CN107299071A - A kind of enteric probiotics preparation - Google Patents
A kind of enteric probiotics preparation Download PDFInfo
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- CN107299071A CN107299071A CN201710700583.2A CN201710700583A CN107299071A CN 107299071 A CN107299071 A CN 107299071A CN 201710700583 A CN201710700583 A CN 201710700583A CN 107299071 A CN107299071 A CN 107299071A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Physiology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Animal Husbandry (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of enteric probiotics preparation, and said preparation is to be prepared from by the aerobic bacteria and anaerobic bacteria of mutualism by mixed fermentation.Aerobic bacteria is inoculated into round first, it is aerobic to cultivate to exponential phase, close oxygen, continue micro-aerobe fermentation, and during micro-aerobe fermentation, be inoculated with anaerobic bacteria, so that aerobic bacteria merges symbiosis with anaerobic bacteria, continue to cultivate to anaerobic bacteria exponential phase later stage or later stage stationary phase under anaerobic, obtain tunning, tunning is obtained into probiotics preparation by centrifugation, spray drying, coating enteric material.Preparation technology of the present invention is simple, and utilization rate of equipment and installations is high, and cost is low, is easy to implement large-scale production；Early stage fusion symbiosis has been carried out in aerobic bacteria and anaerobic bacteria during mixed fermentation, and compatibility becomes excellent；The probiotics preparation pan coating enteric material, makes said preparation just start dissolving under the insoluble environment to intestinal juice under hydrochloric acid in gastric juice and bile environmental condition, can reach the targeting adjustment effect to enteron aisle.
Description
Technical field
The present invention relates to a kind of enteric probiotics preparation, belong to field of microbial fermentation.
Background technology
Probiotics preparation is also known as probiotics, is active bacteria formulation and its metabolism for referring to promote intestinal flora balance
Product.Comprising microorganism living in probiotics preparation, when taking in enough quantity, beneficial health work can be played to host
With.Therefore, probiotics plays more next in the fields closely related with human lives such as medicine, agricultural, industry, food and feed
More important effect.
West Europe and Japan study earliest to probiotics preparation, and product category is more, and market is more perfect.To, Europe in 2004
The value of continent probiotics preparation product is up to 3,000,000,000 dollars, and consumption figure accounts for the 5% of total food consumption total amount；And Japan was in 1998
Ratify health food (FOSHU) 108 May, wherein about 50% is probiotics preparation.By the end of, global Tiny ecosystem system in 2010
The output value of agent has reached 4,700,000,000 dollars.Internal authority mechanism has carried out new regulation to the specification of probiotics preparation product：It is recommended that
Every gram of product is contained 1 × 105Individual effective bacterium is used as physiotherapy standard, the effective bacterium of every gram or every milliliter of probiotic products for health care
Number is not less than 1 × 107It is individual.At present, it is external many around Bacillus acidi lactici, Bifidobacterium, bacillus subtilis and some hammer mushrooms
Probiotics preparation is studied, as shown in table 1.
Table 1
| Trade name | Main component | Manufacturer |
| Probios | A variety of Bacillus acidi lacticis | Nuabs.Division.Pioneer |
| Feed-mate68. | Streptococcus fecalis, lactobacillus acidophilus, Lactobacillus plantarum | Anchor labs st.JosePhMo |
| Strain 40. | Lactobacillus acidophilus | Milwaukee.WI |
| Lactiferm | Streptococcus fecalis | AB.Mediphara.Sweden |
| Toyocorin | Bacillus | Toyo.Joso.co.Ltd.Tokyo |
| ΣMI-5 | Lactic acid bacteria, saccharomycete, actinomyces etc. | Japan |
| The refined clostridium butyricum live bacterial chip of rice | Clostridium butyricum | Japanese Miyarisan Pharmaceutical Co Ltd |
1984, Dalian Medical College of China Kang Bai et al. was developed into " cereobiogen " viable bacteria system with bacillus cereus first
Agent, up to now, production probiotics preparation enterprise of China have exceeded various schools of thinkers, annual nearly 10,000,000,000 yuan of apparent consumption, and on year by year
The trend of liter, in the market has the return of spring life (main component that Dalian Medical College develops using more probiotics preparation：Bifidobacterium), Shen
Positive first pharmaceutical factory develops Zheng Chang Sheng (main component：Bacillus licheniformis) and Chongqing Tai Ping pharmaceutcal corporation, Ltds develop butyric acid
Clostridium viable capsule (main component：Clostridium butyricum) etc..
Probiotics preparation is mainly according to microecology principle, and the preparation studied using microflora strain can be supplemented
Or substantial microorganism species, maintain or adjustment microecological balance, reach the purpose prevented and cured diseases.Clostridium butyricum, Bifidobacterium, plant
Thing lactobacillus, Lactobacillus casei etc. are all the strains of common regulation human body intestinal canal microecological balance, can suppress harmful intestinal tract
Bacterium, eliminates enterotoxin, and prevention of postoperative infection improves immunity of organisms, diminished inflammation, recovers gut flora balance, set up intestines
Road biological barrier.
