CN103704719B - A kind of preparation method of the probiotic microcapsule with high viable count - Google Patents
A kind of preparation method of the probiotic microcapsule with high viable count Download PDFInfo
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- CN103704719B CN103704719B CN201310715323.4A CN201310715323A CN103704719B CN 103704719 B CN103704719 B CN 103704719B CN 201310715323 A CN201310715323 A CN 201310715323A CN 103704719 B CN103704719 B CN 103704719B
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- Prior art keywords
- bifidobacterium
- viable count
- preparation
- bacterium
- probiotic microcapsule
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- 239000003094 microcapsule Substances 0.000 title claims abstract description 71
- 239000006041 probiotic Substances 0.000 title claims abstract description 36
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 36
- 230000000529 probiotic effect Effects 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 241000186000 Bifidobacterium Species 0.000 claims abstract description 51
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 42
- 241000894006 Bacteria Species 0.000 claims description 104
- 239000003292 glue Substances 0.000 claims description 37
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 36
- 239000001963 growth medium Substances 0.000 claims description 34
- 230000004913 activation Effects 0.000 claims description 30
- 230000001580 bacterial effect Effects 0.000 claims description 22
- 238000005406 washing Methods 0.000 claims description 21
- 238000011218 seed culture Methods 0.000 claims description 20
- 239000006228 supernatant Substances 0.000 claims description 19
- 239000000725 suspension Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 235000011187 glycerol Nutrition 0.000 claims description 18
- 239000012530 fluid Substances 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 16
- 238000003756 stirring Methods 0.000 claims description 16
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 11
- 235000015097 nutrients Nutrition 0.000 claims description 11
- 238000004321 preservation Methods 0.000 claims description 11
- 238000001291 vacuum drying Methods 0.000 claims description 11
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 10
- 238000000855 fermentation Methods 0.000 claims description 10
- 230000004151 fermentation Effects 0.000 claims description 10
- 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 9
- 229920001661 Chitosan Polymers 0.000 claims description 9
- 244000000626 Daucus carota Species 0.000 claims description 9
- 235000002767 Daucus carota Nutrition 0.000 claims description 9
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims description 9
- 238000001816 cooling Methods 0.000 claims description 9
- 125000004122 cyclic group Chemical group 0.000 claims description 9
- 239000008246 gaseous mixture Substances 0.000 claims description 9
- 235000013336 milk Nutrition 0.000 claims description 9
- 239000008267 milk Substances 0.000 claims description 9
- 210000004080 milk Anatomy 0.000 claims description 9
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 9
- 229920000053 polysorbate 80 Polymers 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 9
- 235000010413 sodium alginate Nutrition 0.000 claims description 9
- 239000000661 sodium alginate Substances 0.000 claims description 9
- 229940005550 sodium alginate Drugs 0.000 claims description 9
- 150000001875 compounds Chemical class 0.000 claims description 7
- 238000010353 genetic engineering Methods 0.000 claims description 6
- 102000004407 Lactalbumin Human genes 0.000 claims description 5
- 108090000942 Lactalbumin Proteins 0.000 claims description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 235000008939 whole 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
- 235000020251 goat milk Nutrition 0.000 claims description 4
- 239000000413 hydrolysate Substances 0.000 claims description 4
- 235000020232 peanut Nutrition 0.000 claims description 4
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 2
- 230000003213 activating effect Effects 0.000 claims 1
- 238000009461 vacuum packaging Methods 0.000 claims 1
- 239000002775 capsule Substances 0.000 abstract description 13
- 230000012010 growth Effects 0.000 abstract description 10
- 230000036541 health Effects 0.000 abstract description 4
- 238000009395 breeding Methods 0.000 abstract description 3
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- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 3
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- 239000003102 growth factor Substances 0.000 abstract description 2
- 238000011534 incubation Methods 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 9
- 229910052760 oxygen Inorganic materials 0.000 description 9
- 239000001301 oxygen Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 241000726221 Gemma Species 0.000 description 7
- 238000009792 diffusion process Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 238000003825 pressing Methods 0.000 description 5
- 239000002585 base Substances 0.000 description 4
- 235000013402 health food Nutrition 0.000 description 4
- 235000013406 prebiotics Nutrition 0.000 description 4
- 239000013049 sediment Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 241000186018 Bifidobacterium adolescentis Species 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- 235000019764 Soybean Meal Nutrition 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 239000004455 soybean meal Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 241000276408 Bacillus subtilis subsp. subtilis str. 168 Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000009604 anaerobic growth Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000019628 coolness Nutrition 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical class [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 235000008476 powdered milk Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 108010046845 tryptones Proteins 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
Abstract
The invention belongs to probiotic microcapsule preparing technical field, the preparation method of a kind of probiotic microcapsule with high viable count.The present invention uses aerobism hay bacillus and anaerobic Bifidobacterium mixed culture and optimal control in expanding incubation, adds the growth factor promoting bifidobacterium growth breeding absorbed and the suitable microorganism dormancy condition of not decomposed by human body in capsule manufacturing process.The hay bacillus microcapsules of preparation have the feature of high viable count, high resistance to cold and diseases in shelf life.Its product can be applied not only to field of health care food, it is possible to is applied to field of medicaments and field of fodder.
