CN1641014A - Acidophilic lactobacillus and its use - Google Patents
Acidophilic lactobacillus and its use Download PDFInfo
- Publication number
- CN1641014A CN1641014A CNA2004100007670A CN200410000767A CN1641014A CN 1641014 A CN1641014 A CN 1641014A CN A2004100007670 A CNA2004100007670 A CN A2004100007670A CN 200410000767 A CN200410000767 A CN 200410000767A CN 1641014 A CN1641014 A CN 1641014A
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- China
- Prior art keywords
- lactobacterium acidophilum
- milk
- preparation
- lactobacterium
- capsule
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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Abstract
The present invention relates to microbe and its application, is especially one kind of acidophilic lactobacillus and its application. The acidophilic lactobacillus obtained by the inventor through long term screening and culturing has been applied in serial beneficial products. The strain of the acidophilic lactobacillus is preserved in Beijing in the preservation number of CGMCC No. 1084. The acidophilic lactobacillus of the present invention may be used in preparing acidophilic lactobacillus capsule, acidophilic lactobacillus tablet, fermented acidophilic lactobacillus vegetable and fruit juice, acidophilic lactobacillus microcapsule, acidophilic lactobacillus milk powder, acidophilic lactobacillus sour milk and acidophilic lactobacillus milk beverage. The present invention has unique predominance in acid tolerance, choline tolerance, pathogen inhibition and other aspects.
Description
[technical field]
The present invention relates to a kind of microorganism and application thereof, particularly relate to a kind of Lactobacterium acidophilum and application thereof.Belong to field of biological product.
[background technology]
The inventor has obtained Lactobacterium acidophilum of the present invention through secular screening and cultivation, and a series of products that prepare through product Lactobacterium acidophilum among the present invention all have the effect very useful to human body.
[summary of the invention]
The objective of the invention is to obtain a kind of Lactobacterium acidophilum (Lactobacillus acidophilus);
Another object of the present invention is the application of product Lactobacterium acidophilum of the present invention (Lactobacillus acidophilus).
The preserving number of Lactobacterium acidophilum of the present invention: CGMCC No.1084.Preservation date: on January 6th, 2004.Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.Preservation place: China. Beijing. the Zhong Guan-cun.
Product of the present invention has the better and more function than prior art really.At acidproof bile tolerance, suppress pathogenic bacterium etc. and have special advantage aspect a lot.
Technical scheme of the present invention is as follows:
The preparation method of capsule:
Capsule is a kind of important formulation of Lactobacterium acidophilum goods.Capsule has its characteristics: production technique is simpler, transportation and easy to carry, can cover the unpleasant odor of preparation, can there be different colours to be convenient to difference, the bioavailability height of medicine because need not add tackiness agent when making, is not exerted pressure yet, thereby be easy to disperse, can reduce Lactobacterium acidophilum with the opaque capsule shell and be subjected to light, heat and air influence.Its shortcoming is that aqua and bibulous preparation should not directly be made capsule.In addition, capsule is unsuitable for children taking.
Capsule is made up of capsule shell filling preparation, and capsule shell has many kinds, selects suitable softgel shell extremely important to the Pro-Bionate.
(1) selection of capsulae vacuus
Capsule divides hard capsule, soft capsule and enteric coated capsule.Hard capsule is benefit to be given birth to army's agent make in powder or small-particle filling and the capsulae vacuus, and softgel shell claims Capsules or capsulae vacuus.Capsulae vacuus is made up of gelatin, plasticising machine, sanitas, tinting material and the saturating agent of screening, through colloidal sol, dip in operations such as glue, drying, the demoulding, cut and fit and form.Owing to the tinting material difference, can be made into the softgel shell of different colours, also available type printer lettering on softgel shell.Soft capsule means that a certain amount of liquid formulation is sealed in spherical or the oval-shaped soft capsule material, and the capsule material is made by gelatin, glycerine or other suitable medicinal materials.Enteric coated capsule means that the capsule visitor is insoluble to gastric juice, but can be in intestinal juice disintegration discharge content in the capsule, hard capsule or soft capsule are arranged.With formaldehyde and gelatin manufacturing, formaldehyde and gelatin play the ammonia aldolization to enteric coated capsule, make gelatin molecule crosslinked mutually in the past, form formaldehyde-gelatin, do not had amino in the molecule, lose and sour binding ability, so still contain carboxyl,, content discharged so can in alkaline intestinal juice, dissolve.Because this seed capsules material has unstable.