In the industrial fermentation of probiotics preparation both domestic and external, microbial inoculum is generally made or by list using single bacterial strain purification of fermentation
One microbial inoculum is composited again, and such as patent CN102517238B discloses a kind of sour bacillus cereus of production and its application, the invention
Bacillus bacterium powder is obtained by first order seed, secondary seed, three-level seed and fermentation tank culture, and bacillus bacterium powder is independent
Probiotics, or the sour bacillus cereus bacterium powder of production and other at least one are mixed and made into as active ingredient and cornstarch
Plant active ingredient and cornstarch is mixed and made into compound micro-ecological preparation, program complex process, cost is high, and will be multiple
Single culture is developed into composite probiotics preparations, and it is bad there is also acting synergistically in intestinal microenvironment, and poor compatibility etc. is asked
Topic.Therefore, the important development trend that mixed fungus fermentation is domestic and international probiotics preparation is turned to by single bacteria fermentation, but mixes bacterium at present
The bacterium of fermentation is made up of obligate anaerobe mostly, is typically to be sent out by two or more Intestinal flora by mixing anaerobic
Ferment is formed.As CN102240303B discloses a kind of production method for the compound probiotic for preventing and treating intestinal cancer, by by clostridium butyricum
Culture medium is accessed with lactobacillus acidophilus, under conditions of 35~40 DEG C, anaerobism is mixed 24~48 hours, is made and is prevented and treated intestinal cancer
Compound probiotic bacterium solution.This hybrid anaerobic fermentation mode equipment is complicated, and technique is cumbersome, it is necessary to strict control anaerobism ring
Border, prepares cost high.
To sum up, at present in the research process of probiotics preparation, lacking mixing that aerobic fermentation and anaerobic fermentation are combined
Probiotics preparation prepared by fermentation process is closed, even if multiple single cultures are developed into composite probiotics preparations, it is micro- in enteron aisle
Bad there is also acting synergistically in environment, poor compatibility, the problems such as cost is high, in addition, probiotics preparation are held when oral
It is vulnerable to the effect of hydrochloric acid in gastric juice and bile, viable count can significantly decline before action position enteron aisle is reached, so as to influence prebiotic
The curative effect or health-care effect of bacterium product.
The content of the invention
In order to solve the above problems, the invention provides a kind of enteric probiotics preparation, by the aerobic of mutualism relation
Bacterium and anaerobic bacteria, the mixed fermentation mode being combined by anaerobic fermentation after first aerobic fermentation are prepared from, and optimize fermentation
Condition causes two kinds of far different bacterial strains of aerobic fermentation and anaerobic fermentation to be completed in integral fermentation system.By which
Symbiosis is merged in the enteric probiotics preparation fermentation process of preparation, and compatibility becomes excellent.And the probiotics preparation pan coating intestines
Molten material, makes said preparation just start dissolving under the insoluble environment to intestinal juice under hydrochloric acid in gastric juice and bile environmental condition, can reach to intestines
Road targets the purpose of adjustment effect.
The present invention provides a kind of enteric probiotics preparation, and specific solution is as follows：
A kind of enteric probiotics preparation, it is characterised in that：Said preparation is the aerobic bacteria and anaerobic bacteria by mutualism relation
It is prepared from by mixed fermentation；The aerobic bacteria and anaerobic bacteria are all bacillus；
The aerobic bacteria is inoculated into round first, and aerobic culture closes oxygen to exponential phase, described aerobic
Bacterium irritability it is transformed into brood-gemma, continues micro-aerobe fermentation, and during the micro-aerobe fermentation, is inoculated with anaerobic bacteria so that institute
State aerobic bacteria and merge symbiosis with the anaerobic bacteria, continue to cultivate under anaerobic to the anaerobic bacteria exponential phase later stage or
In later stage stationary phase, tunning is obtained, the tunning is obtained by centrifuging, being spray-dried, be coated with enteric material
The enteric probiotics preparation；
Aerobic bacterium number is more than or equal to 1.5 × 10 in the tunning8Cfu/ml, anaerobism bacterium number is more than or equal to
1.2×108cfu/ml；
Effective bacterium number is more than or equal to 10 in the enteric probiotics preparation9cfu/g。
Further, the aerobic bacteria is bacillus cereus, bacillus coagulans or bacillus subtilis, the anaerobic bacteria
It is clostridium butyricum.
Preferably, the aerobic bacteria is bacillus cereus, and the anaerobic bacteria is clostridium butyricum.Bacillus cereus is normal
The strain for the regulation human body intestinal canal microecological balance seen, can produce antibacterial material, suppress harmful microbe breeding, improve human body
With animal intestinal micro-ecology environment.Clostridium butyricum is one of normal bowel bacterium of human body, is that safely, effectively, poison is not secondary for one kind
The probiotics of effect, finds that the mechanism of action that clostridium butyricum prevents and treats intestines problem is mainly following side by studying
Face：1. butyric acid is secreted, nutrition enteron aisle repairs mucous membrane；2. butyric acid, controlling gene expression are secreted；3. suppress harmful intestinal tract bacteria, eliminate
Enterotoxin, prevention of postoperative infection；4. kininase is produced, cancerous tissue is dissolved；5. immunity of organisms is improved；6. diminish inflammation；⑦
Recover gut flora balance, set up enteron aisle biological barrier.
Preferably, the enteric probiotics preparation be coated with the tablet of enteric material, anther sac agent, granule, pill, suppository,
One or more in capsule, powder.
It is preferred that, the enteric material is acrylic resin, cellulose acetate-phthalate, Diclofenac, shellac, poly-
Vinyl alcohol acetate phthalate ester, 1,2,4- benzenetricarboxylic acid cellulose acetates, Hydroxypropyl Methylcellulose Phathalate, 1,2,
One in 4- benzenetricarboxylic acids hydroxypropyl methyl cellulose, acetate succinate cellulose, HPMCAS
Plant or a variety of.Said preparation is just started dissolving under the insoluble environment to intestinal juice under hydrochloric acid in gastric juice and bile environmental condition, can reach pair
The purpose of intestinal-specific adjustment effect.