Description
Technical field
The present invention relates to probiotic microcapsule preparing technical field, a kind of probio with high viable count
The preparation method of microcapsules.
Background technology
Bifidobacterium (Bifidobacterium) is a kind of anaerobic gram positive bacillus, does not moves, does not produce gemma,
Form is the most inconsistent, end usually bifurcated, therefore named Bifidobacterium.Bifidobacterium has 32 hypotypes, and growth requirement often has multiple
Vitamin, a few kind can be containing 10%CO2Air in grow, tunning is mainly acetic acid and lactic acid, does not produce CO2。
Optimum growth temperature is 37～41 DEG C.Bifidobacterium has treatment constipation, anti-curing oncoma, protection liver, prevention and cure of cardiovascular disease
With adjust the pharmacological actions such as function of intestinal canal is disorderly.In health food, Bifidobacterium is one of most important probio.
Hay bacillus (Bacillus subtilis), is a kind of aerobic Gram-positive bacillus, amphitrichous, bacterium
The rough surface that falls is opaque, white or slightly yellow in dirt.In liquid medium within during growth, it is commonly formed wrinkle mould.Hay bacillus pair
Lactose decomposes good effect, it is possible to help milk or the digestion of milk powder and absorption.When entering enteron aisle growth, intestines can be consumed
Oxygen in road, promotes the growth and breeding of the useful anaerobes such as Bifidobacterium.In health food, hay bacillus belongs to prebiotic
Bacterium.
The probio that United Nations Food and Agricultural Organization (FAO) and the World Health Organization (WHO) define is: the micro-life lived
Thing, when taking in appropriate amount, plays wholesome effect to host.Probio in human body mainly has: lactobacillus class, bifid bar
Mushroom, hammer mushroom, hay bacillus and partial yeast bacterium etc..Along with the raising of people's living standard with to healthy concern, benefit
The research of raw bacterium deepens continuously, and many probio health products put goods on the market.But, probiotic composition is from production, preservation, fortune
During defeated, oral administration crosses alimentary canal arrival enteron aisle, need to stand the impact of a series of poor environment factor, cause arriving intestines
The number of viable in road greatly reduces, and has become as the bottleneck that probiotic products produces.
Microencapsulation is a kind of method that the world today solves that this technical barrier is the most effective and ripe, probio
In the existing many successfully report of application.But the short problem of anaerobic probio shelf life does not still have much progress, mainly
Problem is: bacterial strain self resistance poor (sensitive to envirment factors such as oxygen, acid, alkali, heat, especially oxygen), capsule resistance and wall
Contradiction between material dissolubility.The main outlet solving these problems is to reduce the envirment factor impact on bacterial strain.Therefore, this is special
Profit will utilize the intergrowth relation of aerobic hay bacillus and anaerobic bifido bacteria, reduces the envirment factor impact on microbe survival；
Use the microorganism dormancy condition of optimization to keep the biologically active of thalline in prepared by microcapsules；Add in microcapsules simultaneously
The compound sugar growth factor of human body indissoluble rush bifidobacterium growth.
Summary of the invention
The present invention discloses the preparation method of a kind of probiotic microcapsule with high viable count, it is intended to improve probiotics viable bacteria
Resistance, preservation term, reduce viable bacteria through the gastric juice destruction of stomach, make the probio of work as much as possible arrive enteron aisle
Field planting, plays prebiotic health-care effect.
For solving above-mentioned technical problem, the present invention is by the following technical solutions.
The preparation method of a kind of probiotic microcapsule with high viable count, it is characterised in that comprise the following steps:
(1) activation of bacterial strain: the seed that the Bifidobacterium glycerine pipe being stored under the conditions of about-20 DEG C is preserved and guarantor
It is stored in the hay bacillus list bacterium colony under the conditions of about 4 DEG C to be inoculated in respectively in seed culture medium, under the conditions of 32-45 DEG C, bifid
Bacillus anaerobism quiescent culture 24-36 h, hay bacillus 100-300 rpm shaken cultivation 12-24 h, carry out primary activation；
Then respectively Bifidobacterium and the hay bacillus nutrient solution of primary activation are accessed seed again in the ratio of 1-5%
Carrying out second time in culture medium and activate 4-12 h, activation condition is identical with primary activation；Bifid is mixed by the volume ratio of about 1:1
Bacillus and the nutrient solution of hay bacillus, obtain activated seed mixed liquor；
(2) expand cultivate: by activated seed mixed liquor in 1-10%(V/V) ratio be inoculated in equipped with pH 6.5-8.5's
In the fermentation tank of 2-20% native organism raw material culture medium, the liquid amount of native organism raw material culture medium is fermenter volume
50-80%, quiescent culture 24-48 h under the conditions of 35-45 DEG C, collect bacterium solution, its total viable count >=2 × 109Cfu/ml, must send out
Ferment liquid；
(3) washing: by zymotic fluid centrifugal 5-30 min under 3000-8000 rpm, abandon supernatant, by volume 1:1-5
The vibration of bacterial sediment sterilized water being mixed, centrifugal supernatant of abandoning, cyclic washing 1-3 time, subsequently with 1-5% lactoalbumin soln weight
Newly suspend thalline, its total viable count >=1 × 1010Cfu/ml, obtains bacteria suspension；
(4) prepared by bacterium glue: in proportion by 1-10%(W/V) sodium alginate, 0.5-1%(W/V) carboxymethyl chitosan, 0.1-
1 %(W/V) compound sugar, 1-5 %(W/V) carrot, 0.5-2%(V/V) glycerine and 0.1-1%(V/V) Tween 80 joins bacteria suspension
In, stir and evenly mix, anaerobic room deoxidation 30-120 min, obtain bacterium glue；
(5) prepared by microcapsules: bacterium glue injects with 10-30 ml/min flow velocity the 1-of 1-10 DEG C of cooling with syringe pump
In calcium chloride solution 10%(M/V), dropping limit, limit is stirred with the speed of 100-300 rpm, solidifies 30-60 min, aseptic washing
Wash three times, obtain wet microcapsules；
(6) drying preservation: be vacuum-packed after wet microcapsules are carried out low-temperature vacuum drying, obtains probio and does micro-glue
Capsule.