Product of the present invention is made end clothing with PVP, and CAP, beeswax etc. carry out outer dressing again, can further improve quality.
The specification of capsulae vacuus is descending eight kinds of specifications such as to be divided into 000,00,0,1,2,3,4 and No. 5, generally commonly used is 0～No. 5, should select corresponding minimum model capsule for use according to density, granular size and the shared volume of Lactobacterium acidophilum agent during use.
(2) capsular filling
Can load by hand when general a small amount of prepares, then load during large-scale industrial production with automatic filling machine.Automatic filling machine can be divided into four types at present, and the preparation that is suitable for different physical behaviors is loaded.The flowability of preparation is the physical behavior that directly influences weighting material, and I, II type filling machine are because there is mechanical measure to be suitable for loading flowability preparation preferably; The III type is suitable for form of free flowing powders, for improving liquidity, can add and be lower than 2% lubricant such as glycol ester, poly-copper silicon, silicon-dioxide, stearate, stearic acid, talcum powder, methylcellulose gum and starch etc., selecting principle for use is survival rate and the detection thereof that does not influence Lactobacterium acidophilum; IV type machine is suitable for strong needle crystal of aggregation and the hygroscopic preparation of tool, can add mineral oil, edible oil or Microcrystalline Cellulose etc., makes small-particle and loads.The whole process that the Lactobacterium acidophilum capsule is loaded requires to carry out under aseptic condition, and to reduce secondary pollution, the relative humidity of Laminar Flow Room requires to be lower than 40%.
(3) packing of capsule and storage
The Lactobacterium acidophilum capsulation has its particular requirement, and particularly as anerobes such as Lactobacterium acidophilums, the most important condition is to avoid contacting with air the effect of minimizing oxygen as far as possible.Therefore, wrapping material should be selected gas impermeable material for use, and are to pack with vacuum or inflated with nitrogen.Also the same in storage with other capsules, be subject to the influence of envrionment temperature and humidity, should under shady and cool drying conditions, store.When temperature surpasses room temperature, relative humidity was greater than 45% o'clock, and Lactobacterium acidophilum is easily dead, and the capsule deliquescing is clamminess, and expands, and fungi grows easily, if store under high humidity for a long time, then disintegration time also can prolong.
Lactobacterium acidophilum bacterium method for preparing tablet thereof
At present tablet also is a big class of Lactobacterium acidophilum goods, and it is to add suitable auxiliary material with the Lactobacterium acidophilum agent, the Formulation of making by preparation technique.It is accurate that tablet has dosage, and steady quality (so reduce with contact surfaces such as light, air, moisture) is taken with easy to carry, is convenient to identification and advantage such as with low cost.Its shortcoming is that children are difficult for swallowing, and storage condition was not perishable at that time.
(1) preparation of oral tablet
Tablet preparation promptly is that bacterium powder and proper supplementary material are mixed, and makes it have good flowability and compressibility, then by tabletting machine machinery tablet forming.The flowability of raw material is in order to make mixture with the nib of dosage inflow accurately, make the tablet size consistent smoothly.For the Lactobacterium acidophilum tablet, good compressibility is most important, and compressibility is good more, and required pressure is just more little during compressing tablet, can depress to the satisfactory tablet of hardness at the pressure of appropriateness, because the pressure during film-making directly influences the survival rate of bacterium in the tablet.
Compressing tablet divides compressing dry granulation and wet granule compression tablet.Compressing dry granulation divides dry method direct compression and dry granulation compressing tablet again.Compressing dry granulation is suitable for most of Lactobacterium acidophilum tablet preparation.Can direct compression to mobile and mixture that compressibility is good, what have then need granulate in advance, promptly adds tamanori and makes sheet earlier and be ground into film-making behind the particle again.Wet granule compression tablet is that mixture is carried out wet granulation compressing tablet again.Wet granulation has several method: the one, compound is made softwood, and sieve or otherwise granulate, dry back compressing tablet; The 2nd, fluidized-bed spray granulation, promptly further pelletizing press sheet and rotation pelletizing press sheet etc.The purpose of granulating is except that improving liquidity, also can increase the tightness of material, the effusion air, the pine that reduces tablet is split phenomenon, avoid influencing the accuracy of the dosage in the tablet, avoid fine powder to fly upward and attach to punch head surface and cause glutinous dashing and the drawing-die phenomenon because of the powder layering.The influence of the microbial inoculum of baking stage and oxygen thermo-labile to some is bigger in the wet-granulation process, suitable careful usefulness.Most Lactobacterium acidophilums can be adopted and earlier auxiliary material be made terrified particle, mix the method film-making of back compressing tablet again with the bacterium powder, to reduce the death of bacterium in the pelletization.