Present invention also offers a kind of preparation method of enteric probiotics preparation, comprise the following steps：
(1) aerobic bacteria is sequentially passed through after inclined-plane culture, Shaking culture and seed tank culture, be inoculated into equipped with sterilizing
In the round of fermentation medium, be passed through filtrated air at a temperature of 37 ± 1 DEG C, stir culture 24-32h, obtain in pair
The aerobic bacteria in number growth period；
(2) anaerobic bacteria is sequentially passed through into inclined-plane culture, the culture of anaerobism bottle and seeding tank Anaerobic culturel, in incubation
Middle holding anaerobic environment, Anaerobic culturel obtains seeding tank anaerobism seed；
(3) after the step (1) obtains the aerobic bacteria in exponential phase, filtrated air, the aerobic bacteria are closed
Irritability it is transformed into brood-gemma, continues micro-aerobe fermentation 4-8h, and during the micro-aerobe fermentation, step (2) is obtained
Product is inoculated into the round so that the aerobic bacteria merges symbiosis 18-32h with the anaerobic bacteria, is cultivated to described
Anaerobic bacteria exponential phase later stage or later stage stationary phase, obtain tunning；
(4) step (3) is obtained into mixed fermentation product by centrifuging, being spray-dried, be coated with enteric material, institute is made
The enteric probiotics preparation stated.
Preferably, step (1) stir culture refers to, with 100-130r/min stir cultures, be passed through filtrated air volume
With the volume ratio 1 of the fermentation medium:0.5-0.6.
Preferably, the inoculum concentration of the aerobic bacteria is the 6~15% of the fermentation medium cumulative volume, the anaerobic bacteria
Inoculum concentration is the 10~20% of the fermentation medium cumulative volume.
Preferably, the fermentation medium main component is：Contain beancake powder 3%, corn flour 3%, wheat bran in per 100g water
1%th, glucose 1%, tryptone 1%, yeast extract 0.5%, beef extract 0.3%, sodium chloride 0.2%, dipotassium hydrogen phosphate
0.2%th, potassium dihydrogen phosphate 0.015%, ferrous sulfate 0.1%, manganese sulfate 0.02%, calcium carbonate 0.1%, Tween.80
0.02%, the percentage is all mass percent.
Present invention also offers enteric probiotics preparation obtained by a kind of preparation method of enteric probiotics preparation in people or poultry
Application in medicine, health food or the feed addictive of intestines problem.
Beneficial effects of the present invention are：
(1) the enteric probiotics preparation that the present invention is provided, aerobic bacteria and anaerobic bacteria by mutualism relation, by first good
The mixed fermentation mode that anaerobic fermentation is combined after aerobe fermentation is prepared from, and technique is simple, is easy to implement large-scale production.
(2) present invention cleverly make use of aerobic and anaerobism bacterial strain to the difference of oxygen psychological need, and it is mutually held in the mouth
Ground connection is incorporated into same fermentation, and optimizes fermentation condition so that two kinds of far different bacterial strains of aerobic fermentation and anaerobic fermentation
Completed in integral fermentation system, not only saved fermentation resource such as fermentation medium and round etc., and improve
Utilization rate of equipment and installations, shortens the whole production technology time, preparation cost is at least lowered half.
(3) early stage fusion symbiosis has been carried out in the aerobic bacteria and anaerobic bacteria that the present invention is provided during mixed fermentation,
Complementary not antagonism, compatibility becomes excellent, is conducive to probiotics preparation is mutually coordinated in same environment to play a role, so as to play
Human body or animal intestinal micro-ecology balance, the harmful intestinal tract bacteria that suppresses are adjusted, enterotoxin is eliminated, improves immunity of organisms, recovers
Gut flora balances and set up the effect of enteron aisle biological barrier.
(4) the enteric probiotics preparation that the present invention is provided can be with tablet, anther sac agent, granule, pill, suppository, capsule
The various states such as agent are present, and form of diverse is easy to carry, instant.
(5) the enteric probiotics preparation pan coating enteric material that the present invention is provided, makes said preparation in hydrochloric acid in gastric juice and bile ring
Just start dissolving under the conditions of border under the insoluble environment to intestinal juice, the targeting adjustment effect to enteron aisle can be reached.
Below by accompanying drawing, the present invention will be further described.
Brief description of the drawings
Fig. 1 is a kind of preparation technology schematic flow sheet for enteric probiotics preparation that the present invention is provided.
Embodiment
Below by way of specific specific implementation explanation embodiments of the present invention, those skilled in the art can be by this specification
Disclosed content understands other advantages and effect of the present invention easily.
Embodiment 1
The present embodiment provides a kind of enteric probiotics preparation, and said preparation is the waxy gemma of aerobic bacteria by mutualism relation
Bacillus and anaerobic bacteria clostridium butyricum are prepared from by mixed fermentation.To make bacillus cereus and clostridium butyricum in same system
In it is mutually coordinated play a role, before the mixing of two kinds of bacterium, in mixing, mixed growth conditions and inoculum concentration during mixing it is non-
It is often important.
The preparation method for the enteric probiotics preparation that the present embodiment is provided, its process flow diagram are as shown in figure 1, specific
Step is as follows：
1st, the bacillus cereus in exponential phase is obtained.
