CN103704719A - Preparation method for probiotics microcapsule with high viable count - Google Patents
Preparation method for probiotics microcapsule with high viable count Download PDFInfo
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- CN103704719A CN103704719A CN201310715323.4A CN201310715323A CN103704719A CN 103704719 A CN103704719 A CN 103704719A CN 201310715323 A CN201310715323 A CN 201310715323A CN 103704719 A CN103704719 A CN 103704719A
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- bifidobacterium
- viable count
- hay bacillus
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- 239000003094 microcapsule Substances 0.000 title claims abstract description 81
- 238000002360 preparation method Methods 0.000 title claims abstract description 51
- 239000006041 probiotic Substances 0.000 title claims abstract description 37
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 37
- 241000186000 Bifidobacterium Species 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 7
- 241000894006 Bacteria Species 0.000 claims description 97
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 50
- 239000001963 growth medium Substances 0.000 claims description 38
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 36
- 230000000529 probiotic effect Effects 0.000 claims description 32
- 230000001580 bacterial effect Effects 0.000 claims description 30
- 239000003292 glue Substances 0.000 claims description 27
- 238000005406 washing Methods 0.000 claims description 27
- 238000011218 seed culture Methods 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 21
- 230000004913 activation Effects 0.000 claims description 20
- 239000006228 supernatant Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 239000012530 fluid Substances 0.000 claims description 18
- 235000011187 glycerol Nutrition 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 18
- 239000000725 suspension Substances 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 17
- 238000004321 preservation Methods 0.000 claims description 13
- 238000000855 fermentation Methods 0.000 claims description 12
- 230000004151 fermentation Effects 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 11
- 235000015097 nutrients Nutrition 0.000 claims description 11
- 238000001291 vacuum drying Methods 0.000 claims description 11
- 238000009461 vacuum packaging Methods 0.000 claims description 11
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 10
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 10
- 239000002994 raw material Substances 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
- 244000000626 Daucus carota Species 0.000 claims description 9
- 235000002767 Daucus carota Nutrition 0.000 claims description 9
- 150000001875 compounds Chemical class 0.000 claims description 9
- 238000001816 cooling Methods 0.000 claims description 9
- 125000004122 cyclic group Chemical group 0.000 claims description 9
- 238000009792 diffusion process Methods 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
- 239000013049 sediment 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
- 102000004407 Lactalbumin Human genes 0.000 claims description 6
- 108090000942 Lactalbumin Proteins 0.000 claims description 6
- 238000010353 genetic engineering Methods 0.000 claims description 6
- 239000000413 hydrolysate Substances 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
- 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 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 235000020251 goat milk Nutrition 0.000 claims description 4
- 235000020232 peanut Nutrition 0.000 claims description 4
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 3
- 235000013399 edible fruits Nutrition 0.000 claims description 3
- 238000003825 pressing Methods 0.000 claims description 2
- 230000012010 growth Effects 0.000 abstract description 8
- 244000005700 microbiome Species 0.000 abstract description 5
- 235000014469 Bacillus subtilis Nutrition 0.000 abstract description 3
- 239000002775 capsule Substances 0.000 abstract description 3
- 230000005059 dormancy Effects 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 239000003102 growth factor Substances 0.000 abstract description 3
- 244000063299 Bacillus subtilis Species 0.000 abstract description 2
- 235000013305 food Nutrition 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 241001148470 aerobic bacillus Species 0.000 abstract 1
- 229940079593 drug Drugs 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 239000000047 product Substances 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 7
- 239000002054 inoculum Substances 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 235000013402 health food Nutrition 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 2
- 241000186018 Bifidobacterium adolescentis Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 235000019764 Soybean Meal Nutrition 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 235000013406 prebiotics Nutrition 0.000 description 2
- 239000004455 soybean meal Substances 0.000 description 2
- 229920001817 Agar Polymers 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
- 241000726221 Gemma Species 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
- 241000235342 Saccharomycetes Species 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
- 239000003795 chemical substances by application Substances 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
- 201000010099 disease Diseases 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 230000000968 intestinal effect Effects 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
- 210000004185 liver Anatomy 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000012856 packing 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
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 108010046845 tryptones Proteins 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
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Abstract
The invention belongs to the technical field of preparation of probiotics microcapsules and in particular relates to a preparation method for the probiotics microcapsule with high viable count. In an enlarging cultivation process, aerobic bacillus subtilis and anaerobic bifidobacterium are cultivated in a mixed manner, and optimized control is executed; in a capsule preparation process, growth factors for promoting growth and reproduction of the bifidobacterium which cannot be decomposed and absorbed by a human body are added, and a proper microorganism dormancy condition is created. A prepared bacillus subtilis microcapsule has the characteristics of high viable count and high stress resistance during shelf life. A product can be applied to the field of health-protection food and also applied to the fields of medicines and feeds.
