CN103704718B - There is in a kind of shelf-lives the preparation method of the Bacillus subtilus microcapsule of high viable count - Google Patents
There is in a kind of shelf-lives the preparation method of the Bacillus subtilus microcapsule of high viable count Download PDFInfo
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- 239000003094 microcapsule Substances 0.000 title claims abstract description 113
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 67
- 238000002360 preparation method Methods 0.000 title claims abstract description 44
- 241000894006 Bacteria Species 0.000 claims abstract description 56
- 238000009461 vacuum packaging Methods 0.000 claims abstract description 14
- 230000001580 bacterial effect Effects 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 9
- 230000008569 process Effects 0.000 claims abstract description 6
- 239000002994 raw material Substances 0.000 claims abstract description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000001301 oxygen Substances 0.000 claims abstract description 4
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 4
- 238000004519 manufacturing process Methods 0.000 claims abstract description 3
- 239000007788 liquid Substances 0.000 claims description 40
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 239000001963 growth medium Substances 0.000 claims description 24
- 235000013336 milk Nutrition 0.000 claims description 24
- 239000008267 milk Substances 0.000 claims description 24
- 210000004080 milk Anatomy 0.000 claims description 24
- 239000000843 powder Substances 0.000 claims description 23
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 22
- 238000011218 seed culture Methods 0.000 claims description 21
- 230000004913 activation Effects 0.000 claims description 20
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- 238000004321 preservation Methods 0.000 claims description 19
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 18
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 18
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- 238000000855 fermentation Methods 0.000 claims description 18
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- 238000003756 stirring Methods 0.000 claims description 16
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 11
- 235000011187 glycerol Nutrition 0.000 claims description 11
- 238000001291 vacuum drying Methods 0.000 claims description 11
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 10
- 229920001661 Chitosan Polymers 0.000 claims description 10
- 239000002253 acid Substances 0.000 claims description 10
- 235000010413 sodium alginate Nutrition 0.000 claims description 10
- 239000000661 sodium alginate Substances 0.000 claims description 10
- 229940005550 sodium alginate Drugs 0.000 claims description 10
- 235000008939 whole milk Nutrition 0.000 claims description 10
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 9
- 238000001816 cooling Methods 0.000 claims description 9
- 238000009792 diffusion process Methods 0.000 claims description 9
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- 239000000243 solution Substances 0.000 claims description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 235000019764 Soybean Meal Nutrition 0.000 claims description 6
- 239000006027 corn-soybean meal Substances 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 239000002504 physiological saline solution Substances 0.000 claims description 6
- 239000004455 soybean meal Substances 0.000 claims description 6
- 235000020251 goat milk Nutrition 0.000 claims description 5
- 235000017060 Arachis glabrata Nutrition 0.000 claims description 4
- 244000105624 Arachis hypogaea Species 0.000 claims description 4
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 4
- 235000018262 Arachis monticola Nutrition 0.000 claims description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 235000012054 meals Nutrition 0.000 claims description 4
- 235000020232 peanut Nutrition 0.000 claims description 4
- 235000020183 skimmed milk Nutrition 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 2
- 238000007711 solidification Methods 0.000 claims description 2
- 230000008023 solidification Effects 0.000 claims description 2
- 238000003805 vibration mixing Methods 0.000 claims 1
- 239000006041 probiotic Substances 0.000 abstract description 16
- 235000018291 probiotics Nutrition 0.000 abstract description 16
- 230000000529 probiotic effect Effects 0.000 abstract description 15
- 239000003814 drug Substances 0.000 abstract description 10
- 235000013305 food Nutrition 0.000 abstract description 10
- 239000002775 capsule Substances 0.000 abstract description 4
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- 229930014626 natural product Natural products 0.000 abstract description 2
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- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- 231100000719 pollutant Toxicity 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 13
- 230000001681 protective effect Effects 0.000 description 8
- 241000726221 Gemma Species 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 241000276408 Bacillus subtilis subsp. subtilis str. 168 Species 0.000 description 5
- 235000019628 coolness Nutrition 0.000 description 5
- 230000000968 intestinal effect Effects 0.000 description 4
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- 244000005700 microbiome Species 0.000 description 3
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
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- 108010046845 tryptones Proteins 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 244000215068 Acacia senegal Species 0.000 description 1
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- 108010062877 Bacteriocins Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
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- 229920002148 Gellan gum Polymers 0.000 description 1