1. inclined-plane culture：The bacillus cereus strain (CGMCC NO.8614) of preservation is transferred to slant medium,
Ensure under aseptic condition, be inoculated with oese in plate, 37 DEG C of culture 24h.Switching three generations inclined-plane is repeated, is often trained for slant strains
After the completion of supporting, dyeing, with micro- sem observation bacillus cereus growing state, selects without miscellaneous bacteria and the excellent strain of growth is standby
With.
The slant medium is PDA culture medium：Potato 200.0g, 20.0g, agar 20.0g, water 1.0L, natural pH
Value.
2. Shaking culture：Inclined plane inoculating, 500mL triangular flask loading amounts 100mL, 37 DEG C, 200r/min shaking table cultures 16~
24h, access seeding tank expands culture.
3. seed tank culture：5L triangular flask loading amount 1L, inoculum concentration 6% (v/v), 37 DEG C, 200r/min shaking table cultures 18~
24h。
Culture medium used in the Shaking culture and seed tank culture：Peptone 10.0g, yeast extract 3.0g, NaCl
5.0g, water 1.0L, natural ph.
Bacillus cereus is sequentially passed through after above-mentioned inclined-plane culture, Shaking culture and seed tank culture, is inoculated into and is equipped with
In the 250L stirred-tank fermenters of 150L sterilization fermentation culture mediums, the inoculum concentration of bacillus cereus is fermentation medium cumulative volume
6~15%, be passed through filtrated air at a temperature of 37 ± 1 DEG C, with 100-130r/min stir culture 24-32h, be passed through sterile sky
The volume ratio 1 of air volume and the fermentation medium：0.5-0.6, obtains the bacillus cereus in exponential phase.
The fermentation medium components are：Contain beancake powder 3%, corn flour 3%, wheat bran 1%, glucose in per 100g water
1%th, tryptone 1%, yeast extract 0.5%, beef extract 0.3%, sodium chloride 0.2%, dipotassium hydrogen phosphate 0.2%, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.015%, ferrous sulfate 0.1%, manganese sulfate 0.02%, calcium carbonate 0.1%, Tween.80 0.02%, the percentage
All it is mass percent.
(2) clostridium butyricum seeding tank Anaerobic culturel seed is obtained
1. inclined-plane culture：The clostridium butyricum strain (CICC NO.10390) of preservation is transferred to slant medium, ensured
Under anaerobic condition, by the way of sterile working, inclined-plane is inoculated with oese, in 37 DEG C in anaerobic culture box culture 24h, with aobvious
Micro mirror observes bacillus cereus growing state, selects without miscellaneous bacteria and the excellent strain of growth is standby.
The slant medium：Beef extract 3.0g, peptone 10.0g, NaCl 5.0g, agar 20.0g, water 1.0L, pH=
7.2±0.2。
2. Shaking culture：1ml bacterium suspensions, which are drawn, with aseptic straw is inoculated into the 250ml triangular flasks equipped with 50ml culture mediums
In, in 37 DEG C in anaerobic culture box static gas wave refrigerator 24h, and mixing is rocked every 5h, switching is attached to 450ml culture mediums 2500ml tri-
In the bottle of angle, 37 DEG C of constant temperature static gas wave refrigerator 24h, and rock mixing every 5h.
3. seeding tank Anaerobic culturel：Anaerobism bottle seed microscopy without transferring in the seeding tank equipped with 4.5L culture mediums again after miscellaneous bacteria
In, the volume ratio of sterile nitrogen and culture medium is 1:1, Anaerobic culturel 24h.
Culture medium used in above-mentioned Shaking culture and seeding tank Anaerobic culturel：Gravy 10.0g, peptone 3.0g, yeast is taken out
Extract 3.0g, NaCl 5.0g, glucose 5.0g, starch 3.0g, NaAc 3.0g, L-cysteine hydrochloride 0.5g, water 1.0L,
PH=6.8 ± 0.2.
Clostridium butyricum is sequentially passed through into above-mentioned inclined-plane culture, the culture of anaerobism bottle and seeding tank Anaerobic culturel, in incubation
In sterile nitrogen is passed through to keep anaerobic environment, Anaerobic culturel obtains seeding tank Anaerobic culturel seed.
(3) after the step (1) obtains the bacillus cereus in exponential phase, filtrated air is closed, it is described
Bacillus cereus irritability it is transformed into brood-gemma, continues micro-aerobe fermentation 4-8h, and during the micro-aerobe fermentation, will walk
Suddenly the product that (2) are obtained is inoculated into the round, and the inoculum concentration of the anaerobic bacteria is the fermentation medium cumulative volume
10~20%.So that the bacillus cereus merges symbiosis 18-32h, culture to the butyric acid shuttle with the clostridium butyricum
Bacterium exponential phase later stage or later stage stationary phase, obtain tunning.
(4) step (3) is obtained into mixed fermentation product by centrifuging, being spray-dried, be coated with enteric material, institute is made
The enteric probiotics preparation stated.
The present embodiment using colony counting method determine tunning in bacillus cereus number be more than or equal to 1.5 ×
108Cfu/ml, clostridium butyricum number is more than or equal to 1.2 × 108cfu/ml；Effective bacterium number is big in gained enteric probiotics preparation
In or equal to 109cfu/g。
Enteric probiotics preparation in the present embodiment 1 is the capsule for being coated with acrylic resin, meanwhile, the enteric material is also
It can be cellulose acetate-phthalate, Diclofenac, shellac, PVAP, 1,2,4- benzene front threes
Sour cellulose acetate, Hydroxypropyl Methylcellulose Phathalate, 1,2,4- benzenetricarboxylic acid hydroxypropyl methyl celluloses, butanedioic acid
One or more in cellulose acetate, HPMCAS；The form of said preparation can also be tablet,
Anther sac agent, granule, pill, suppository, the one or more of powder.