Description
Technical field
The present invention relates to probiotic microcapsule preparing technical field, specifically a kind of preparation method with the probiotic microcapsule of high viable count.
Background technology
Bifidobacterium (
bifidobacterium) be a kind of anaerobic Gram-positive bacillus, do not move, do not produce gemma, form is very inconsistent, and end is bifurcated usually, therefore named Bifidobacterium.Bifidobacterium has 32 hypotypes, and growth requires to have multivitamin often, and a few kind can contain 10%CO
2air in grow, tunning is mainly acetic acid and lactic acid, does not produce CO
2.Optimum growth temperature is 37～41 ℃.Bifidobacterium has the pharmacological actions such as treatment constipation, anti-curing oncoma, protection liver, prevention and cure of cardiovascular disease and the disorder of adjustment function of intestinal canal.In health food, Bifidobacterium is one of most important probio.
Hay bacillus (
bacillus subtilis), be a kind of aerobic Gram-positive bacillus, amphitrichous, bacterium colony rough surface is opaque, is dirty white or micro-yellow.While growing in liquid medium within, the normal wrinkle mould that forms.Hay bacillus is decomposed and has good effect lactose, can help the digestion of milk or milk powder and absorption.While entering enteron aisle growth, can consume the oxygen in enteron aisle, promote the growth and breeding of the useful anaerobes such as Bifidobacterium.In health food, hay bacillus belongs to probio.
The probio of United Nations Food and Agricultural Organization (FAO) and the World Health Organization (WHO) definition is: the microorganism living, when taking in appropriate amount, plays wholesome effect to host.Probio in human body mainly contains: lactobacillus class, Bifidobacterium class, hammer mushroom, hay bacillus and part saccharomycete etc.Along with the raising of people's living standard with to healthy concern, probio research deepens continuously, and many probio health products put goods on the market.Yet, probiotic composition is during crossing the process of alimentary canal arrival enteron aisle from production, preservation, transportation, oral administration, need to stand the impact of a series of poor environment factors, cause the number of viable that arrives enteron aisle to greatly reduce, become the bottleneck that probiotic products is produced.
Microencapsulation technology is that the world today solves a kind of comparatively effective and ripe method of this technical barrier, the existing many successful reports of application in probio.But the short problem of anaerobic probio shelf life does not still have much progress, and subject matter is: bacterial strain self resistance poor (to the envirment factor sensitivities such as oxygen, acid, alkali, heat, especially oxygen), the contradiction between capsule resistance and wall material dissolubility.The main outlet addressing these problems is to reduce the impact of envirment factor on bacterial strain.Therefore, this patent will utilize the intergrowth relation of aerobic hay bacillus and anaerobic bifido bacteria, reduces the impact of envirment factor on microorganism existence; In microcapsules preparation, use the microorganism dormancy condition of optimizing to keep the biologically active of thalline; The compound sugar growth factor of adding the indissoluble short bifidobacterium growth of human body in microcapsules simultaneously.
Summary of the invention
The present invention discloses a kind of preparation method with the probiotic microcapsule of high viable count, be intended to improve resistance, the preservation term of probiotics viable bacteria, gastric juice destruction while reducing viable bacteria through stomach, makes the probio of work as much as possible arrive enteron aisle field planting, plays prebiotic health-care effect.
For solving the problems of the technologies described above, the present invention by the following technical solutions.
