenomena of agglomeration, precipitation and delamination, while the composite protective agent obtained in the embodiment 24 has the problems of sedimentation and delamination.
TABLE 1
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in other forms, and any person skilled in the art may modify or change the technical content of the above disclosure into equivalent embodiments with equivalent changes, but all those simple modifications, equivalent changes and modifications made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the present invention.
Claims (10)
1. The composite protectant for the freeze-dried powder of the bifidobacteria is characterized by comprising, by weight, 10-30% of skimmed milk powder, 0.1-5.0% of amino acid and salt thereof, 3-25% of disaccharide or/and polysaccharide, 1-5% of small molecular polyol and the balance of phosphate buffer solution, wherein the pH value of the composite protectant is 6.5-8.5.
2. A lyophilized powder composite protectant for Bifidobacterium according to claim 1, wherein the amino acid is selected from one or more of glycine, alanine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, glutamine, methionine, serine, threonine, cysteine, proline, histidine, and arginine.
3. The bifidobacterium lyophilized powder composite protectant according to claim 1 or 2, characterized in that the disaccharide is selected from one or more of trehalose, sucrose, maltose and lactose; preferably, the polysaccharide is selected from any one or more of starch, cellulose, inulin, pectin, pollen polysaccharide, stigma Maydis polysaccharide, fructus Jujubae polysaccharide, fructus fici polysaccharide, tea polysaccharide, Aloe polysaccharide, folium Mori polysaccharide, longan polysaccharide, tea polysaccharide, Aloe polysaccharide, folium Mori polysaccharide, and longan polysaccharide.
4. A bifidobacterium freeze-dried powder composite protective agent as claimed in any one of claims 1 to 3, wherein the preparation method of the composite protective agent comprises the following steps:
(1) dissolving skimmed milk powder and micromolecular polyalcohol in phosphate buffer solution, and sterilizing at high temperature to obtain solution A;
(2) dissolving disaccharide or/and polysaccharide in phosphate buffer solution, and sterilizing at high temperature to obtain solution B;
(3) dissolving amino acid and its salt in phosphate buffer solution, filtering with filter membrane for sterilization to obtain solution C;
(4) mixing solution A, solution B and solution C under aseptic condition.
5. A freeze-dried powder of bifidobacterium infantis, which is characterized in that the compound protective agent is the freeze-dried powder compound protective agent of bifidobacterium in any one of claims 1 to 4.
6. A preparation method of bifidobacterium infantis freeze-dried powder according to claim 5, characterized in that the bifidobacterium infantis freeze-dried powder is prepared by mixing wet bacterial sludge and a compound protective agent, balancing at 30-45 ℃ for 20-60 min, pre-freezing at-20 to-60 ℃ for 3-10 h, and then freeze-drying.
7. The method for preparing bifidobacterium infantis freeze-dried powder according to claim 6, wherein the weight ratio of the wet bacterial sludge to the composite protective agent is (0.2-3.5): 1.
8. a method for preparing lyophilized powder of Bifidobacterium infantis as claimed in claim 6 or 7, wherein the wet bacterial sludge is prepared by activating strains, culturing primary strains, culturing secondary strains, fermenting, and collecting strains.
9. The method for preparing bifidobacterium infantis freeze-dried powder according to claim 8, wherein a fermentation medium used for fermentation culture contains an auxiliary agent, the auxiliary agent comprises a second component amino acid, a second component polyol and distilled water, and the relative molecular weight of the second component polyol is 100-400 g/mol.