Meanwhile, in addition to the culture medium that embodiment 1 is mentioned, other are any suitable for Bacillus cereus and clostridium butyricum growth
Solid medium or fluid nutrient medium may be applicable to the preparation of the enteric probiotics preparation of the present embodiment 1.
The enteric probiotics preparation that embodiment 1 is provided, aerobic bacteria and anaerobic bacteria by mutualism relation, by first aerobic
The mixed fermentation mode that anaerobic fermentation is combined after fermentation is prepared from, and technique is simple, is easy to implement large-scale production；This implementation
Example cleverly make use of aerobic bacteria and anaerobic bacteria to the difference of oxygen psychological need, and be incorporated into same hair with being mutually linked
In ferment, and fermentation condition is optimized so that two kinds of far different bacterial strains of aerobic fermentation and anaerobic fermentation are with integral fermentation system
It is middle to complete, fermentation resource such as fermentation medium and round etc. have not only been saved, and utilization rate of equipment and installations is improved, shorten
The whole production technology time, preparation cost is set at least to lower half；The aerobic bacteria and anaerobic bacteria that the present embodiment is provided are sent out in mixing
Early stage fusion symbiosis has been carried out during ferment, complementary not antagonism, compatibility becomes excellent, is conducive to probiotics preparation in same ring
It is mutually coordinated in border to play a role, so as to play regulation human body or animal intestinal micro-ecology balance, suppress harmful intestinal tract bacteria, disappear
Except enterotoxin, immunity of organisms is improved, recover gut flora balance and sets up the effect of enteron aisle biological barrier；The present embodiment is carried
The enteric probiotics preparation of confession can exist with various states such as tablet, anther sac agent, granule, pill, suppository, capsules, shape
State is various, is easy to carry, instant；The enteric probiotics preparation pan coating enteric material that the present embodiment is provided, makes the system
Agent just starts dissolving under hydrochloric acid in gastric juice and bile environmental condition under the insoluble environment to intestinal juice, can reach that the targeting regulation to enteron aisle is made
With.
Embodiment 2
The present embodiment provides a kind of enteric probiotics preparation, and said preparation is to condense gemma by the aerobic bacteria of mutualism relation
Bacillus and anaerobic bacteria clostridium butyricum are prepared from by mixed fermentation.To make bacillus coagulans and clostridium butyricum in same system
In it is mutually coordinated play a role, before the mixing of two kinds of bacterium, in mixing, mixed growth conditions and inoculum concentration during mixing it is non-
It is often important.
The preparation method for the enteric probiotics preparation that the present embodiment is provided, its process flow diagram are as shown in figure 1, specific
Step is as follows：
1st, the bacillus coagulans in exponential phase is obtained.
1. inclined-plane culture：The bacillus coagulans strain (CICC NO.21736) of preservation is transferred to slant medium,
Ensure under aseptic condition, the streak inoculation on nutrient agar culture medium, in cultivating 20h at 37 DEG C.
Slant medium：Peptone 10.0g, beef extract 3.0g, NaCl 5.0g, agar 15.0g, water 1.0L, pH 7.0.
2. Shaking culture：Take in 250ml triangular flasks of the ring inclined-plane access equipped with 25ml MRS culture mediums, 40 DEG C, 140r/
Min cultivates 20h, as primary seed solution, is accessed primary seed solution equipped with 120ml MRS culture mediums with 5% inoculum concentration
In 500ml triangular flasks, 40 DEG C, 180r/min cultures 18h.
The MRS culture mediums：Peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, Tween-80 1ml, citric acid three
Ammonium 2.0g, K2HPO42.0g, glucose 20.0g, sodium acetate 5.0g, MgSO4·7H2O 0.1g, MnSO40.05g, water 1.0L,
pH7.0。
3. seed tank culture：Accessed with 5% inoculum concentration in the 5L Fermentations equipped with 3.5L culture mediums, set liquid amount
For 70%, starting speed of agitator is 100r/min, and throughput is 1：1, it is 100% now to determine dissolved oxygen；When dissolved oxygen be down to 30% with
When lower, rotating speed is improved to control dissolved oxygen more than 30%, 40 DEG C of culture 48h of temperature.
Culture medium used in seed tank culture：Beancake powder 10.0g, corn starch 10.0g, glucose 6.0g, MgSO4·
7H2O 1.0g, K2HPO41.0g, MnSO450ppm, water 1.0L, pH7.0.
Bacillus coagulans is sequentially passed through after above-mentioned inclined-plane culture, Shaking culture and seed tank culture, is inoculated into and is equipped with
In the 250L stirred-tank fermenters of 150L sterilization fermentation culture mediums, the inoculum concentration of bacillus coagulans is fermentation medium cumulative volume
6~15%, be passed through filtrated air at a temperature of 37 ± 1 DEG C, with 100-130r/min stir culture 24-32h, be passed through sterile sky
The volume ratio 1 of air volume and the fermentation medium：0.5-0.6, obtains the bacillus coagulans in exponential phase.
The fermentation medium components are：Contain beancake powder 3%, corn flour 3%, wheat bran 1%, glucose in per 100g water
1%th, tryptone 1%, yeast extract 0.5%, beef extract 0.3%, sodium chloride 0.2%, dipotassium hydrogen phosphate 0.2%, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.015%, ferrous sulfate 0.1%, manganese sulfate 0.02%, calcium carbonate 0.1%, Tween.80 0.02%, the percentage
All it is mass percent.