A preparation method with the probiotic microcapsule of high viable count, is characterized in that, comprises the following steps:
(1) activation of bacterial strain: be inoculated in respectively in seed culture medium by being stored in the seed of-20 ℃ of Bifidobacterium glycerine pipes preservations under the condition of left and right and being stored in 4 ℃ of single bacterium colonies of the hay bacillus under the condition of left and right, under 32-45 ℃ of condition, the standing cultivation of Bifidobacterium anaerobism 24-36 h, hay bacillus 100-300 rpm shaken cultivation 12-24 h, activate for the first time;
Then in the ratio of 1-5%, respectively the Bifidobacterium of activation for the first time and hay bacillus nutrient solution are again accessed and in seed culture medium, activate for the second time 4-12 h, activation condition with activate for the first time identical; The volume ratio of pressing 1:1 left and right is mixed the nutrient solution of Bifidobacterium and hay bacillus, obtains activated seed mixed liquor;
(2) expand to cultivate: by activated seed mixed liquor in 1-10%(V/V) ratio be inoculated in the fermentation tank of the natural organism raw material of the 2-20% culture medium that pH 6.5-8.5 is housed, the liquid amount of natural organism raw material culture medium is fermentation tank volume 50-80%, standing cultivation 24-48 h under 35-45 ℃ of condition, collect bacterium liquid, its total viable count>=2 * 10
9cfu/ml, obtains zymotic fluid;
(3) washing: by zymotic fluid centrifugal 5-30 min under 3000-8000 rpm, abandon supernatant, 1:1-5 is mixed bacterial sediment with sterilized water vibration by volume, the centrifugal supernatant of abandoning, cyclic washing 1-3 time, uses 1-5% lactoalbumin soln Eddy diffusion thalline, its total viable count>=1 * 10 subsequently
10cfu/ml, obtains bacteria suspension;
(4) bacterium glue preparation: in proportion by 1-10%(W/V) sodium alginate, 0.5-1%(W/V) CMC, 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, obtains bacterium glue;
(5) calcium chloride solution microcapsules preparation: bacterium glue is injected to 1-10 ℃ of cooling 1-10%(M/V with 10-30 ml/min flow velocity with syringe pump), stir with the speed of 100-300 rpm on dropping limit, limit, solidify 30-60 min, sterilized water washing three times, obtains wet microcapsules;
(6) drying preservation: the microcapsules that will wet carry out carrying out vacuum packaging after low-temperature vacuum drying, obtain the dry microcapsules of probio.
The preparation method of the described probiotic microcapsule with high viable count, is characterized in that, step (1)-step (5) is all undertaken by sterile working rules.
The preparation method of the described probiotic microcapsule with high viable count, is characterized in that, it is 40 that wet microcapsules described in step (5) are carried out to low-temperature vacuum drying
oany drying mode below C, and carry out vacuum packaging.
The preparation method of the described probiotic microcapsule with high viable count, it is characterized in that, bifidobacterium strain described in step (1) refers to one or more in wild type Bifidobacterium, genetic engineering Bifidobacterium, and described hay bacillus bacterial strain refers to one or more in wild type hay bacillus, genetic engineering hay bacillus; Described seed culture medium is the property improved PYTG culture medium.
The preparation method of the described probiotic microcapsule with high viable count, is characterized in that, the described Anaerobic cultural methods of step (1) is N
2and N
2, CO
2, H
2gaseous mixture carries out any of deoxidation.
The preparation method of the described probiotic microcapsule with high viable count, is characterized in that, the described natural organism raw material of step (2) is any 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, is characterized in that, the described lactalbumin of step (3) is the lactalbumin in milk.
The preparation method of the described probiotic microcapsule with high viable count, is characterized in that, the described compound sugar of step (4) is that human body can not utilize and Bifidobacterium is easy to any of the FOS that absorbs and oligomeric fruit glucose and derivative thereof.
A preparation method for probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: the seed that the Bifidobacterium glycerine pipe that is stored in-20 ℃ is preserved and 4 ℃ of single bacterium colonies of hay bacillus are inoculated in respectively in seed culture medium, under 32-45 ℃ of condition, the standing cultivation of Bifidobacterium anaerobism 24-36 h, hay bacillus 100-300 rpm shaken cultivation 12-24 h, activate for the first time; Then by the inoculum concentration of 1-5%, access for the second time in seed culture medium, with first activation condition, cultivate 4-12 h respectively and again activate, by the volume ratio of 1:1, mix the nutrient solution of Bifidobacterium and hay bacillus, obtain activated seed mixed liquor.
Described bifidobacterium strain and hay bacillus bacterial strain are any in wild type Bifidobacterium, genetic engineering Bifidobacterium, wild type hay bacillus and genetic engineering hay bacillus; Described seed culture medium is the property improved PYTG culture medium; Described Anaerobic cultural methods is N
2and N
2, CO
2, H
2gaseous mixture carries out any of deoxidation.
(2) expand to cultivate: by activated seed liquid in 1-10%(V/V) ratio be inoculated in (liquid amount is 50-80%) in the fermentation tank that the natural organism raw material of 2-20% culture medium (pH 6.5-8.5) is housed, standing cultivation 24-48 h under 35-45 ℃ of condition, collect bacterium liquid (bacterium is dense>=2 * 10
9cfu/ml), obtain zymotic fluid.
Described natural organism raw material is any in whole milk, goat milk, mare's milk, Soy hydrolysate, peanut hydrolyzate and prepared by yeast hydrolysate.