The preparation method of the described probiotic microcapsule with high viable count, it is characterised in that step (1)-step (5)
All carry out by sterile working code.
The preparation method of the described probiotic microcapsule with high viable count, it is characterised in that described in step (5)
It is 40 that wet microcapsules carry out low-temperature vacuum dryingoAny drying mode of below C, and be vacuum-packed.
The preparation method of the described probiotic microcapsule with high viable count, it is characterised in that described in step (1)
Bifidobacterium strain refers to one or more in wild type Bifidobacterium, genetic engineering Bifidobacterium, described withered grass bar
Bacteria strain refers to one or more in wild type B, genetic engineering hay bacillus；Described seed culture medium is for changing
The property entered PYTG culture medium.
The preparation method of the described probiotic microcapsule with high viable count, it is characterised in that detesting described in step (1)
Oxygen method is N2And N2、CO2、H2Gaseous mixture carries out any one of deoxidation.
The preparation method of the described probiotic microcapsule with high viable count, it is characterised in that step (2) is described natural
Organism raw material is any one in whole milk, goat milk, mare's milk, Soy hydrolysate, peanut hydrolyzate and prepared by yeast hydrolysate.
The preparation method of the described probiotic microcapsule with high viable count, it is characterised in that step (3) described whey
Albumen is the lactalbumin in milk.
The preparation method of the described probiotic microcapsule with high viable count, it is characterised in that step (4) is described oligomeric
For human body can not utilize, Bifidobacterium is prone to FOS and any one of oligomeric high fructose corn and derivative thereof absorbed to sugar.
The preparation method of a kind of probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: the seed and the 4 DEG C of hay bacilluses that are preserved by the Bifidobacterium glycerine pipe being stored in-20 DEG C are single
Bacterium colony is inoculated in seed culture medium respectively, under the conditions of 32-45 DEG C, and Bifidobacterium anaerobism quiescent culture 24-36 h, withered grass
Bacillus 100-300 rpm shaken cultivation 12-24 h, carries out primary activation；Then the inoculum concentration second time pressing 1-5% accesses seed
In culture medium, again activate with primary activation CMC model 4-12 h respectively, by 1:1 volume ratio mix Bifidobacterium with
The nutrient solution of hay bacillus, obtains activated seed mixed liquor.
Described bifidobacterium strain and B. subtilis strain are wild type Bifidobacterium, genetic engineering Bifidobacterium, wild
Any one in type hay bacillus and genetic engineering hay bacillus；Described seed culture medium is the property improved PYTG culture medium；Described
Anaerobic cultural methods is N2And N2、CO2、H2Gaseous mixture carries out any one of deoxidation.
(2) expand cultivate: by activated seed liquid in 1-10%(V/V) ratio be inoculated in equipped with 2-20% native organism former
In the fermentation tank of material culture medium (pH 6.5-8.5) (liquid amount is 50-80%), quiescent culture 24-48 under the conditions of 35-45 DEG C
H, collect bacterium solution (bacterium is dense >=2 × 109Cfu/ml), zymotic fluid is obtained.
Described native organism raw material is whole milk, goat milk, mare's milk, Soy hydrolysate, peanut hydrolyzate and yeast water
Solve any one in liquid.
(3) washing: by zymotic fluid centrifugal 5-30 min under 3000-8000 rpm, abandoning supernatant, 1:1-5 will by volume
The vibration of bacterial sediment sterilized water is mixed, centrifugal supernatant of abandoning, cyclic washing 1-3 time, subsequently with 1-5% lactoalbumin soln again
Suspension thalline (bacterium is dense >=and 1 × 1010Cfu/ml), bacteria suspension is obtained.
Described lactalbumin is to obtain after the lactalbumin in milk, typically Yoghourt are centrifuged (6000 rpm, 20 min)
The supernatant arrived.