(2) clostridium butyricum seeding tank Anaerobic culturel seed is obtained；
Step is identical with the preparation method step (2) for the enteric probiotics preparation that embodiment 1 is provided.
(3) after the step (1) obtains the bacillus coagulans in exponential phase, filtrated air is closed, it is described
Bacillus coagulans irritability it is transformed into brood-gemma, continues micro-aerobe fermentation 4-8h, and during the micro-aerobe fermentation, will walk
Suddenly the product that (2) are obtained is inoculated into the round, and the inoculum concentration of the anaerobic bacteria is the fermentation medium cumulative volume
10~20% so that the bacillus coagulans merges symbiosis 18-32h with the clostridium butyricum, culture to the butyric acid shuttle
Bacterium exponential phase later stage or later stage stationary phase, obtain tunning.
(4) step (3) is obtained into mixed fermentation product by centrifuging, being spray-dried, be coated with enteric material, institute is made
The enteric probiotics preparation stated.
The present embodiment using colony counting method determine tunning in bacillus coagulans number be more than or equal to 1.5 ×
108Cfu/ml, clostridium butyricum number is more than or equal to 1.2 × 108cfu/ml；Effective bacterium number is big in gained enteric probiotics preparation
In or equal to 109cfu/g。
Enteric probiotics preparation in the present embodiment 2 is the pill for being coated with shellac, meanwhile, the enteric material can also be adjacent
Cellulose Acetate Phthalate, Diclofenac, acrylic resin, PVAP, 1,2,4- benzenetricarboxylic acids
Cellulose acetate, Hydroxypropyl Methylcellulose Phathalate, 1,2,4- benzenetricarboxylic acid hydroxypropyl methyl celluloses, butanedioic acid second
One or more in acid cellulose, HPMCAS；The form of said preparation can also be tablet, medicine
Wafer, granule, capsule, suppository, the one or more of powder.
Meanwhile, in addition to the culture medium that embodiment 2 is mentioned, other are any suitable for Bacillus cereus and clostridium butyricum growth
Solid medium or fluid nutrient medium may be applicable to the preparation of the enteric probiotics preparation of the present embodiment 2.
The enteric probiotics preparation that embodiment 2 is provided, aerobic bacteria and anaerobic bacteria by mutualism relation, by first aerobic
The mixed fermentation mode that anaerobic fermentation is combined after fermentation is prepared from, and technique is simple, is easy to implement large-scale production；This implementation
Example cleverly make use of aerobic bacteria and anaerobic bacteria to the difference of oxygen psychological need, and be incorporated into same hair with being mutually linked
In ferment, and fermentation condition is optimized so that two kinds of far different bacterial strains of aerobic fermentation and anaerobic fermentation are with integral fermentation system
It is middle to complete, fermentation resource such as fermentation medium and round etc. have not only been saved, and utilization rate of equipment and installations is improved, shorten
The whole production technology time, preparation cost is set at least to lower half；The aerobic bacteria and anaerobic bacteria that the present embodiment is provided are sent out in mixing
Early stage fusion symbiosis has been carried out during ferment, complementary not antagonism, compatibility becomes excellent, is conducive to probiotics preparation in same ring
It is mutually coordinated in border to play a role, so as to play regulation human body or animal intestinal micro-ecology balance, suppress harmful intestinal tract bacteria, disappear
Except enterotoxin, immunity of organisms is improved, recover gut flora balance and sets up the effect of enteron aisle biological barrier；The present embodiment is carried
The enteric probiotics preparation of confession can exist with various states such as tablet, anther sac agent, granule, pill, suppository, capsules, shape
State is various, is easy to carry, instant；The enteric probiotics preparation pan coating enteric material that the present embodiment is provided, makes the system
Agent just starts dissolving under hydrochloric acid in gastric juice and bile environmental condition under the insoluble environment to intestinal juice, can reach that the targeting regulation to enteron aisle is made
With.
Embodiment 3
The present embodiment provides a kind of enteric probiotics preparation, and said preparation is the aerobic bacteria withered grass gemma by mutualism relation
Bacillus and anaerobic bacteria clostridium butyricum are prepared from by mixed fermentation.To make bacillus subtilis and clostridium butyricum in same system
In it is mutually coordinated play a role, before the mixing of two kinds of bacterium, in mixing, mixed growth conditions and inoculum concentration during mixing it is non-
It is often important.
The preparation method for the enteric probiotics preparation that the present embodiment is provided, its process flow diagram are as shown in figure 1, specific
Step is as follows：
1st, the bacillus subtilis in exponential phase is obtained.
1. inclined-plane culture：Bacillus subtilis is inoculated with (CICC NO.10732) inclined-plane training is carried out on slant medium
Support, control slant medium pH6.8-7.2, it is 36~38 DEG C to control temperature, cultivates 24~48h.
Slant medium：Peptone 10.0g, yeast extract powder 5.0g, glucose 5.0g, sodium chloride 10.0g, agar
20.0g, water 1.0L, natural ph.
2. Shaking culture：Bacillus subtilis after inclined-plane culture is inoculated on culture medium, controls pH6.8-7.2, control
Temperature is 36~38 DEG C, 10~16h of 180r/min shaking table cultures.
Shaking culture used medium：Peptone 10.0g, yeast extract powder 5.0g, glucose 5.0g, sodium chloride
10.0g, water 1.0L, natural ph.