(3) washing: by zymotic fluid centrifugal 5-30 min under 3000-8000 rpm, abandon supernatant, 1:1-5 is mixed bacterial sediment with sterilized water vibration by volume, the centrifugal supernatant of abandoning, cyclic washing 1-3 time, use subsequently 1-5% lactoalbumin soln Eddy diffusion thalline (bacterium is dense>=1 * 10
10cfu/ml), obtain bacteria suspension.
Described lactalbumin is the lactalbumin in milk, is typically the supernatant obtaining after Yoghourt centrifugal (6000 rpm, 20 min).
(4) bacterium glue preparation: in proportion by 1-10%(W/V) sodium alginate, 0.5-1%(W/V) CMC, 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, obtains bacterium glue.
Described compound sugar is that human body can not utilize and Bifidobacterium is easy to any of the FOS that absorbs and oligomeric fruit glucose and derivative thereof.
(5) calcium chloride solution microcapsules preparation: bacterium glue is injected to 1-10 ℃ of cooling 1-10%(M/V with 10-30 ml/min flow velocity with syringe pump), while dripping, add stirring (100-300 rpm), solidify 30-60 min, sterilized water washing three times, obtains wet microcapsules;
(6) drying preservation: the microcapsules that will wet carry out carrying out vacuum packaging after low-temperature vacuum drying, obtain the dry microcapsules of probio.
The above-described prepared probiotic microcapsule of preparation method with the probiotic microcapsule of high viable count can be applicable to health food, medicine and field of fodder.
The invention has the beneficial effects as follows: when expanding cultivation, adopt the condition of culture of optimizing, comprise dissolved oxygen, temperature, pH, time and culture medium, particularly by the anaerobic growth environment that anaerobic bifido bacteria growth provides that is grown to of the aerobic hay bacillus in Mixed culture; In microcapsules preparation, do not add and decomposed the growth factor of the promotion bifidobacterium growth breeding absorbing and suitable microorganism dormancy condition by human body.Improve resistance, the shelf life of probiotics viable bacteria, thereby made probio as much as possible arrive enteron aisle field planting, played prebiotic health care.
The present invention has advantages of high viable count, high resistance to cold and diseases in shelf life by integrating the probiotic microcapsule of method for culturing microbes and the production of capsule technology of preparing.Its product not only can be applicable to health food, medicine and field of fodder.Bacterial strain of the present invention is probio, and raw material and production process are all nontoxic, can realize non-pollutant discharge, green, safety, environmental protection.
The specific 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 is described further and is illustrated.
Following examples are used following material:
Institute's water all meets country's " drinking water sanitary standard " (GB 5749-2006).
Wild mushroom: bifidobacterium adolescentis
bifidobacterium adolescentisCGMCC1.2190, hay bacillus
b. subtilis168.
Engineering bacteria: bifidobacterium adolescentis
bifidobacterium adolescentisiBL291, hay bacillus
b. subtilisiBL 241 [WB800N/pHCMC05-svu022].
Natural 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 extracts, 10.0 g glucose, 40 ml salting liquids (L-0.5 g cysteine, 0.2 g calcium chloride, 0.48 g magnesium sulfate, 1.0 g dipotassium hydrogen phosphates), pH value 7.0 ± 0.1, packing (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
A preparation method for probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: the bacterial classification that the wild type Bifidobacterium glycerine pipe that is stored in-20 ℃ is preserved is inoculated in respectively in seed culture medium with the single bacterium colony of the wild type hay bacillus that is stored in 4 ℃, under 35-43 ℃ of condition, wild type Bifidobacterium anaerobism (N
2, CO
2, H
2gaseous mixture deoxidation) standing cultivation 24-28 h, wild type hay bacillus 100-300 rpm shaken cultivation 18-24 h, activate for the first time; Then by the inoculum concentration of 2-4%, access for the second time in seed culture medium, with first activation condition, cultivate 8-10 h respectively and again activate, by the volume ratio of 1:1, mix the nutrient solution of wild type Bifidobacterium and wild type hay bacillus, obtain activated seed mixed liquor;
(2) expand to cultivate: by activated seed mixed liquor in 2-5%(V/V) ratio be inoculated in (liquid amount is fermentation tank volume 60-70%) in the fermentation tank that 5-15% Soy hydrolysate culture medium (pH 6.5-7.5) is housed, standing cultivation 30-36 h under 37-41 ℃ of condition, collect bacterium liquid (bacterium is dense>=5 * 10
9cfu/ml), obtain zymotic fluid;
(3) washing: by zymotic fluid centrifugal 10-15 min under 5000 rpm, abandon supernatant, 1:1-2 is mixed bacterial sediment with sterilized water vibration by volume, the centrifugal supernatant of abandoning, cyclic washing 2 times, uses 2% lactoalbumin soln Eddy diffusion thalline (viable count>=2 * 10 subsequently
10cfu/ml), obtain bacteria suspension;
(4) bacterium glue preparation: in proportion by 4%(W/V) sodium alginate, 0.5%(W/V) CMC, 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 in bacteria suspension, stir and evenly mix anaerobic room N
2, CO
2, H
2gaseous mixture deoxidation 60-90 min, obtains bacterium glue;
(5) calcium chloride solution microcapsules preparation: bacterium glue is injected to 4-5 ℃ of cooling 2-4%(M/V with 20 ml/min flow velocitys with syringe pump), adds stirring (200 rpm) while dripping, solidify 40-50 min, and sterilized water washing three times, obtains wet microcapsules;
(6) drying preservation: the microcapsules that will wet carry out carrying out vacuum packaging after low-temperature vacuum drying, obtain the dry microcapsules of probio.