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- 241001465754 Metazoa Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- ZNOZWUKQPJXOIG-XSBHQQIPSA-L [(2r,3s,4r,5r,6s)-6-[[(1r,3s,4r,5r,8s)-3,4-dihydroxy-2,6-dioxabicyclo[3.2.1]octan-8-yl]oxy]-4-[[(1r,3r,4r,5r,8s)-8-[(2s,3r,4r,5r,6r)-3,4-dihydroxy-6-(hydroxymethyl)-5-sulfonatooxyoxan-2-yl]oxy-4-hydroxy-2,6-dioxabicyclo[3.2.1]octan-3-yl]oxy]-5-hydroxy-2-( Chemical compound O[C@@H]1[C@@H](O)[C@@H](OS([O-])(=O)=O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H]2OC[C@H]1O[C@H](O[C@H]1[C@H]([C@@H](CO)O[C@@H](O[C@@H]3[C@@H]4OC[C@H]3O[C@H](O)[C@@H]4O)[C@@H]1O)OS([O-])(=O)=O)[C@@H]2O ZNOZWUKQPJXOIG-XSBHQQIPSA-L 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
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- 210000004051 gastric juice Anatomy 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 235000020252 horse milk Nutrition 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 235000008476 powdered milk Nutrition 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
- 239000000230 xanthan gum Substances 0.000 description 1
- 235000010493 xanthan gum Nutrition 0.000 description 1
- 229940082509 xanthan gum Drugs 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
-
- 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
- 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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Polymers & Plastics (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to probiotic microcapsule preparing technical field, specifically there is in a kind of shelf-lives the preparation method of the Bacillus subtilus microcapsule of high viable count.The present invention is optimized control to the temperature in enlarged culturing process, dissolved oxygen, pH, time and substratum, adopts bilayered microcapsule in conjunction with vacuum packaging in capsule manufacturing process.The Bacillus subtilus microcapsule produced have the advantage of high viable count, high resistance to cold and diseases in shelf-lives.Its product not only can be applicable to field of health care food, also can be applicable to field of medicaments.Bacterial strain of the present invention is probiotic bacterium, and raw material is nontoxic natural compounds, can realize non-pollutant discharge, green, safety, environmental protection.
Description
Technical field
The present invention relates to probiotic microcapsule preparing technical field, specifically there is in a kind of shelf-lives the preparation method of the Bacillus subtilus microcapsule of high viable count.
Background technology
Bacillus subtilus (Bacillussubtilis) is a kind of aerobic gram-positive microorganism, amphitrichous.The statospore of Bacillus subtilus is high temperature resistant (120 ° of C can survive 20min), acid and alkali-resistance (viable bacteria of 100% through intestine and small intestine).After entering digestive tube, to be brought back to life by dormant state rapidly in a short time, be multiplied into the dominant population of high bacteria containing amount.And then the oxygen consumed in enteron aisle, produce hydrogen peroxide, bacteriocin, promote the breeding of useful anaerobion, suppress the growth of unwanted bacteria (intestinal bacteria, Salmonella), set up microecological balance.Thus the pre-intestines and stomach such as anti-diarrhea, diarrhea disease.Bacillus subtilus belongs to the beneficial microorganism of security on clinical medicine, in protective foods, belong to probiotic bacterium.
Probiotic bacterium is colonizated in human intestinal, in reproductive system, can produces the beneficial microorganism general name of the life of definite health efficacy.Along with going deep into of understanding probiotic bacterium people, pay attention to and study, probiotic bacterium just extends from health care to medical field.But, probiotic composition is being crossed digestive tube arrive in the process of enteron aisle from production, preservation, transport, oral administration, need the impact standing a series of poor environment factor, cause the number of viable arriving enteron aisle to greatly reduce, limit the performance of probiotic bacterium physiological function.Probiotic bacterium is embedded in the impact that can alleviate environment undesirable element in microcapsule, thus improves the survival rate of probiotic bacterium.
The material that can be used as microcapsule wall material is a lot.There are natural polymers, semi-synthetic high molecular polymer and synthesising macromolecule copolymer, mainly comprise: carbohydrate, as sodium alginate, chitosan, starch; Vegetable jelly class, as gum arabic, kappa-carrageenan; Animal proteinum, as milk, gelatin; Bacterial polysaccharides, as gellan, xanthan gum.The wall material being used for embedding probiotic bacterium should possess following feature usually: good biocompatibility and film-forming properties, suitable viscosity, perviousness, wetting ability and solvability etc.; And there is stable in properties, can coordinate with core, can biological degradation etc.Many reports are had to the research of probiotic microcapsule, but also have a lot of deficiency in aerobic probiotic microcapsule, as: the problem that poor heat resistance, ventilation property are strong and survival time is short.The main outlet addressed these problems is the resistance and the capsule tolerance that improve bacterial strain.
Summary of the invention
The present invention discloses the preparation method of the Bacillus subtilus microcapsule in a kind of shelf-lives with high viable count, be intended to the resistance, the preservation term that improve Bacillus subtilus, significantly reduce the mortality ratio of viable bacteria through digestive tube, make viable bacteria as much as possible arrive intestinal colonisation, play prebiotic health-care effect.
For solving the problems of the technologies described above, the present invention by the following technical solutions.