10. The method for preparing bifidobacterium infantis freeze-dried powder according to claim 8, wherein the auxiliary comprises 0.01 to 0.5 percent of second component polyol, 0.01 to 0.3 percent of second component amino acid and the balance of distilled water by weight percent.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113005040A (en) * | 2021-01-22 | 2021-06-22 | 武汉微康益生菌研究院有限公司 | Bifidobacterium lactis freeze-drying protective agent and using method thereof |
| CN113575961A (en) * | 2021-07-30 | 2021-11-02 | 宁波大学 | Tea polyphenol probiotic lactic acid bacteria powder and preparation method thereof |
| CN113621535A (en) * | 2021-07-14 | 2021-11-09 | 湖南省中医药研究院 | Composite protective agent for improving survival rate of probiotic freeze-dried powder and preparation method thereof |
| CN114246341A (en) * | 2021-12-23 | 2022-03-29 | 瘦哪儿(广东)生物科技有限公司 | Probiotic composition for improving body fat rate and preparation method thereof |
| CN114292788A (en) * | 2021-12-30 | 2022-04-08 | 江苏德禧生物科技有限公司 | Preparation method of active probiotics by freeze drying |
| CN115558620A (en) * | 2022-09-30 | 2023-01-03 | 微康益生菌(苏州)股份有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
| CN116574611A (en) * | 2023-04-26 | 2023-08-11 | 甘肃普诺贝康生物科技有限责任公司 | Freeze-drying protective agent for lactobacillus buchneri and method for preparing lactobacillus buchneri freeze-drying powder by using freeze-drying protective agent |
| CN117070425A (en) * | 2023-10-10 | 2023-11-17 | 广东益可维生物技术有限公司 | Technological method for improving metabolic stability of probiotics in organism and probiotics freeze-dried powder |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016527A (en) * | 2007-02-02 | 2007-08-15 | 丽珠医药集团股份有限公司 | Bifidobacteria viable bacteria preparation and special-purpose protective agent thereof |
| CN101338295A (en) * | 2008-08-09 | 2009-01-07 | 甘肃省科学院生物研究所 | Bacillus bifidus freeze concentration leaven and method for preparing same |
| CN101649293A (en) * | 2009-09-02 | 2010-02-17 | 中国农业大学 | Microbial freezing drying protective agent and application thereof |
| CN102021162A (en) * | 2009-09-19 | 2011-04-20 | 菲伯纳生物医药有限责任公司 | High-activity bifidobacterium powder and preparation method thereof |
| CN102358884A (en) * | 2011-10-13 | 2012-02-22 | 陕西农产品加工技术研究院 | Bifidobacterium bifidum lyophilization composite protection agent and application method thereof |
-
2020
- 2020-06-18 CN CN202010561524.3A patent/CN111647510A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016527A (en) * | 2007-02-02 | 2007-08-15 | 丽珠医药集团股份有限公司 | Bifidobacteria viable bacteria preparation and special-purpose protective agent thereof |
| CN101338295A (en) * | 2008-08-09 | 2009-01-07 | 甘肃省科学院生物研究所 | Bacillus bifidus freeze concentration leaven and method for preparing same |
| CN101649293A (en) * | 2009-09-02 | 2010-02-17 | 中国农业大学 | Microbial freezing drying protective agent and application thereof |
| CN102021162A (en) * | 2009-09-19 | 2011-04-20 | 菲伯纳生物医药有限责任公司 | High-activity bifidobacterium powder and preparation method thereof |
| CN102358884A (en) * | 2011-10-13 | 2012-02-22 | 陕西农产品加工技术研究院 | Bifidobacterium bifidum lyophilization composite protection agent and application method thereof |
Non-Patent Citations (3)
| Title |
|---|
| F MARTIN-DEJARDIN ET AL: "A way to follow the viability of encapsulated Bifidobacterium bifidum subjected to a freeze-drying process in order to target the colon: Interest of flow cytometry", 《 EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES 》 * |
| 曹永梅等: "保护剂在冷冻干燥双歧杆菌中的作用", 《食品与发酵工业》 * |
| 陈合等: "山梨醇、甘露醇及甜菜碱等对两歧双歧杆菌冻干的影响", 《食品科技》 * |
Cited By (12)
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|---|---|---|---|---|
| CN113005040A (en) * | 2021-01-22 | 2021-06-22 | 武汉微康益生菌研究院有限公司 | Bifidobacterium lactis freeze-drying protective agent and using method thereof |
| CN113621535A (en) * | 2021-07-14 | 2021-11-09 | 湖南省中医药研究院 | Composite protective agent for improving survival rate of probiotic freeze-dried powder and preparation method thereof |
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