Shelf-life>=2 year of the dry microcapsule product of this probio, viable count>=1 * 10
11individual/g, Ya Bao≤60%; Faint yellow, free from extraneous odour.
Embodiment 2
A preparation method for probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: the bacterial classification that the Bifidobacterium engineering bacteria glycerine pipe that is stored in-20 ℃ is preserved is inoculated in respectively in seed culture medium with the hay bacillus engineering bacteria list bacterium colony that is stored in 4 ℃, under 37-41 ℃ of condition, Bifidobacterium anaerobism (N
2deoxidation) standing cultivation 24-28 h, hay bacillus engineering bacteria 100-300 rpm shaken cultivation 18-24 h, activate for the first time; Then by the inoculum concentration of 2-4%, access for the second time in seed culture medium, with first activation condition, cultivate 8-10 h respectively and again activate, by the volume ratio of 1:1, mix the nutrient solution of Bifidobacterium engineering bacteria and hay bacillus engineering bacteria, obtain activated seed mixed liquor;
(2) expand to cultivate: by activated seed mixed liquor in 2-5%(V/V) ratio be inoculated in (liquid amount is 60-70%) in the fermentation tank that 5-10% whole milk culture medium (pH 7.0) are housed, standing cultivation 30-36 h under 37-41 ℃ of condition, collect bacterium liquid (bacterium is dense>=1 * 10
10cfu/ml), obtain zymotic fluid;
(3) washing: by zymotic fluid centrifugal 10-15 min under 5000 rpm, abandon supernatant, 1:1-2 is mixed bacterial sediment with sterilized water vibration by volume, the centrifugal supernatant of abandoning, cyclic washing 2 times, use subsequently 2% lactoalbumin soln Eddy diffusion thalline (bacterium is dense>=5 * 10
10cfu/ml), obtain bacteria suspension;
(4) bacterium glue preparation: in proportion by 5%(W/V) sodium alginate, 1.5%(W/V) CMC, 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 in bacteria suspension, stir and evenly mix anaerobic room N
2qi exhaustion oxygen 60-90 min, obtains bacterium glue;
(5) calcium chloride solution microcapsules preparation: bacterium glue is injected to 4-5 ℃ of cooling 2-4%(M/V with 20 ml/min flow velocitys with syringe pump), adds stirring (200 rpm) while dripping, solidify 40-50 min, and sterilized water washing three times, obtains wet microcapsules;
(6) drying preservation: the microcapsules that will wet carry out carrying out vacuum packaging after low-temperature vacuum drying, obtain the dry microcapsules of probio.
Shelf-life>=2 year of the dry microcapsule product of this hay bacillus, viable count>=2 * 10
11individual/g, Ya Bao≤60%; Faint yellow, free from extraneous odour.