There is in shelf-lives a preparation method for the Bacillus subtilus microcapsule of high viable count, it is characterized in that, comprise the following steps:
(1) activation of bacterial strain: the single colony inoculation of the Bacillus subtilus being less than month will be preserved under the condition of about 4 DEG C in seed culture medium, at 32-45 DEG C, cultivate 12-24h under 100-300rpm condition and carry out primary activation, then the inoculum size second time of 1-5% is pressed by the Bacillus subtilus nutrient solution of primary activation access seed culture medium, cultivate 4-10h under the same conditions again to activate, obtain activated seed liquid;
(2) enlarged culturing: activated seed liquid is inoculated in the fermentor tank of the milk powder be equipped with-dregs of beans aseptic culture medium of pH6.5-8.5 by the inoculum size of 1-10%, at 35-45 DEG C, Rong Xie Yang≤35% condition bottom fermentation 18-24h, continuation to endogenous spore generation Shuai≤50%, obtains fermented liquid at 25-35 DEG C of fermentation 18-48h;
(3) wash: by fermented liquid centrifugal 5-30min under 3000-10000rpm speed, remove supernatant liquor, the vibration of the thalline sterilized water of precipitation is mixed by the ratio of 1:1-5 by volume, centrifugal segregation supernatant liquor under condition as hereinbefore, repetitive scrubbing 1-3 time, use 1-5% aseptic milk powder solution Eddy diffusion thalline subsequently, its viable count>=10
10cfu/ml, obtains bacteria suspension;
(4) Single-layer microcapsules preparation: in 1-5%(W/V) ratio by the sodium alginate of aseptically process, 0.1-1%(W/V) forulic acid and 1-2%(W/V) glycerine add in bacteria suspension, under aseptic condition, abundant stirring and evenly mixing, obtains bacterium glue; With 10-30ml/min flow velocity syringe pump bacterium glue injected the 1-10%(M/V of aseptic 1-10 DEG C of cooling) calcium chloride solution, dropping limit, limit is stirred under 100--300rpm, solidification 30-60min, and sterilized water washs three times, obtains individual layer and to wet microcapsule;
(5) bilayered microcapsule preparation: wet gained individual layer microcapsule and overlay film 30-60min in the 0.5-5% chitosan of use aseptically process, and sterilized water washs three times, obtains double-deck wet microcapsule;
(6) drying preservation: the microcapsule that wet by bilayer aseptically carry out vacuum packaging after carrying out low-temperature vacuum drying, obtains the dry microcapsule of Bacillus subtilus.
State the preparation method of Bacillus subtilus microcapsule, it is characterized in that, carrying out low-temperature vacuum drying to wet microcapsule double-deck described in step (5) is 40
oany one drying mode of below C, and vacuum packaging.
The preparation method of described Bacillus subtilus microcapsule, is characterized in that, the B. subtilis strain described in step (1) refer in wild type B, engineered Bacillus subtilus one or both.
The preparation method of described Bacillus subtilus microcapsule, is characterized in that, the described milk powder raw material of step (2) is any one in whole milk, skimmed milk, goat milk and mare's milk, and described dregs of beans is any one of corn-soybean meal, soybean meal and peanut meal.
The preparation method of described Bacillus subtilus microcapsule, is characterized in that, described aseptic procedure is any one in high-temperature sterilization and filtration sterilization.
The preparation method of described Bacillus subtilus microcapsule, is characterized in that, the sterilized water in step (3) (4) (5) is any one of physiological saline, phosphoric acid buffer, tap water and distilled water.
A preparation method for Bacillus subtilus microcapsule, comprises the following steps:
(1) activation of bacterial strain: the single colony inoculation of Bacillus subtilus 4 DEG C of preservations being less than month, in seed culture medium, is cultivated 12-24h and carried out primary activation under 32-45 DEG C, 100-300rpm condition; Then press in the inoculum size second time access seed culture medium of 1-5%, cultivate 4-10h under the same conditions and again activate, obtain activated seed liquid;
Described B. subtilis strain is any one in wild type B and engineered Bacillus subtilus; Described seed culture medium is improvement LB substratum: 10g Tryptones, 10g sodium-chlor, and 5g yeast extract powder, 5g glycerine, adding water is settled to 1000mL, and adjust pH to 7.0-7.4, packing (solid medium adds 1.5% agar), 115
osterilizing 20min under C, agents useful for same is analytical pure.
(2) enlarged culturing: activated seed liquid is inoculated in by the inoculum size of 1-10% in the fermentor tank of aseptic milk powder-dregs of beans substratum that pH6.5-8.5 is housed, at 35-45 DEG C, Rong Xie Yang≤35% condition bottom fermentation 18-24h, continuation to endogenous spore generation Shuai≤50%, obtains fermented liquid at 25-35 DEG C of fermentation 18-48h;
Described milk powder is any one in whole milk, skimmed milk, goat milk and mare's milk, and described dregs of beans is any one of corn-soybean meal, soybean meal and peanut meal, and described aseptic procedure is any one in high-temperature sterilization and filtration sterilization.Milk powder-dregs of beans substratum: cross 100 mesh sieve bean cake powder 10-20 gram and be dissolved in 1000 ml waters, 60 DEG C are incubated 30 minutes, get supernatant and obtain dregs of beans substratum after the centrifugal 10min of 4000 turns/min; Add in 20-200 gram of milk powder to dregs of beans substratum and obtain milk powder-dregs of beans substratum.