(2) external application tablet---the preparation of effervescent tablet
Be used for the treatment of vaginopathy or improve microecology in vaginas equilibrated Pro-Bionate, can be made into effervescent tablet.After this tablet was put into vagina, reacting with vaginal fluids promptly produced a large amount of bubbles, and the effervescent tablet disintegration makes Lactobacterium acidophilum be distributed to the vaginal mucosa surface uniformly and plays a role.
Effervescent tablet is made up of Lactobacterium acidophilum bacterium powder and gas-producing disintegrant.Soda acid system in the effervescent tablet and water mutual reactance generate carbon dioxide and speeding up disintegration of tablet dissolving and formation foam.The used bronsted lowry acids and bases bronsted lowry of disintegrating agent should be granulated respectively, mixes compressing tablet immediately with the bacterium powder before compressing tablet.The most frequently used bronsted lowry acids and bases bronsted lowry system is Citric Acid and tartaric mixture, and carbonate or saleratus, the acid of glycine carbonic acid, sodium and concentrated crystal soda etc.
Effervescent tablet requires the bubble of generation tiny and lasting, fully disperses to reach Lactobacterium acidophilum.Effervescent tablet should adopt airtight waterproof, and the wrapping material of lucifuge pack, and is rotten to prevent the moisture absorption.Lactobacterium acidophilum electuary preparation method
Electuary also is a big class of Lactobacterium acidophilum goods.The electuary production technique is simple, does not need complex apparatus, and is easy to carry and use.Electuary is with compositions such as bacterium powder, weighting agent, stablizer and seasoningss.Electuary can be divided into solubility electuary and suspension granule, and the Lactobacterium acidophilum electuary mostly is the latter.Lactobacterium acidophilum electuary major ingredient is dry bacterium powder, and auxiliary material is milk powder and starch etc., also can add seasoningss such as some sweeting agents, essence according to product requirement, some nutrition-fortifying agents of also adding that have such as VITAMIN etc.In the Lactobacterium acidophilum electuary, can also add bifidus factor such as oligose.Make particle or pulvis through thorough mixing, use the composite aluminium film vacuum packaging, by contained bacterium number different size is arranged, every gram contains bacterium number from several hundred million to 10,000,000,000.
The Lactobacterium acidophilum electuary forms commodity already in Japan and Europe, domestic also existing analogous products listing.In preservation period, how to keep the viable count in the product to be and important problem, because viable count is unique index of this tired product.Can not use boiling water when this series products is taken, can only take after mixing it with water with being lower than 50 ℃ of warm water.
Lactobacterium acidophilum oral liquid series products preparation method
Oral liquid also is a big class of Lactobacterium acidophilum goods.Many products occurred in the nineties in 20th century, and the bigger market share is arranged, but for various reasons, some product sales volumes fall sharply in recent years.However, the rise of China's Lactobacterium acidophilum oral liquid is crossed human consumer's understanding and understanding Lactobacterium acidophilum and nourishing function thereof to me and has been played active effect, has promoted the research and development of Lactobacterium acidophilum goods.Along with going deep into of fundamental research and product development, its effect of realistic propaganda is eliminated understanding and some theoretic deviation, and the Lactobacterium acidophilum goods comprise that the Lactobacterium acidophilum oral liquid will have bright prospect.
The Lactobacterium acidophilum oral liquid has multiple formulations, has to contain bifidus factor, the herbal medicine that adds food medicine dual-purpose is arranged, also have to contain enhanced nutrient, or the like.As be the healthcare food oral liquid, its formula material must be the herbal medicine of the food medicine dual-purpose of food and Ministry of Health's approved announcement, must not arbitrarily add other drug.Nutrient substance also will amount in accordance with regulations add.