(4) prepared by bacterium glue: in proportion by 1-10%(W/V) sodium alginate, 0.5-1%(W/V) carboxymethyl chitosan, 0.1-
1 %(W/V) compound sugar, 1-5 %(W/V) carrot, 0.5-2%(V/V) glycerine and 0.1-1%(V/V) Tween 80 joins bacteria suspension
In, stir and evenly mix, anaerobic room deoxidation 30-120 min, obtain bacterium glue.
Described compound sugar be human body can not utilize and Bifidobacterium be prone to absorb FOS and oligomeric high fructose corn and
Any one of derivative.
(5) prepared by microcapsules: bacterium glue injects with 10-30 ml/min flow velocity the 1-of 1-10 DEG C of cooling with syringe pump
In calcium chloride solution 10%(M/V), drip while add stirring (100-300 rpm), solidify 30-60 min, sterilized water washing three
Secondary, obtain wet microcapsules；
(6) drying preservation: be vacuum-packed after wet microcapsules are carried out low-temperature vacuum drying, obtains probio and does micro-glue
Capsule.
The probiotic microcapsule prepared by preparation method of the above-described probiotic microcapsule with high viable count can
It is applied to health food, medicine and field of fodder.
The invention has the beneficial effects as follows: expand cultivate time use optimize condition of culture, including dissolved oxygen, temperature, pH, time
Between and culture medium, the anaerobic bifido bacteria growth that is grown to especially by the aerobic hay bacillus in mixed culture provides
Anaerobic growth environment；Microcapsules prepare in add not by human body decompose absorb promote bifidobacterium growth breeding growth because of
Sub and suitable microorganism dormancy condition.Improve the resistance of probiotics viable bacteria, shelf life, so that as much as possible prebiotic
Bacterium arrives intestinal colonisation, plays prebiotic health care.
The probiotic microcapsule that the present invention produces by integrating method for culturing microbes and capsule technology of preparing has goods
The advantage of high viable count, high resistance to cold and diseases in the frame phase.Its product can be applied not only to health food, medicine and field of fodder.This
Bright bacterial strain is probio, and raw material is the most nontoxic with production process, can realize non-pollutant discharge, green, safe and environment-friendly.
Detailed description of the invention
In order to more fully understand the technology contents of the present invention, below in conjunction with specific embodiment, technical scheme is made
It is further described and illustrates.
Following example following material of use:
Water used all meets country's " standards for drinking water quality " (GB 5,749 2006).
Wild mushroom: bifidobacterium adolescentis Bifidobacterium adolescentisCGMCC 1.2190, hay bacillus
B. subtilis 168。
Engineering bacteria: bifidobacterium adolescentis Bifidobacterium adolescentis IBL291, hay bacillus B.
subtilis IBL 241 [WB800N/pHCMC05-svu022]。
Native organism 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 Co., Ltd.
Seed culture medium is the property improved PYTG culture medium (g/L): 10.0 g soybean meal powder, 5.0 g tryptones, 10.0
G yeast extract, 10.0 g glucose, 40 ml salting liquids (L-0.5 g cysteine, 0.2 g calcium chloride, 0.48 g sulfuric acid
Magnesium, 1.0 g dipotassium hydrogen phosphates), pH value 7.0 ± 0.1, dispense (solid medium adds 1.5% agar, 1 ml resazurin),
115 oSterilizing 20 min under C, it is pure that agents useful for same is analysis.
Embodiment 1
The preparation method of a kind of probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: by be stored in-20 DEG C wild type Bifidobacterium glycerine pipe preserve bacterial classification and be stored in 4
DEG C wild type B list bacterium colony be inoculated in respectively in seed culture medium, under the conditions of 35-43 DEG C, wild type bifid bar
Bacterium anaerobism (N2、CO2、H2Gaseous mixture deoxidation) quiescent culture 24-28 h, wild type B 100-300 rpm shaken cultivation
18-24 h, carries out primary activation；Then the inoculum concentration second time pressing 2-4% accesses in seed culture medium, uses primary activation respectively
CMC model 8-10 h activates again, mixes wild type Bifidobacterium and wild type B by the volume ratio of 1:1
Nutrient solution, obtains activated seed mixed liquor；
(2) expand cultivate: by activated seed mixed liquor in 2-5%(V/V) ratio be inoculated in equipped with 5-15% soy hydrolysate
In the fermentation tank of liquid culture medium (pH 6.5-7.5) (liquid amount is fermenter volume 60-70%), stand under the conditions of 37-41 DEG C
Cultivate 30-36 h, collect bacterium solution (bacterium is dense >=5 × 109Cfu/ml), zymotic fluid is obtained；
(3) washing: by zymotic fluid centrifugal 10-15 min under 5000 rpm, abandoning supernatant, 1:1-2 is by thalline by volume
The vibration of precipitation sterilized water is mixed, and is centrifuged and abandons supernatant, and cyclic washing 2 times, subsequently with 2% lactoalbumin soln Eddy diffusion thalline
(viable count >=2 × 1010Cfu/ml), bacteria suspension is obtained；
(4) prepared by bacterium glue: in proportion by 4%(W/V) sodium alginate, 0.5%(W/V) carboxymethyl chitosan, 0.2 %(W/
V) FOS, (W/V), 2-3 %(W/V) carrot, 0.8-1%(V/V) glycerine and 0.2%(V/V) Tween 80 joins bacteria suspension
In, stir and evenly mix, anaerobic room N2、CO2、H2Gaseous mixture deoxidation 60-90 min, obtains bacterium glue；
(5) prepared by microcapsules: bacterium glue injects with 20 ml/min flow velocitys the 2-4%(M/ of 4-5 DEG C of cooling with syringe pump
V) in calcium chloride solution, dripping while add stirring (200 rpm), solidify 40-50 min, sterilized water washs three times, obtains wet micro-glue
Capsule；
(6) drying preservation: be vacuum-packed after wet microcapsules are carried out low-temperature vacuum drying, obtains probio and does micro-glue
Capsule.