(3) wash: by fermented liquid centrifugal 5-30min under 3000-10000rpm speed, remove supernatant, the vibration of the thalline sterilized water of precipitation is mixed by the ratio of 1:1-5 by volume, centrifugal segregation supernatant liquor under condition as hereinbefore, repetitive scrubbing 1-3 time, uses the aseptic milk powder solution Eddy diffusion thalline of 1-5% (viable count>=10 subsequently
10cfu/ml), bacteria suspension is obtained.
Described sterilized water is any one of physiological saline, phosphoric acid buffer, tap water and distilled water.
(4) Single-layer microcapsules preparation: in 1-5%(W/V) ratio by sodium alginate, 0.1-1%(W/V) forulic acid and 1-2%(W/V) glycerine adds in bacteria suspension, under aseptic condition, abundant stirring and evenly mixing, obtains bacterium glue; With 10-30ml/min flow velocity syringe pump bacterium glue injected the 1-10%(M/V of aseptic 1-10 DEG C of cooling) calcium chloride solution, drip while add stirrings (100--300rpm), solidify 30-60min, sterilized water washs three times, obtains individual layer and to wet microcapsule;
(5) bilayered microcapsule preparation: overlay film 30-60min in gained individual layer is wet microcapsule and 0.5-5% chitosan, sterilized water washs three times, obtains double-deck wet microcapsule;
(6) drying preservation: the microcapsule that wet by bilayer aseptically carry out vacuum packaging after carrying out low-temperature vacuum drying, obtains the dry microcapsule of Bacillus subtilus.
Can be N for the gas of deoxidation in described vacuum packaging
2or N
2, CO
2, H
2gas mixture.
Bacillus subtilus microcapsule prepared by the preparation method of the above Bacillus subtilus microcapsule are applied to protective foods and field of medicaments.
The invention has the beneficial effects as follows: adopt the culture condition optimized when enlarged culturing, comprise temperature, dissolved oxygen, pH, time and substratum, particularly two temperature section cultivation, makes it cell amount reproduction and spore is formed in a large number; In capsule preparation, adopt bilayered microcapsule to make cell be in good dormancy environment in conjunction with vacuum packaging.Improve resistance, the preservation term of probiotics viable bacteria, significantly reduce the destruction of viable bacteria through stomach gastric juice, make viable bacteria as much as possible arrive intestinal colonisation, play prebiotic nourishing function.
The present invention has the advantage of high viable count, high resistance to cold and diseases in shelf-lives by the Bacillus subtilus microcapsule that integrator cell physiological characteristic and capsule technology of preparing are produced.Its product not only can be applicable to field of health care food, also can be applicable to field of medicaments.Bacterial strain of the present invention is probiotic bacterium, and raw material is nontoxic natural compounds, can realize non-pollutant discharge, green, safety, environmental protection.
Embodiment
In order to more fully understand technology contents of the present invention, below in conjunction with specific embodiment, technical scheme of the present invention being described further and being illustrated.
Following examples use following material:
Water used all meets country " drinking water sanitary standard " (GB5749-2006).
Wild mushroom: subtilis
b.subtilis168.
Engineering bacteria: subtilis
b.subtilisiBL241 [WB800N/pHCMC05-svu022].
Culture medium raw material: whole milk powder, Nestle full cream powdered milk, Nestle Shuangcheng Ltd; Dregs of beans, soybean meal, Shandong Jia Guan oil and fat chemical company limited.
Seed culture medium is improvement LB seed culture medium (1L): 10g Tryptones, 10g sodium-chlor, 5g yeast extract powder, 5g glycerine, and adding water is settled to 1000mL, and adjust pH to 7.0-7.4, packing (solid medium adds 1.5% agar), 115
osterilizing 20min under C, agents useful for same is analytical pure.