The production technique of Lactobacterium acidophilum oral liquid depends on the type of oral liquid.Above-mentioned first kind oral liquid technology is compared with general liquid-state fermentation technology, only after fermentation ends, increase filling process, can requires to carry out under aseptic condition, therefore, fermentor tank is tightly connected by pipeline and filling apparatus, racking room requires to have certain cleanliness factor, and one avoids polluting.Second series products then divides two portions: the one, and cultivation of Lactobacterium acidophilum and fungicide preparation; The 2nd,, then the two is mixed on request capable again can by formulated oral liquid base-material.
The Lactobacterium acidophilum fermented fruits and vegetables juice
The Lactobacterium acidophilum fermented fruits and vegetables juice is a class new formulation, it is to utilize some the refining garden spgarden stuff that is of high nutritive value, the liquid product of making through suitable Lactobacterium acidophilum fermentation, its advantage is the beneficial effect that has Lactobacterium acidophilum and metabolite and the high nutrition of garden spgarden stuff concurrently, can not add any manual member, reach the requirement of All Pure Nature.
Material choice: Radix Dauci Sativae should select that redness, fine and tender taste, fiber are few, the fine quality of no hard-core.
The Lactobacterium acidophilum fermented fruits and vegetables juice is made in all available Lactobacterium acidophilum fermentation of the present invention of fruits and vegetables such as Radix Dauci Sativae and tomato.
The fermentation of Lactobacterium acidophilum garden spgarden stuff is to carry out in fermentor tank.Need the pH value and the bacterium number of timing sampling detection fermented liquid in the fermenting process, with timely understanding fermentation situation.The discrimination standard of fermentation termination, the one, pH value, general pH value reaches 4.2～4.5; The 2nd, viable count reaches 10
8More than the CFU/ml.According to the particular requirement of Lactobacterium acidophilum product, after the fermentation ends immediately below the cool to room temperature, can refrigeration rapidly under aseptic condition then.
The Lactobacterium acidophilum microcapsule formulation
One of most important sign of Lactobacterium acidophilum quality of item is a viable count, is even more important the product of unique index with viable count merely concerning those particularly.Using maximum in the Lactobacterium acidophilum is Lactobacterium acidophilum and milk-acid bacteria etc., and how their inactivations very easily in product is preserved prolong a great problem that preservation period has become this series products.Both at home and abroad all wait and improve the resistivity of bacterial classification attempting screening by genetic modification or acidproof oxytolerant bacterial strain acid and oxygen; On Technology, unfavorable factors such as bacterium and oxygen and acid are isolated, to improve the survival rate of bacterium.
1. the preparation technology Lactobacterium acidophilum bacterial classification liquid or solid culture medium culturing of the capsule heart and capsule material is collected and the washing thalline, adds then that starch, milk powder or other protective materials are mixed into suspension or lyophilize becomes the bacterium powder; With suitable capsule material wiring solution-forming, after sterilizing, mix with the capsule heart.Solidifying agent adopts the calcium chloride solution sterilization postcooling of 0.1～0.3mol/L stand-by usually.
2. the preparation technology of microcapsule is squeezed into the mixture of heartwood and capsule material and carefully drips or spray in the curing agent solution, slowly stirs and solidifies, and collects microcapsule then, promptly gets the Lactobacterium acidophilum microcapsule that wet three times with the damping fluid washing.
Wet microcapsule can be directly used in the various preparations of production and add in the food-drink.Dried microcapsule also can be made into capsule, tablets and other formulations except that adding the food-drink, its preservation period is longer.
The drying of wet microcapsule has dual mode, and the one, in wet capsule, add some weighting agents such as starch, lime carbonate, talcum powder etc. and mix thoroughly, be not higher than 40 ℃ of oven dry down then; The 2nd, carry out drying with spray-drying process.Spray-dired advantage is can be to make sample drying than fast speeds, and its shortcoming is that the mortality ratio of Lactobacterium acidophilum is higher.When inlet temperature is 140～160 ℃, raise with temperature of outgoing air, mortality ratio also raises, and 70～73 ℃ is the thalline stage the most responsive to temperature, and then mortality ratio changes little from 72 ℃ to 82 ℃.Consider that from the angle of survival rate best temperature is 68～70 ℃, but the sample moisture that obtain this moment is too high, it is comparatively suitable generally to select about 80 ℃.Under such temperature of outgoing air, if the capsule material formula is selected better then can obtain higher survival rate.Under optimal conditions, after the microcapsule spraying drying, the survival rate of milk-acid bacteria is about 20%.The principal element that influences microcapsule survival rate of bacterium in spraying drying is that spraying is the prescription of turnover wind-warm syndrome degree and capsule material.Lactobacterium acidophilum is owing to the oxygen sensitivity, should not carry out drying with spray-drying process.Survival rate of bacterium and protective material, moisture content etc. have substantial connection in the dry microcapsule.