Embodiment 3
A preparation method for probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: the bacterial classification that the wild Bifidobacterium glycerine pipe that is stored in-20 ℃ is preserved is inoculated in respectively in seed culture medium with the hay bacillus engineering bacteria list bacterium colony that is stored in 4 ℃, under 37-39 ℃ of condition, wild type Bifidobacterium anaerobism (N
2, CO
2, H
2gaseous mixture deoxidation) standing cultivation 24-28 h, hay bacillus engineering bacteria 200 rpm shaken cultivation 18-24 h, activate for the first time; Then by the inoculum concentration of 2-4%, access for the second time in seed culture medium, with first activation condition, cultivate 8-10 h respectively and again activate, by the volume ratio of 1:1, mix the nutrient solution of wild type Bifidobacterium and hay bacillus engineering bacteria, obtain activated seed mixed liquor;
(2) expand to cultivate: by activated seed mixed liquor in 2-5%(V/V) ratio be inoculated in (liquid amount is 60-70%) in the fermentation tank that 5-15% goat milk culture medium (pH 7.0) are housed, standing cultivation 30-36 h under 37-39 ℃ of condition, collect bacterium liquid (bacterium is dense>=5 * 10
9cfu/ml), obtain zymotic fluid;
(3) washing: by zymotic fluid centrifugal 10-15 min under 5000 rpm, abandon supernatant, 1:2 is mixed bacterial sediment with sterilized water vibration by volume, the centrifugal supernatant of abandoning, cyclic washing 2 times, use subsequently 2% lactoalbumin soln Eddy diffusion thalline (bacterium is dense>=2 * 10
10cfu/ml), obtain bacteria suspension;
(4) bacterium glue preparation: in proportion by 4%(W/V) sodium alginate, 1.0%(W/V) CMC, 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 N
2, CO
2, H
2gaseous mixture deoxidation 60-90 min, obtains bacterium glue;
(5) calcium chloride solution microcapsules preparation: bacterium glue is injected to 4-5 ℃ of cooling 2-4%(M/V with 20 ml/min flow velocitys with syringe pump), adds stirring (200 rpm) while dripping, solidify 60 min, and sterilized water washing three times, obtains wet microcapsules;
(6) drying preservation: the microcapsules that will wet carry out carrying out vacuum packaging after low-temperature vacuum drying, obtain the dry microcapsules of probio.
Shelf-life>=2 year of the dry microcapsule product of this hay bacillus, viable count>=5 * 10
10individual/g, Ya Bao≤60%; Faint yellow, free from extraneous odour.
Embodiment 4
A preparation method for probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: the bacterial classification that the Bifidobacterium engineering bacteria glycerine pipe that is stored in-20 ℃ is preserved is inoculated in respectively in seed culture medium with the single bacterium colony of the wild hay bacillus that is stored in 4 ℃, under 37-39 ℃ of condition, engineering bacteria Bifidobacterium anaerobism (N
2, CO
2, H
2gaseous mixture deoxidation) standing cultivation 24-28 h, wild type hay bacillus 200 rpm shaken cultivation 18-24 h, activate for the first time; Then by the inoculum concentration of 2-4%, access for the second time in seed culture medium, with first activation condition, cultivate 8-10 h respectively and again activate, by the volume ratio of 1:1, mix the nutrient solution of wild type Bifidobacterium and hay bacillus engineering bacteria, obtain activated seed mixed liquor;
(2) expand to cultivate: by activated seed mixed liquor in 2-5%(V/V) ratio be inoculated in (liquid amount is 60-70%) in the fermentation tank that 5-15% mare's milk culture medium (pH 7.0) are housed, standing cultivation 30-36 h under 37-39 ℃ of condition, collect bacterium liquid (bacterium is dense>=1 * 10
10cfu/ml), obtain zymotic fluid;
(3) washing: by zymotic fluid centrifugal 10-15 min under 5000 rpm, abandon supernatant, 1:2 is mixed bacterial sediment with sterilized water vibration by volume, the centrifugal supernatant of abandoning, cyclic washing 2 times, use subsequently 2% lactoalbumin soln Eddy diffusion thalline (bacterium is dense>=3 * 10
10cfu/ml), obtain bacteria suspension;
(4) bacterium glue preparation: in proportion by 5%(W/V) sodium alginate, 1.0%(W/V) CMC, 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 in bacteria suspension, stir and evenly mix anaerobic room N
2, CO
2, H
2gaseous mixture deoxidation 60-90 min, obtains bacterium glue;
(5) calcium chloride solution microcapsules preparation: bacterium glue is injected to 4-5 ℃ of cooling 2-4%(M/V with 20 ml/min flow velocitys with syringe pump), adds stirring (200 rpm) while dripping, solidify 60 min, and sterilized water washing three times, obtains wet microcapsules;
(6) drying preservation: the microcapsules that will wet carry out carrying out vacuum packaging after low-temperature vacuum drying, obtain the dry microcapsules of probio.
Shelf-life>=2 year of the dry microcapsule product of this hay bacillus, viable count>=1 * 10
11individual/g, Ya Bao≤60%; Faint yellow, free from extraneous odour.