Embodiment 1
A preparation method for Bacillus subtilus microcapsule, comprises the following steps:
(1) activation of bacterial strain: by the Bacillus subtilus being stored in 4 DEG C of preservations and being less than month
b.subtilis168 single colony inoculations, in seed culture medium, at 37 DEG C, are cultivated 16-20h and are carried out primary activation under 200rpm condition; Then by the inoculum size second time access seed culture medium of 2%, cultivate 4-10h under the same conditions and again activate, obtain activated seed liquid;
(2) enlarged culturing: activated seed liquid is inoculated in the fermentor tank of the substratum (pH7.5) of 115 DEG C of high-temperature sterilizations that milk powder-dregs of beans (5% skim-milk and the centrifugal supernatant of 1% soybean meal 4000rpm) is housed by the inoculum size of 4-6%, at 37 DEG C, Rong Xie Yang≤30% condition bottom fermentation 20-24h, continue to be formed in a large number (gemma generation Shuai≤50%) to endogenous spore at 25 DEG C of fermentation 24-36h, obtain fermented liquid;
(3) wash: by fermented liquid centrifugal 15min under 6000rpm speed, remove supernatant, the vibration of the thalline aseptic water of precipitation is mixed by the ratio of 1:2 by volume, centrifugal segregation supernatant liquor under condition as hereinbefore, repetitive scrubbing 2 times, uses 1% sterile absorbent milk powder solution Eddy diffusion thalline (viable count>=10 subsequently
10cfu/ml), bacteria suspension is obtained;
(4) Single-layer microcapsules preparation: in 2%(W/V) ratio by sodium alginate, 0.5%(W/V) forulic acid and 1%(W/V) glycerine adds in bacteria suspension, under aseptic condition, abundant stirring and evenly mixing, obtains bacterium glue; With 20ml/min flow velocity syringe pump bacterium glue injected the 5%(M/V of 4 DEG C of aseptic coolings) calcium chloride solution, drip while add stirrings (200rpm), solidify 40min, aseptic water washs three times, obtains individual layer and to wet microcapsule;
(5) bilayered microcapsule preparation: overlay film 45min in gained individual layer is wet microcapsule and 1% chitosan, aseptic water washs three times, obtains double-deck wet microcapsule;
(6) drying preservation: the microcapsule that wet by bilayer aseptically carry out vacuum packaging after carrying out low-temperature vacuum drying, obtains the dry microcapsule of Bacillus subtilus.
The dry microcapsule product of this Bacillus subtilus quality guaranteed period>=2 years, viable count>=1 × 10
9individual/g, Ya Bao≤60%; Faint yellow, free from extraneous odour.
The more than obtained dry microcapsule product of Bacillus subtilus can be applicable to protective foods and field of medicaments.
Embodiment 2
A preparation method for Bacillus subtilus microcapsule, comprises the following steps:
(1) activation of bacterial strain: 4 DEG C of preservations will be stored in and be less than a month Bacillus subtilus
b.subtilis168 single colony inoculations, in seed culture medium, at 39 DEG C, are cultivated 16-20h and are carried out primary activation under 200rpm condition; Then by the inoculum size second time access seed culture medium of 2%, cultivate 4-10h under the same conditions and again activate, obtain activated seed liquid;
(2) enlarged culturing: activated seed liquid is inoculated in the fermentor tank of the substratum (pH8.0) of 115 DEG C of high-temperature sterilizations that milk powder-dregs of beans (5% whole milk powder and 1% corn-soybean meal) is housed by the inoculum size of 4-6%, at 39 DEG C, Rong Xie Yang≤40% condition bottom fermentation 20-24h, continue to be formed in a large number (gemma generation Shuai≤50%) to endogenous spore at 30 DEG C of fermentation 36-48h, obtain fermented liquid;
(3) wash: by fermented liquid centrifugal 20min under 4000rpm speed, remove supernatant, the vibration of the thalline stroke-physiological saline solution (0.9%NaCl) of precipitation is mixed by the ratio of 1:3 by volume, centrifugal segregation supernatant liquor under condition as hereinbefore, repetitive scrubbing 2 times, uses 1% aseptic whole milk powder liquid Eddy diffusion thalline (viable count>=10 subsequently
10cfu/ml), bacteria suspension is obtained;
(4) Single-layer microcapsules preparation: in 3%(W/V) ratio by sodium alginate, 0.5%(W/V) forulic acid and 2%(W/V) glycerine adds in bacteria suspension, under aseptic condition, abundant stirring and evenly mixing, obtains bacterium glue; With 20ml/min flow velocity syringe pump bacterium glue injected the 4%(M/V of 4 DEG C of aseptic coolings) calcium chloride solution, drip while add stirrings (200rpm), solidify 40min, stroke-physiological saline solution washs three times, obtains individual layer and to wet microcapsule;
(5) bilayered microcapsule preparation: overlay film 45min in gained individual layer is wet microcapsule and 2% chitosan, stroke-physiological saline solution washs three times, obtains double-deck wet microcapsule;
(6) drying preservation: the microcapsule that wet by bilayer aseptically carry out vacuum packaging after carrying out low-temperature vacuum drying, obtains the dry microcapsule of Bacillus subtilus.
The dry microcapsule product of this Bacillus subtilus quality guaranteed period>=2 years, viable count>=1 × 10
10individual/g, Ya Bao≤80%; Faint yellow, free from extraneous odour.
The more than obtained dry microcapsule product of Bacillus subtilus can be applicable to protective foods and field of medicaments.