Shelf-life >=2 year of the dry microcapsule product of this probio, viable count >=1 × 1011Individual/g, gemma 60%；Yellowish
Look, free from extraneous odour.
Embodiment 2
The preparation method of a kind of probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: by be stored in-20 DEG C Bifidobacterium engineering bacteria glycerine pipe preserve bacterial classification and be stored in 4
DEG C hay bacillus engineering bacteria list bacterium colony be inoculated in respectively in seed culture medium, under the conditions of 37-41 DEG C, Bifidobacterium anaerobism
(N2Deoxidation) quiescent culture 24-28 h, hay bacillus engineering bacteria 100-300 rpm shaken cultivation 18-24 h, live for the first time
Change；Then the inoculum concentration second time pressing 2-4% accesses in seed culture medium, carries out with primary activation CMC model 8-10 h respectively
Again activate, mix the nutrient solution of Bifidobacterium engineering bacteria and hay bacillus engineering bacteria by the volume ratio of 1:1, obtain activated seed and mix
Close liquid；
(2) expand cultivate: by activated seed mixed liquor in 2-5%(V/V) ratio be inoculated in equipped with 5-10% whole milk
In the fermentation tank of culture medium (pH 7.0) (liquid amount is 60-70%), quiescent culture 30-36 h under the conditions of 37-41 DEG C, collects
Bacterium solution (bacterium is dense >=and 1 × 1010Cfu/ml), zymotic fluid is obtained；
(3) washing: by zymotic fluid centrifugal 10-15 min under 5000 rpm, abandoning supernatant, 1:1-2 is by thalline by volume
The vibration of precipitation sterilized water is mixed, and is centrifuged and abandons supernatant, and cyclic washing 2 times, subsequently with 2% lactoalbumin soln Eddy diffusion thalline
(bacterium is dense >=and 5 × 1010Cfu/ml), bacteria suspension is obtained；
(4) prepared by bacterium glue: in proportion by 5%(W/V) sodium alginate, 1.5%(W/V) carboxymethyl chitosan, 0.2 %(W/
V) FOS, (W/V), 2-3 %(W/V) carrot, 0.8-1%(V/V) glycerine and 0.2%(V/V) Tween 80 joins bacteria suspension
In, stir and evenly mix, anaerobic room N2Qi exhaustion oxygen 60-90 min, obtains bacterium glue；
(5) prepared by microcapsules: bacterium glue injects with 20 ml/min flow velocitys the 2-4%(M/ of 4-5 DEG C of cooling with syringe pump
V) in calcium chloride solution, dripping while add stirring (200 rpm), solidify 40-50 min, sterilized water washs three times, obtains wet micro-glue
Capsule；
(6) drying preservation: be vacuum-packed after wet microcapsules are carried out low-temperature vacuum drying, obtains probio and does micro-glue
Capsule.
Shelf-life >=2 year of the dry microcapsule product of this hay bacillus, viable count >=2 × 1011Individual/g, gemma 60%；Yellowish
Look, free from extraneous odour.
Embodiment 3
The preparation method of a kind of probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: by be stored in-20 DEG C wild Bifidobacterium glycerine pipe preserve bacterial classification and be stored in 4 DEG C
Hay bacillus engineering bacteria list bacterium colony be inoculated in respectively in seed culture medium, under the conditions of 37-39 DEG C, wild type Bifidobacterium
Anaerobism (N2、CO2、H2Gaseous mixture deoxidation) quiescent culture 24-28 h, hay bacillus engineering bacteria 200 rpm shaken cultivation 18-24 h,
Carry out primary activation；Then the inoculum concentration second time pressing 2-4% accesses in seed culture medium, uses primary activation CMC model respectively
8-10 h activates again, mixes the nutrient solution of wild type Bifidobacterium and hay bacillus engineering bacteria by the volume ratio of 1:1,
Activated seed mixed liquor；
(2) expand cultivate: by activated seed mixed liquor in 2-5%(V/V) ratio be inoculated in equipped with 5-15% goat milk cultivation
In the fermentation tank of base (pH 7.0) (liquid amount is 60-70%), quiescent culture 30-36 h under the conditions of 37-39 DEG C, collects bacterium solution
(bacterium is dense >=and 5 × 109Cfu/ml), zymotic fluid is obtained；
(3) washing: by zymotic fluid centrifugal 10-15 min under 5000 rpm, abandoning supernatant, thalline is sunk by 1:2 by volume
Shallow lake sterilized water vibration is mixed, and is centrifuged and abandons supernatant, and cyclic washing 2 times, subsequently with 2% lactoalbumin soln Eddy diffusion thalline (bacterium
Dense >=2 × 1010Cfu/ml), bacteria suspension is obtained；
(4) prepared by bacterium glue: in proportion by 4%(W/V) sodium alginate, 1.0%(W/V) carboxymethyl chitosan, 0.2 %(W/
V) FOS, (W/V), 5 %(W/V) carrot, 0.8%(V/V) glycerine and 0.2%(V/V) Tween 80 joins in bacteria suspension,
Stir and evenly mix, anaerobic room N2、CO2、H2Gaseous mixture deoxidation 60-90 min, obtains bacterium glue；
(5) prepared by microcapsules: bacterium glue injects with 20 ml/min flow velocitys the 2-4%(M/ of 4-5 DEG C of cooling with syringe pump
V) in calcium chloride solution, dripping while add stirring (200 rpm), solidify 60 min, sterilized water washs three times, obtains wet microcapsules；
(6) drying preservation: be vacuum-packed after wet microcapsules are carried out low-temperature vacuum drying, obtains probio and does micro-glue
Capsule.