Embodiment 5
A preparation method for probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: the bacterial classification that the wild type Bifidobacterium glycerine pipe that is stored in-20 ℃ is preserved is inoculated in respectively in seed culture medium with the single bacterium colony of the wild type hay bacillus that is stored in 4 ℃, under 45 ℃ of conditions, standing cultivation 24 h of wild type Bifidobacterium anaerobism, wild type hay bacillus 100 rpm shaken cultivation 12 h, activate for the first time; Then by 1% inoculum concentration, access for the second time in seed culture medium, with first activation condition, cultivate 4 h respectively and again activate, by the volume ratio of 1:1, mix the nutrient solution of wild type Bifidobacterium and wild type hay bacillus, obtain activated seed mixed liquor;
(2) expand to cultivate: by activated seed mixed liquor in 10%(V/V) ratio be inoculated in (liquid amount is 80%) in the fermentation tank that 20% peanut hydrolyzate culture medium (pH 6.5) is housed, standing cultivation 24 h under 45 ℃ of conditions, collect bacterium liquid (bacterium is dense>=2 * 10
9cfu/ml), obtain zymotic fluid;
(3) washing: by zymotic fluid centrifugal 30 min under 3000 rpm, abandon supernatant, 1:1 is mixed bacterial sediment with sterilized water vibration by volume, the centrifugal supernatant of abandoning, cyclic washing 3 times, use subsequently 5% lactoalbumin soln Eddy diffusion thalline (bacterium is dense>=5 * 10
9cfu/ml), obtain bacteria suspension;
(4) bacterium glue preparation: in proportion by 1%(W/V) sodium alginate, 1%(W/V) CMC, 1%(W/V) compound 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, anaerobic room deoxidation 120 min, obtain bacterium glue;
(5) calcium chloride solution microcapsules preparation: bacterium glue is injected to 1 ℃ of cooling 10%(M/V with 30 ml/min flow velocitys with syringe pump), adds stirring (100 rpm) while dripping, solidify 60 min, and sterilized water washing three times, obtains wet microcapsules;
(6) drying preservation: the microcapsules that will wet carry out carrying out vacuum packaging after low-temperature vacuum drying, obtain the dry microcapsules of probio.
Shelf-life>=2 year of the dry microcapsule product of this probio, viable count>=5 * 10
9individual/g, Ya Bao≤80%; Faint yellow, free from extraneous odour.
Embodiment 6
A preparation method for probiotic microcapsule, comprises the following steps:
(1) activation of bacterial strain: the bacterial classification that the Bifidobacterium engineering bacteria glycerine pipe that is stored in-20 ℃ is preserved is inoculated in respectively in seed culture medium with the hay bacillus engineering bacteria list bacterium colony that is stored in 4 ℃, under 32 ℃ of conditions, standing cultivation 36 h of Bifidobacterium engineering bacteria anaerobism, hay bacillus engineering bacteria 100 rpm shaken cultivation 24 h, activate for the first time; Then by 5% inoculum concentration, access for the second time in seed culture medium, with first activation condition, cultivate 12 h respectively and again activate, by the volume ratio of 1:1, mix the nutrient solution of Bifidobacterium engineering bacteria and hay bacillus engineering bacteria, obtain activated seed mixed liquor;
(2) expand to cultivate: by activated seed mixed liquor in 1%(V/V) ratio be inoculated in (liquid amount is 50%) in the fermentation tank that 2% prepared by yeast hydrolysate culture medium (pH 8.5) are housed, standing cultivation 48 h under 35 ℃ of conditions, collection bacterium liquid (bacterium is dense>=1 * 10
9cfu/ml), obtain zymotic fluid;
(3) washing: by zymotic fluid centrifugal 5 min under 8000 rpm, abandon supernatant, 1:5 is mixed bacterial sediment with sterilized water vibration by volume, the centrifugal supernatant of abandoning, cyclic washing 1 time, use subsequently 1% lactoalbumin soln Eddy diffusion thalline (bacterium is dense>=2 * 10
9cfu/ml), obtain bacteria suspension;
(4) bacterium glue preparation: in proportion by 10%(W/V) sodium alginate, 0.5%(W/V) CMC, 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, stir and evenly mix, anaerobic room deoxidation 30 min, obtain bacterium glue;
(5) calcium chloride solution microcapsules preparation: bacterium glue is injected to 10 ℃ of cooling 1%(M/V with 10 ml/min flow velocitys with syringe pump), adds stirring (300 rpm) while dripping, solidify 30 min, and sterilized water washing three times, obtains wet microcapsules;
(6) drying preservation: the microcapsules that will wet carry out carrying out vacuum packaging after low-temperature vacuum drying, obtain the dry microcapsules of probio.
Shelf-life>=2 year of the dry microcapsule product of this probio, viable count>=1 * 10
9individual/g, Ya Bao≤80%; Faint yellow, free from extraneous odour.