Embodiment 3
A preparation method for Bacillus subtilus microcapsule, comprises the following steps:
(1) activation of bacterial strain: by the Bacillus subtilus being stored in 4 DEG C of preservations and being less than month
b.subtilis168 single colony inoculations, in seed culture medium, at 37 DEG C, are cultivated 16-20h and are carried out primary activation under 200rpm condition; Then by the inoculum size second time access seed culture medium of 2%, cultivate 4-10h under the same conditions and again activate, obtain activated seed liquid;
(2) enlarged culturing: activated seed liquid is inoculated in the fermentor tank of the substratum (pH7.0) of 115 DEG C of high-temperature sterilizations that milk powder-dregs of beans (10% whole milk powder and 1% corn-soybean meal) is housed by the inoculum size of 4-6%, at 39 DEG C, Rong Xie Yang≤30% condition bottom fermentation 20-24h, continue to be formed in a large number (gemma generation Shuai≤50%) to endogenous spore at 28 DEG C of fermentation 36-48h, obtain fermented liquid;
(3) wash: by fermented liquid centrifugal 20min under 4000rpm speed, remove supernatant, the vibration of the thalline aseptic water of precipitation is mixed by the ratio of 1:3 by volume, centrifugal segregation supernatant liquor under condition as hereinbefore, repetitive scrubbing 2 times, uses 1% aseptic whole milk powder liquid Eddy diffusion thalline (viable count>=10 subsequently
10cfu/ml), bacteria suspension is obtained;
(4) Single-layer microcapsules preparation: in 3%(W/V) ratio by sodium alginate, 0.5%(W/V) forulic acid and 1%(W/V) glycerine adds in bacteria suspension, under aseptic condition, abundant stirring and evenly mixing, obtains bacterium glue; With 20ml/min flow velocity syringe pump bacterium glue injected the 3%(M/V of 4 DEG C of aseptic coolings) calcium chloride solution, drip while add stirrings (200rpm), solidify 40min, aseptic water washs three times, obtains individual layer and to wet microcapsule;
(5) bilayered microcapsule preparation: overlay film 45min in gained individual layer is wet microcapsule and 2% chitosan, aseptic water washs three times, obtains double-deck wet microcapsule;
(6) drying preservation: the microcapsule that wet by bilayer aseptically carry out vacuum packaging after carrying out low-temperature vacuum drying, obtains the dry microcapsule of Bacillus subtilus.
The dry microcapsule product of this Bacillus subtilus quality guaranteed period>=2 years, viable count>=2 × 10
10individual/g, Ya Bao≤80%; Faint yellow, free from extraneous odour.
The more than obtained dry microcapsule product of Bacillus subtilus can be applicable to protective foods and field of medicaments.
Embodiment 4
A preparation method for Bacillus subtilus microcapsule, comprises the following steps:
(1) activation of bacterial strain: by the Bacillus subtilus being stored in 4 DEG C of preservations and being less than month
b.subtilis241 single colony inoculations, in seed culture medium, at 37 DEG C, are cultivated 16-20h and are carried out primary activation under 200rpm condition; Then by the inoculum size second time access seed culture medium of 2%, cultivate 4-10h under the same conditions and again activate, obtain activated seed liquid;
(2) enlarged culturing: activated seed liquid is inoculated in the fermentor tank of the substratum (pH7.0) of the 110-115 DEG C of high-temperature sterilization that milk powder-dregs of beans (10% whole milk powder and 2% corn-soybean meal) is housed by the inoculum size of 4-6%, at 39 DEG C, Rong Xie Yang≤30% condition bottom fermentation 20-24h, continue to be formed in a large number (gemma generation Shuai≤50%) to endogenous spore at 28 DEG C of fermentation 36-48h, obtain fermented liquid;
(3) wash: by fermented liquid centrifugal 20min under 4000rpm speed, remove supernatant, the vibration of the thalline aseptic water of precipitation is mixed by the ratio of 1:3 by volume, centrifugal segregation supernatant liquor under condition as hereinbefore, repetitive scrubbing 2 times, uses 1% aseptic whole milk powder liquid Eddy diffusion thalline (viable count>=10 subsequently
10cfu/ml), bacteria suspension is obtained;
(4) Single-layer microcapsules preparation: in 2.5%(W/V) ratio by sodium alginate, 0.5%(W/V) forulic acid and 1%(W/V) glycerine adds in bacteria suspension, under aseptic condition, abundant stirring and evenly mixing, obtains bacterium glue; With 20ml/min flow velocity syringe pump bacterium glue injected the 3%(M/V of 4 DEG C of aseptic coolings) calcium chloride solution, drip while add stirrings (200rpm), solidify 60min, aseptic water washs three times, obtains individual layer and to wet microcapsule;
(5) bilayered microcapsule preparation: overlay film 45min in gained individual layer is wet microcapsule and 2% chitosan, aseptic water washs three times, obtains double-deck wet microcapsule;
(6) drying preservation: the microcapsule that wet by bilayer aseptically carry out vacuum packaging after carrying out low-temperature vacuum drying, obtains the dry microcapsule of Bacillus subtilus.
The dry microcapsule product of this Bacillus subtilus quality guaranteed period>=2 years, viable count>=5 × 10
10individual/g, Ya Bao≤90%; Faint yellow, free from extraneous odour.
The more than obtained dry microcapsule product of Bacillus subtilus can be applicable to protective foods and field of medicaments.