Shelf-life >=2 year of the dry microcapsule product of this hay bacillus, viable count >=5 × 1010Individual/g, gemma 60%；Yellowish
Look, free from extraneous odour.
Embodiment 4
The preparation method of a kind of probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: by be stored in-20 DEG C Bifidobacterium engineering bacteria glycerine pipe preserve bacterial classification and be stored in 4
DEG C wild hay bacillus list bacterium colony be inoculated in respectively in seed culture medium, under the conditions of 37-39 DEG C, engineering bacteria Bifidobacterium
Anaerobism (N2、CO2、H2Gaseous mixture deoxidation) quiescent culture 24-28 h, wild type B 200 rpm shaken cultivation 18-24 h,
Carry out primary activation；Then the inoculum concentration second time pressing 2-4% accesses in seed culture medium, uses primary activation CMC model respectively
8-10 h activates again, mixes the nutrient solution of wild type Bifidobacterium and hay bacillus engineering bacteria by the volume ratio of 1:1,
Activated seed mixed liquor；
(2) expand cultivate: by activated seed mixed liquor in 2-5%(V/V) ratio be inoculated in equipped with 5-15% mare's milk cultivation
In the fermentation tank of base (pH 7.0) (liquid amount is 60-70%), quiescent culture 30-36 h under the conditions of 37-39 DEG C, collects bacterium solution
(bacterium is dense >=and 1 × 1010Cfu/ml), zymotic fluid is obtained；
(3) washing: by zymotic fluid centrifugal 10-15 min under 5000 rpm, abandoning supernatant, thalline is sunk by 1:2 by volume
Shallow lake sterilized water vibration is mixed, and is centrifuged and abandons supernatant, and cyclic washing 2 times, subsequently with 2% lactoalbumin soln Eddy diffusion thalline (bacterium
Dense >=3 × 1010Cfu/ml), bacteria suspension is obtained；
(4) prepared by bacterium glue: in proportion by 5%(W/V) sodium alginate, 1.0%(W/V) carboxymethyl chitosan, 0.2 %(W/
V) FOS, (W/V), 3.0 %(W/V) carrot, 1.0%(V/V) glycerine and 0.2%(V/V) Tween 80 joins bacteria suspension
In, stir and evenly mix, anaerobic room N2、CO2、H2Gaseous mixture deoxidation 60-90 min, obtains bacterium glue；
(5) prepared by microcapsules: bacterium glue injects with 20 ml/min flow velocitys the 2-4%(M/ of 4-5 DEG C of cooling with syringe pump
V) in calcium chloride solution, dripping while add stirring (200 rpm), solidify 60 min, sterilized water washs three times, obtains wet microcapsules；
(6) drying preservation: be vacuum-packed after wet microcapsules are carried out low-temperature vacuum drying, obtains probio and does micro-glue
Capsule.
Shelf-life >=2 year of the dry microcapsule product of this hay bacillus, viable count >=1 × 1011Individual/g, gemma 60%；Yellowish
Look, free from extraneous odour.