3. seed tank culture：Seed liquor through shake-flask seed culture is inoculated on culture medium, is in 35~38 DEG C, rotating speed
6-8h is cultivated in 200r/min, pH value control between 6.0-6.5, controls to cultivate 18h in 7.0-7.4 thereafter.
Seed tank culture used medium：Dregs of beans 30.0g, corn flour 10.0g, dusty yeast 0.2g, potassium dihydrogen phosphate 1.5g,
Dipotassium hydrogen phosphate 3.0g, magnesium sulfate 0.5gL, ferrous sulfate 0.1g, calcium carbonate 0.1g, manganese sulfate 0.2g, sodium chloride 5.0g, water
1.0L, pH are 7.0.
Bacillus subtilis is sequentially passed through after above-mentioned inclined-plane culture, Shaking culture and seed tank culture, is inoculated into and is equipped with
In the 250L stirred-tank fermenters of 150L sterilization fermentation culture mediums, the inoculum concentration of bacillus subtilis is fermentation medium cumulative volume
6~15%, be passed through filtrated air at a temperature of 37 ± 1 DEG C, with 100-130r/min stir culture 24-32h, be passed through sterile sky
The volume ratio 1 of air volume and the fermentation medium：0.5-0.6.Obtain the bacillus subtilis in exponential phase.
The fermentation medium components are：Contain beancake powder 3%, corn flour 3%, wheat bran 1%, glucose in per 100g water
1%th, tryptone 1%, yeast extract 0.5%, beef extract 0.3%, sodium chloride 0.2%, dipotassium hydrogen phosphate 0.2%, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.015%, ferrous sulfate 0.1%, manganese sulfate 0.02%, calcium carbonate 0.1%, Tween.80 0.02%, the percentage
All it is mass percent.
(2) clostridium butyricum seeding tank Anaerobic culturel seed is obtained；
The step is identical with the preparation method step (2) for the enteric probiotics preparation that embodiment 1 is provided.
(3) after the step (1) obtains the bacillus subtilis in exponential phase, filtrated air is closed, it is described
Bacillus subtilis irritability it is transformed into brood-gemma, continues micro-aerobe fermentation 4-8h, and during the micro-aerobe fermentation, will walk
Suddenly the product that (2) are obtained is inoculated into the round, and the inoculum concentration of the anaerobic bacteria is the fermentation medium cumulative volume
10~20% so that the bacillus subtilis merges symbiosis 18-32h with the clostridium butyricum, culture to the butyric acid shuttle
Bacterium exponential phase later stage or later stage stationary phase, obtain tunning.
(4) step (3) is obtained into mixed fermentation product by centrifuging, being spray-dried, be coated with enteric material, institute is made
The enteric probiotics preparation stated.
The present embodiment using colony counting method determine tunning in bacillus subtilis bacterium number be more than or equal to 1.5 ×
108Cfu/ml, clostridium butyricum number is more than or equal to 1.2 × 108cfu/ml；Effective bacterium number is big in gained enteric probiotics preparation
In or equal to 109cfu/g。
The enteric probiotics preparation of the present embodiment 3 is the tablet for being coated with cellulose acetate-phthalate, meanwhile, the enteric
Material can also be acrylic resin, Diclofenac, shellac, PVAP, 1,2,4- benzenetricarboxylic acid second
Acid cellulose, Hydroxypropyl Methylcellulose Phathalate, 1,2,4- benzenetricarboxylic acid hydroxypropyl methyl celluloses, acetate succinate
One or more in cellulose, HPMCAS；The form of said preparation can also be capsule, medicine
Wafer, granule, pill, suppository, the one or more of powder.
Meanwhile, in addition to the culture medium that embodiment 3 is arrived, other are any to grow suitable for Bacillus cereus and clostridium butyricum
Solid medium or fluid nutrient medium may be applicable to the preparation of the enteric probiotics preparation of the present embodiment 3.
The enteric probiotics preparation that embodiment 3 is supplied, aerobic bacteria and anaerobic bacteria by mutualism relation pass through first aerobic hair
The mixed fermentation mode that anaerobic fermentation is combined after ferment is prepared from, and technique is simple, is easy to implement large-scale production；The present embodiment
Aerobic bacteria and anaerobic bacteria cleverly be make use of to the difference of oxygen psychological need, and be incorporated into same fermentation with being mutually linked
In, and fermentation condition is optimized so that two kinds of far different bacterial strains of aerobic fermentation and anaerobic fermentation are in integral fermentation system
Complete, not only saved fermentation resource such as fermentation medium and round etc., and improve utilization rate of equipment and installations, shorten whole
The individual production technology time, preparation cost is set at least to lower half；The aerobic bacteria of the present embodiment offer and anaerobic bacteria are in mixed fermentation
During early stage fusion symbiosis has been carried out, complementary not antagonism, compatibility becomes excellent, is conducive to probiotics preparation in same environment
In it is mutually coordinated play a role so that play regulation human body or animal intestinal micro-ecology balance, suppress harmful intestinal tract bacteria, eliminate
Enterotoxin, raising immunity of organisms, recovery gut flora balance and the effect for setting up enteron aisle biological barrier；The present embodiment is provided
Enteric probiotics preparation can exist with various states such as tablet, anther sac agent, granule, pill, suppository, capsules, form
It is various, it is easy to carry, instant；The enteric probiotics preparation pan coating enteric material that the present embodiment is provided, makes said preparation
Just start dissolving under the insoluble environment to intestinal juice under hydrochloric acid in gastric juice and bile environmental condition, can reach that the targeting regulation to enteron aisle is made
With.