Embodiment 5
A preparation method for Bacillus subtilus microcapsule, comprises the following steps:
(1) activation of bacterial strain: by the Bacillus subtilus being stored in 4 DEG C of preservations and being less than month
b.subtilis168 single colony inoculations, in seed culture medium, at 32 DEG C, are cultivated 24h and are carried out primary activation under 100rpm condition; Then by the inoculum size second time access seed culture medium of 1%, cultivate 10h under the same conditions and again activate, obtain activated seed liquid;
(2) enlarged culturing: by activated seed liquid by 1% inoculum size be inoculated in the fermentor tank of the substratum (pH6.5) of the 110-115 DEG C of high-temperature sterilization that milk powder-dregs of beans (2% goat milk powder and 1% soybean meal) is housed, at 35 DEG C, Rong Xie Yang≤35% condition bottom fermentation 24h, continue to be formed in a large number (gemma generation Shuai≤50%) to endogenous spore at 25 DEG C of fermentation 48h, obtain fermented liquid;
(3) wash: by fermented liquid centrifugal 30min under 3000rpm speed, remove supernatant, the vibration of the thalline sterile distilled water of precipitation is mixed by the ratio of 1:1 by volume, centrifugal segregation supernatant liquor under condition as hereinbefore, repetitive scrubbing 1 time, uses 1% aseptic goat milk powder liquid Eddy diffusion thalline (viable count>=10 subsequently
9cfu/ml), bacteria suspension is obtained;
(4) Single-layer microcapsules preparation: in 1%(W/V) ratio by sodium alginate, 0.1%(W/V) forulic acid and 1%(W/V) glycerine adds in bacteria suspension, under aseptic condition, abundant stirring and evenly mixing, obtains bacterium glue; With 10ml/min flow velocity syringe pump bacterium glue injected the 1%(M/V of 1 DEG C of aseptic cooling) calcium chloride solution, drip while add stirrings (100rpm), solidify 30min, sterile distilled water washs three times, obtains individual layer and to wet microcapsule;
(5) bilayered microcapsule preparation: overlay film 60min in gained individual layer is wet microcapsule and 0.5% chitosan, sterile distilled water washs three times, obtains double-deck wet microcapsule;
(6) drying preservation: the microcapsule that wet by bilayer aseptically carry out vacuum packaging after carrying out low-temperature vacuum drying, obtains the dry microcapsule of Bacillus subtilus.
The dry microcapsule product of this Bacillus subtilus quality guaranteed period>=2 years, viable count>=1 × 10
8individual/g, Ya Bao≤70%; Faint yellow, free from extraneous odour.
The more than obtained dry microcapsule product of Bacillus subtilus can be applicable to protective foods and field of medicaments.
Embodiment 6
A preparation method for Bacillus subtilus microcapsule, comprises the following steps:
(1) activation of bacterial strain: by the Bacillus subtilus being stored in 4 DEG C of preservations and being less than month
b.subtilisthe mono-colony inoculation of IBL241, in seed culture medium, at 45 DEG C, is cultivated 12h and is carried out primary activation under 300rpm condition; Then by the inoculum size second time access seed culture medium of 5%, cultivate 4h under the same conditions and again activate, obtain activated seed liquid;
(2) enlarged culturing: by activated seed liquid by 10% inoculum size be inoculated in the fermentor tank of the substratum (pH6.5) after the filtration sterilization that milk powder-dregs of beans (20% mare's milk powder and 2% peanut meal) is housed, at 45 DEG C, Rong Xie Yang≤35% condition bottom fermentation 18h, continue to be formed in a large number (gemma generation Shuai≤50%) to endogenous spore at 35 DEG C of fermentation 18h, obtain fermented liquid;
(3) wash: by fermented liquid centrifugal 5min under 10000rpm speed, remove supernatant, the vibration of the thalline sterile phosphate buffer of precipitation is mixed by the ratio of 1:5 by volume, centrifugal segregation supernatant liquor under condition as hereinbefore, repetitive scrubbing 3 times, uses 5% aseptic horse milk powder solution Eddy diffusion thalline (viable count>=10 subsequently
9cfu/ml), bacteria suspension is obtained;
(4) Single-layer microcapsules preparation: in 5%(W/V) ratio by sodium alginate, 1%(W/V) forulic acid and 2%(W/V) glycerine adds in bacteria suspension, under aseptic condition, abundant stirring and evenly mixing, obtains bacterium glue; With 30ml/min flow velocity syringe pump bacterium glue injected the 10%(M/V of 10 DEG C of aseptic coolings) calcium chloride solution, drip while add stirrings (300rpm), solidify 60min, sterile phosphate buffer washs three times, obtains individual layer and to wet microcapsule;
(5) bilayered microcapsule preparation: overlay film 30min in gained individual layer is wet microcapsule and 5% chitosan, sterile phosphate buffer washs three times, obtains double-deck wet microcapsule;
(6) drying preservation: the microcapsule that wet by bilayer aseptically carry out vacuum packaging after carrying out low-temperature vacuum drying, obtains the dry microcapsule of Bacillus subtilus.