Embodiment 5
The preparation method of a kind of probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: by be stored in-20 DEG C wild type Bifidobacterium glycerine pipe preserve bacterial classification and be stored in 4
DEG C wild type B list bacterium colony be inoculated in respectively in seed culture medium, under the conditions of 45 DEG C, wild type is detested by Bifidobacterium
Oxygen quiescent culture 24 h, wild type B 100 rpm shaken cultivation 12 h, carry out primary activation；Then the inoculation of 1% is pressed
Flow control secondary accesses in seed culture medium, again activates, by the volume ratio of 1:1 with primary activation CMC model 4 h respectively
Mixing wild type Bifidobacterium and the nutrient solution of wild type B, obtain activated seed mixed liquor；
(2) expand cultivate: by activated seed mixed liquor in 10%(V/V) ratio be inoculated in equipped with 20% peanut hydrolyzate train
Support in the fermentation tank of base (pH 6.5) (liquid amount is 80%), quiescent culture 24 h under the conditions of 45 DEG C, collect bacterium solution (bacterium is dense >=2
×109Cfu/ml), zymotic fluid is obtained；
(3) washing: by zymotic fluid centrifugal 30 min under 3000 rpm, abandoning supernatant, bacterial sediment is used by 1:1 by volume
Sterilized water vibration is mixed, centrifugal supernatant of abandoning, cyclic washing 3 times, subsequently with 5% lactoalbumin soln Eddy diffusion thalline (bacterium is dense >=
5×109Cfu/ml), bacteria suspension is obtained；
(4) prepared by bacterium glue: in proportion by 1%(W/V) sodium alginate, 1%(W/V) carboxymethyl chitosan, 1%(W/V) oligomeric
Sugar, (W/V), 1 %(W/V) carrot, 0.5%(V/V) glycerine and 1%(V/V) Tween 80 joins in bacteria suspension, stir and evenly mix, detest
Oxygen room deoxidation 120 min, obtains bacterium glue；
(5) prepared by microcapsules: bacterium glue injects with 30 ml/min flow velocitys the 10%(M/V of 1 DEG C of cooling with syringe pump)
In calcium chloride solution, dripping while add stirring (100 rpm), solidify 60 min, sterilized water washs three times, obtains wet microcapsules；
(6) drying preservation: be vacuum-packed after wet microcapsules are carried out low-temperature vacuum drying, obtains probio and does micro-glue
Capsule.
Shelf-life >=2 year of the dry microcapsule product of this probio, viable count >=5 × 109Individual/g, gemma 80%；Yellowish
Look, free from extraneous odour.
Embodiment 6
The preparation method of a kind of probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: by be stored in-20 DEG C Bifidobacterium engineering bacteria glycerine pipe preserve bacterial classification and be stored in 4
DEG C hay bacillus engineering bacteria list bacterium colony be inoculated in respectively in seed culture medium, under the conditions of 32 DEG C, Bifidobacterium engineering bacteria is detested
Oxygen quiescent culture 36 h, hay bacillus engineering bacteria 100 rpm shaken cultivation 24 h, carry out primary activation；Then the inoculation of 5% is pressed
Flow control secondary accesses in seed culture medium, again activates, by the volume ratio of 1:1 with primary activation CMC model 12 h respectively
Mixing Bifidobacterium engineering bacteria and the nutrient solution of hay bacillus engineering bacteria, obtain activated seed mixed liquor；
(2) expand cultivate: by activated seed mixed liquor in 1%(V/V) ratio be inoculated in equipped with 2% prepared by yeast hydrolysate cultivation
In the fermentation tank of base (pH 8.5) (liquid amount is 50%), quiescent culture 48 h under the conditions of 35 DEG C, collect bacterium solution (bacterium is dense >=1 ×
109Cfu/ml), zymotic fluid is obtained；
(3) washing: by zymotic fluid centrifugal 5 min under 8000 rpm, abandoning supernatant, bacterial sediment is used by 1:5 by volume
Sterilized water vibration is mixed, centrifugal supernatant of abandoning, cyclic washing 1 time, subsequently with 1% lactoalbumin soln Eddy diffusion thalline (bacterium is dense >=
2×109Cfu/ml), bacteria suspension is obtained；
(4) prepared by bacterium glue: in proportion by 10%(W/V) sodium alginate, 0.5%(W/V) carboxymethyl chitosan, 0.1%(W/
V) compound sugar, (W/V), 5 %(W/V) carrot, 2%(V/V) glycerine and 0.1%(V/V) Tween 80 joins in bacteria suspension, stirring
Mixing, anaerobic room deoxidation 30 min, obtain bacterium glue；
(5) prepared by microcapsules: bacterium glue injects with 10 ml/min flow velocitys the 1%(M/V of 10 DEG C of coolings with syringe pump)
In calcium chloride solution, dripping while add stirring (300 rpm), solidify 30 min, sterilized water washs three times, obtains wet microcapsules；
(6) drying preservation: be vacuum-packed after wet microcapsules are carried out low-temperature vacuum drying, obtains probio and does micro-glue
Capsule.
Shelf-life >=2 year of the dry microcapsule product of this probio, viable count >=1 × 109Individual/g, gemma 80%；Yellowish
Look, free from extraneous odour.
The above only further illustrates the technology contents of the present invention with embodiment, in order to reader is easier to understand,
But not representing embodiments of the present invention and be only limitted to this, any technology done according to the present invention extends or recreation, all by this
Bright protection.