It is described above, only presently preferred embodiments of the present invention, the principle of the merely exemplary explanation present invention of above-described embodiment and
Its effect, and formal and substantial limitation not any to the present invention.It should be pointed out that for the common skill of the art
Art personnel, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these improve and supplemented
It should be regarded as protection scope of the present invention.All those skilled in the art, are not departing from the feelings of the spirit and scope of the present invention
It is this when the equivalent variations for a little variation, modification and evolution made using disclosed above technology contents under condition
The equivalent embodiment of invention；Meanwhile, it is all to obtain any equivalent variations that substantial technological is made to above-described embodiment according to the present invention
Variation, modification and evolution, in the range of still falling within technical scheme.
Claims (10)
1. a kind of enteric probiotics preparation, it is characterised in that：Said preparation is led to by the aerobic bacteria and anaerobic bacteria of mutualism relation
Mixed fermentation is crossed to be prepared from；Affiliated aerobic bacteria and anaerobic bacteria are all bacillus；
The aerobic bacteria is inoculated into round first, and aerobic culture closes oxygen to exponential phase, and the aerobic bacteria should
It is transformed into brood-gemma with swashing property, continues micro-aerobe fermentation, and during the micro-aerobe fermentation, is inoculated with anaerobic bacteria so that described good
Oxygen bacterium merges symbiosis with the anaerobic bacteria, continues to cultivate to the anaerobic bacteria exponential phase later stage or stably under anaerobic
In later stage phase, tunning is obtained, the tunning is obtained described by centrifuging, being spray-dried, being coated with enteric material
Enteric probiotics preparation；
Aerobic bacterium number is more than or equal to 1.5 × 10 in the tunning8Cfu/ml, anaerobism bacterium number be more than or equal to 1.2 ×
108cfu/ml；
Effective bacterium number is more than or equal to 10 in the enteric probiotics preparation9cfu/g。
2. enteric probiotics preparation according to claim 1, it is characterised in that：The aerobic bacteria be bacillus cereus,
Bacillus coagulans or bacillus subtilis, the anaerobic bacteria are clostridium butyricums.
3. enteric probiotics preparation according to claim 2, it is characterised in that：The aerobic bacteria is bacillus cereus,
The anaerobic bacteria is clostridium butyricum.
4. according to any described enteric probiotics preparations of claim 1-3, it is characterised in that：Said preparation is coating enteric material
Tablet, anther sac agent, granule, pill, suppository, capsule, the one or more in powder.
5. enteric probiotics preparation according to claim 4, it is characterised in that：The enteric material be acrylic resin,
Cellulose acetate-phthalate, Diclofenac, shellac, PVAP, 1,2,4- benzenetricarboxylic acid acetic acid
Cellulose, Hydroxypropyl Methylcellulose Phathalate, 1,2,4- benzenetricarboxylic acid hydroxypropyl methyl celluloses, acetate succinate are fine
One or more in dimension element, HPMCAS.
6. according to a kind of preparation method of any described enteric probiotics preparations of claim 1-5, comprise the following steps：
(1) aerobic bacteria is sequentially passed through after inclined-plane culture, Shaking culture and seed tank culture, is inoculated into equipped with sterilization fermentation training
In the round for supporting base, filtrated air is passed through at a temperature of 37 ± 1 DEG C, stir culture 24-32h obtains being in logarithmic growth
The aerobic bacteria of phase；
(2) anaerobic bacteria is sequentially passed through into inclined-plane culture, the culture of anaerobism bottle and seeding tank Anaerobic culturel, protected in incubation
Anaerobic environment is held, Anaerobic culturel obtains seeding tank anaerobism seed；
(3) after the step (1) obtains the aerobic bacteria in exponential phase, filtrated air is closed, the aerobic bacteria stress
Property be transformed into brood-gemma, continue micro-aerobe fermentation 4-8h, and during the micro-aerobe fermentation, the product that step (2) is obtained
It is inoculated into the round so that the aerobic bacteria merges symbiosis 18-32h, culture to the anaerobism with the anaerobic bacteria
Bacterium exponential phase later stage or later stage stationary phase, obtain tunning；
(4) by step (3) obtain mixed fermentation product by centrifuging, spray drying, coating enteric material, be made described
Enteric probiotics preparation.
7. a kind of preparation method of enteric probiotics preparation according to claim 6, it is characterised in that：The step (1)
Stir culture refers to 100-130r/min stir cultures, is passed through the volume ratio 1 of filtrated air volume and the fermentation medium:
0.5-0.6。
8. a kind of preparation method of enteric probiotics preparation according to claim 6, it is characterised in that：The aerobic bacteria
Inoculum concentration is the 6~15% of the fermentation medium cumulative volume, and the inoculum concentration of the anaerobic bacteria is overall for the fermentation medium
Long-pending 10~20%.
9. a kind of preparation method of enteric probiotics preparation according to claim 6, it is characterised in that：The fermented and cultured
Base main component is：Contain beancake powder 3%, corn flour 3%, wheat bran 1%, glucose 1%, tryptone 1%, ferment in per 100g water
Female cream 0.5%, beef extract 0.3%, sodium chloride 0.2%, dipotassium hydrogen phosphate 0.2%, potassium dihydrogen phosphate 0.015%, sulfuric acid are sub-
Iron 0.1%, manganese sulfate 0.02%, calcium carbonate 0.1%, Tween.80 0.02%, the percentage are all mass percents.
10. enteric probiotics preparation obtained by a kind of preparation method of enteric probiotics preparation according to claim 6-9 exists
Application in medicine, health food or the feed addictive of people or poultry intestines problem.
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