The dry microcapsule product of this Bacillus subtilus quality guaranteed period>=2 years, viable count>=1 × 10
9individual/g, Ya Bao≤80%; Faint yellow, free from extraneous odour.
The more than obtained dry microcapsule product of Bacillus subtilus can be applicable to protective foods and field of medicaments.
The above only further illustrates technology contents of the present invention with embodiment, so that reader is easier to understand, but does not represent embodiments of the present invention and is only limitted to this, and any technology done according to the present invention extends or recreation, all by protection of the present invention.
Claims (6)
1. there is in a shelf-lives preparation method of the Bacillus subtilus microcapsule of high viable count, it is characterized in that, comprise the following steps: the activation of (1) bacterial strain: the single colony inoculation of the Bacillus subtilus being less than month will be preserved under the condition of 4 DEG C in seed culture medium, at 32-45 DEG C, cultivate 12-24h under 100-300rpm condition and carry out primary activation, then the inoculum size second time of 1-5% is pressed by the Bacillus subtilus nutrient solution of primary activation access seed culture medium, cultivate 4-10h under the same conditions again to activate, obtain activated seed liquid;
(2) enlarged culturing: what activated seed liquid was inoculated in pH6.5-8.5 by the inoculum size of 1-10% is equipped with in the fermentor tank of milk powder-dregs of beans aseptic culture medium, at 35-45 DEG C, dissolved oxygen >=35% condition bottom fermentation 18-24h, continuation to endogenous spore production rate >=50%, obtains fermented liquid at 25-35 DEG C of fermentation 18-48h;
(3) wash: by fermented liquid centrifugal 5-30min under 3000-10000rpm speed, remove supernatant liquor, the ratio of 1:1-5 is by the sterilized water vibration mixing of the thalline of precipitation by volume, centrifugal segregation supernatant liquor under condition as hereinbefore, repetitive scrubbing 1-3 time, use 1-5% aseptic milk powder solution Eddy diffusion thalline subsequently, its viable count>=10
10cfu/ml, obtains bacteria suspension;
(4) Single-layer microcapsules preparation: in 1-5%(W/V) ratio by the sodium alginate of aseptically process, 0.1-1%(W/V) forulic acid and 1-2%(W/V) glycerine add in bacteria suspension, under aseptic condition, abundant stirring and evenly mixing, obtains bacterium glue; With 10-30ml/min flow velocity syringe pump bacterium glue injected the 1-10%(M/V of aseptic 1-10 DEG C of cooling) calcium chloride solution, dropping limit, limit is stirred under 100--300rpm, solidification 30-60min, and sterilized water washs three times, obtains individual layer and to wet microcapsule;
(5) bilayered microcapsule preparation: carry out overlay film 30-60min with the 0.5-5% chitosan after aseptically process to the gained individual layer microcapsule that wet, sterilized water washs three times, obtains double-deck wet microcapsule;
(6) drying preservation: the microcapsule that wet by bilayer aseptically carry out vacuum packaging after carrying out low-temperature vacuum drying, obtains the dry microcapsule of Bacillus subtilus.
2. the preparation method of Bacillus subtilus microcapsule according to claim 1, is characterized in that, carries out to wet microcapsule double-deck described in step (5) any one drying mode that low-temperature vacuum drying is less than 40 DEG C, and vacuum packaging.
3. the preparation method of Bacillus subtilus microcapsule according to claim 1, is characterized in that, the B. subtilis strain described in step (1) refer in wild type B, engineered Bacillus subtilus one or both.
4. the preparation method of Bacillus subtilus microcapsule according to claim 3, it is characterized in that, the described milk powder raw material of step (2) is any one in whole milk, skimmed milk, goat milk and mare's milk, and described dregs of beans is any one of corn-soybean meal, soybean meal and peanut meal.
5. the preparation method of Bacillus subtilus microcapsule according to claim 3, is characterized in that, described aseptic procedure is any one in high-temperature sterilization and filtration sterilization.
6. the preparation method of Bacillus subtilus microcapsule according to claim 4 or 5, is characterized in that, the sterilized water in step (3) (4) (5) is any one of physiological saline, phosphoric acid buffer, tap water and distilled water.
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| RU2781792C1 (en) * | 2021-05-13 | 2022-10-18 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Курская государственная сельскохозяйственная академия имени И.И. Иванова" | Method for producing microcapsules of vetom 1 probiotic |
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| CN104431370A (en) * | 2014-12-15 | 2015-03-25 | 南京优帆生物科技有限公司 | Efficient probiotics microcapsule as well as preparation method and application thereof |
| CN104970368B (en) * | 2015-06-30 | 2017-11-17 | 安徽大学 | A kind of preparation method of the stomach juice-resistant type bacillus licheniformis microcapsules with high viability |
| CN105169362A (en) * | 2015-09-02 | 2015-12-23 | 钟华 | Biopharmaceutical method for intestinal diseases |
| CN106343578A (en) * | 2016-08-29 | 2017-01-25 | 河北然生物科技有限公司 | Enteric double-layer seamless solid probiotic microspheres and preparation method and application thereof |
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