Claims (8)
1. the preparation method of a probiotic microcapsule with high viable count, it is characterised in that comprise the following steps:
(1) activation of bacterial strain: seed that Bifidobacterium glycerine pipe under the conditions of-20 DEG C preserves will be stored in and be stored in 4 DEG C of bars
Hay bacillus list bacterium colony under part is inoculated in seed culture medium respectively, and under the conditions of 32-45 DEG C, Bifidobacterium anaerobism stands
Cultivate 24-36 h, hay bacillus 100-300 rpm shaken cultivation 12-24 h, carry out primary activation；Then in the ratio of 1-5%
Respectively Bifidobacterium and the hay bacillus nutrient solution of primary activation are again accessed and carry out in seed culture medium activating 4-for the second time
12 h, activation condition is identical with primary activation；Mix the nutrient solution of Bifidobacterium and hay bacillus by the volume ratio of 1:1, obtain work
Change seed mixed liquor；
(2) expand cultivate: by activated seed mixed liquor in 1-10%(V/V) ratio be inoculated in the 2-20% equipped with pH 6.5-8.5
In the fermentation tank of native organism raw material culture medium, the liquid amount of native organism raw material culture medium is fermenter volume 50-
80%, quiescent culture 24-48 h under the conditions of 35-45 DEG C, collect bacterium solution, its total viable count >=2 × 109Cfu/ml, obtains fermentation
Liquid；
(3) washing: by zymotic fluid centrifugal 5-30 min under 3000-8000 rpm, abandoning supernatant, 1:1-5 is by bacterium by volume
The vibration of body precipitation sterilized water is mixed, and is centrifuged and abandons supernatant, cyclic washing 1-3 time, again hangs with 1-5% lactoalbumin soln subsequently
Floating thalline, its total viable count >=1 × 1010Cfu/ml, obtains bacteria suspension；
(4) prepared by bacterium glue: in proportion by 1-10%(W/V) sodium alginate, 0.5-1%(W/V) carboxymethyl chitosan, 0.1-1 %
(W/V) compound sugar, 1-5 %(W/V) carrot, 0.5-2%(V/V) glycerine and 0.1-1%(V/V) Tween 80 joins in bacteria suspension,
Stir and evenly mix, anaerobic room deoxidation 30-120 min, obtain bacterium glue；
(5) prepared by microcapsules: bacterium glue injects with 10-30 ml/min flow velocity the 1-10%(M/ of 1-10 DEG C of cooling with syringe pump
V) in calcium chloride solution, dropping limit, limit is stirred with the speed of 100-300 rpm, solidifies 30-60 min, sterilized water washing three
Secondary, obtain wet microcapsules；
(6) drying preservation: be vacuum-packed after wet microcapsules are carried out low-temperature vacuum drying, obtains the dry microcapsules of probio.
The preparation method of the probiotic microcapsule with high viable count the most according to claim 1, it is characterised in that step
(1)-step (5) is all carried out by sterile working code.
The preparation method of the probiotic microcapsule with high viable count the most according to claim 1, it is characterised in that right
Wet microcapsules described in step (5) carry out any drying mode that low-temperature vacuum drying is less than 40 DEG C, and carry out vacuum
Packaging.
The preparation method of the probiotic microcapsule with high viable count the most according to claim 1, it is characterised in that step
(1) bifidobacterium strain described in refers to one or more in wild type Bifidobacterium, genetic engineering Bifidobacterium, institute
The B. subtilis strain stated refers to one or more in wild type B, genetic engineering hay bacillus；Described seed
Culture medium is the property improved PYTG culture medium.
The preparation method of the probiotic microcapsule with high viable count the most according to claim 1, it is characterised in that step
(1) Anaerobic cultural methods described in is N2And N2、CO2、H2Gaseous mixture carries out any one of deoxidation.
The preparation method of the probiotic microcapsule with high viable count the most according to claim 1, it is characterised in that step
(2) during described native organism raw material is whole milk, goat milk, mare's milk, Soy hydrolysate, peanut hydrolyzate and prepared by yeast hydrolysate
Any one.
The preparation method of the probiotic microcapsule with high viable count the most according to claim 1, it is characterised in that step
(3) lactalbumin during described lactalbumin is milk.
The preparation method of the probiotic microcapsule with high viable count the most according to claim 1, it is characterised in that step
(4) described compound sugar is that human body can not utilize and Bifidobacterium is prone to the FOS and any one of derivative thereof that absorb.
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| CN104351938B (en) * | 2014-10-21 | 2017-08-01 | 吉林农业大学 | A kind of preparation method of food-grade enteric solubility microcapsules |
| CN105169362A (en) * | 2015-09-02 | 2015-12-23 | 钟华 | Biopharmaceutical method for intestinal diseases |
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| CN107125767B (en) * | 2017-04-28 | 2020-09-29 | 黑龙江大学 | Preparation method and application of bifidobacterium breve, lactobacillus fermentum and lactobacillus plantarum three-bacterium composite micro-particles |
| CN107259577A (en) * | 2017-07-25 | 2017-10-20 | 李立风 | Probiotic gel grain and preparation method thereof |
| CN107823171B (en) * | 2017-12-14 | 2019-01-11 | 深圳职业技术学院 | Preparation method for producing composite coated active probiotic dropping pills by one-step method and dropping pills |
| CN108323571A (en) * | 2018-02-08 | 2018-07-27 | 广州医科大学 | A kind of wall material of probiotic microcapsule and the preparation method of probiotic microcapsule |
| CN113749258A (en) * | 2021-09-16 | 2021-12-07 | 江南大学 | Calcium alginate liquid core microcapsule and preparation method thereof |
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