The above only further illustrates technology contents of the present invention with embodiment, so that reader is easier to understand, but does not represent that embodiments of the present invention only limit to this, and any technology of doing according to the present invention is extended or recreation, is all subject to protection of the present invention.
Claims (8)
1. a preparation method with the probiotic microcapsule of high viable count, is characterized in that, comprises the following steps:
(1) activation of bacterial strain: be inoculated in respectively in seed culture medium by being stored in the seed of-20 ℃ of Bifidobacterium glycerine pipes preservations under the condition of left and right and being stored in 4 ℃ of single bacterium colonies of the hay bacillus under the condition of left and right, under 32-45 ℃ of condition, the standing cultivation of Bifidobacterium anaerobism 24-36 h, hay bacillus 100-300 rpm shaken cultivation 12-24 h, activate for the first time;
Then in the ratio of 1-5%, respectively the Bifidobacterium of activation for the first time and hay bacillus nutrient solution are again accessed and in seed culture medium, activate for the second time 4-12 h, activation condition with activate for the first time identical; The volume ratio of pressing 1:1 left and right is mixed the nutrient solution of Bifidobacterium and hay bacillus, obtains activated seed mixed liquor;
(2) expand to cultivate: by activated seed mixed liquor in 1-10%(V/V) ratio be inoculated in the fermentation tank of the natural organism raw material of the 2-20% culture medium that pH 6.5-8.5 is housed, the liquid amount of natural organism raw material culture medium is fermentation tank volume 50-80%, standing cultivation 24-48 h under 35-45 ℃ of condition, collect bacterium liquid, its total viable count>=2 * 10
9cfu/ml, obtains zymotic fluid;
(3) washing: by zymotic fluid centrifugal 5-30 min under 3000-8000 rpm, abandon supernatant, 1:1-5 is mixed bacterial sediment with sterilized water vibration by volume, the centrifugal supernatant of abandoning, cyclic washing 1-3 time, uses 1-5% lactoalbumin soln Eddy diffusion thalline, its total viable count>=1 * 10 subsequently
10cfu/ml, obtains bacteria suspension;
(4) bacterium glue preparation: in proportion by 1-10%(W/V) sodium alginate, 0.5-1%(W/V) CMC, 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, obtains bacterium glue;
(5) calcium chloride solution microcapsules preparation: bacterium glue is injected to 1-10 ℃ of cooling 1-10%(M/V with 10-30 ml/min flow velocity with syringe pump), stir with the speed of 100-300 rpm on dropping limit, limit, solidify 30-60 min, sterilized water washing three times, obtains wet microcapsules;
(6) drying preservation: the microcapsules that will wet carry out carrying out vacuum packaging after low-temperature vacuum drying, obtain the dry microcapsules of probio.
2. the preparation method with the probiotic microcapsule of high viable count according to claim 1, is characterized in that, step (1)-step (5) is all undertaken by sterile working rules.
3. the preparation method with the probiotic microcapsule of high viable count according to claim 1, is characterized in that, it is 40 that wet microcapsules described in step (5) are carried out to low-temperature vacuum drying
oany drying mode below C, and carry out vacuum packaging.
4. the preparation method with the probiotic microcapsule of high viable count according to claim 1, it is characterized in that, bifidobacterium strain described in step (1) refers to one or more in wild type Bifidobacterium, genetic engineering Bifidobacterium, and described hay bacillus bacterial strain refers to one or more in wild type hay bacillus, genetic engineering hay bacillus; Described seed culture medium is the property improved PYTG culture medium.
5. the preparation method with the probiotic microcapsule of high viable count according to claim 1, is characterized in that, the described Anaerobic cultural methods of step (1) is N
2and N
2, CO
2, H
2gaseous mixture carries out any of deoxidation.
6. the preparation method with the probiotic microcapsule of high viable count according to claim 1, it is characterized in that, the described natural organism raw material of step (2) is any in whole milk, goat milk, mare's milk, Soy hydrolysate, peanut hydrolyzate and prepared by yeast hydrolysate.
7. the preparation method with the probiotic microcapsule of high viable count according to claim 1, is characterized in that, the described lactalbumin of step (3) is the lactalbumin in milk.
8. the preparation method with the probiotic microcapsule of high viable count according to claim 1, it is characterized in that, the described compound sugar of step (4) is that human body can not utilize and Bifidobacterium is easy to any of the FOS that absorbs and oligomeric fruit glucose and derivative thereof.
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| CN116784452A (en) * | 2023-05-04 | 2023-09-22 | 河北同福健康产业有限公司 | Composite fermentation bacteria for old-flour steamed bread and preparation method thereof |
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