Selmer-Olsen etc. (1999) are in the research of lactobacillus helveticus microcapsule, nutrient solution is being lower than 5 ℃ of centrifugal collection thalline down, thalline is suspended in respectively in ribitol, front three ammonia second lactone, glycerine and the skim-milk reconstituted milk, mix with heat treated sodium alginate soln, splash in the calcium lactate solution under the slow stirring, stir 30min, promptly obtain the lactobacillus micro-capsule that wets, with the rinsing of sterilization Ringer liquid, remove unnecessary calcium ion and free cell, preserve down at 2～3 ℃.Wet microcapsule carry out drying by fluidized-bed, and the fluidized-bed inlet temperature is 5 ℃, relative humidity 55% ± 2.5%, the about 3mm of the diameter of microcapsule.In 4 protective materials, ribitol keeps the thalline survival rate relative higher with skimming milk, counts ratio with starter bacteria, is respectively 71% and 57%.Survival rate in its storage is also the highest.Survival rate increases with the water content in the microcapsule and descends.
The quality evalution of Lactobacterium acidophilum microcapsule
The quality evalution of Lactobacterium acidophilum microcapsule comprises three aspects, and promptly the survival rate of embedding efficiency, embedding productive rate and storage period bacterium is promptly stable.Embedding efficiency is meant the quantity that the inside and outside bacterium of microcapsule exists, and the high more explanation embedding efficiency of the inner viable count of microcapsule is high more; The embedding productive rate is meant the ratio of the bacterium number that adds when the viable count in the product prepares with microcapsule; And stability is to represent that with the survival rate of thalline behind certain hour under certain storage condition the high more explanation stability of the survival rate of bacterium is good more.
The micro encapsulation of Lactobacterium acidophilum is to solve one of conservatory important technology of product; should further combine strain improvement, new capsule material and protectant selection and capsule processing technology; to improve the survival rate of Lactobacterium acidophilum, obtain better Pro-Bionate at storage period.
Lactobacterium acidophilum milk powder
Utilize the various milk-acid bacterias various yoghurt products that agent is made through cultivation and fermentation, have very high nutritive value and special medical functions.Protein contained in the goods is easier of human consumption.Use yoghurt product that human body is had very high medical functions, decapacitation promotes people's digestion and strengthens for Digestive tract illnesss such as gastroenteropathy, dysentery characterized by blood in the stool, diarrhoea, constipation, special curative effect being arranged outside the metabolism.Because the metabolism of milk-acid bacteria can produce lactic acid and antibiotics, can suppress the breeding and the growth of spoilage microorganisms in the large intestine, improve micropopulation in the enteron aisle.
Feed with common milk powder if the baby feeds for a long time, then, can cause detrimentally affect baby's digestion and growth owing to lack necessary milk-acid bacteria; If baby's digestive function can be treated and improve to the yoghurt product of nurture to make through the pure culture starter then, the promoting digestion effect adds excitometabolic.
Yoghurt product has many types, Lactobacterium acidophilum breast owing to employed milk-acid bacteria is different be the highest a kind of of medical value, and employed milk-acid bacteria is a Lactobacterium acidophilum.This bacterium has higher vigor than lactobacillus bulgaricus in intestines.
Use Lactobacterium acidophilum pure culture starter to make the Lactobacterium acidophilum breast, various countries are very general at present.This Lactobacterium acidophilum breast is a kind of evenly tender corpus mamillare, only inconvenient prolonged preservation and transportation.Therefore, begin to make the milk powder that contains this Lactobacterium acidophilum in China recently, like this, not only be convenient to store and transportation, and the vigor of former bacterium can be in over a long time unlikely weakening.
With product Lactobacterium acidophilum of the present invention, the Lactobacterium acidophilum milk powder for preparing not only can keep the effect of Lactobacterium acidophilum to human body, and is convenient to store and transportation.
Can also prepare a kind of pure culture starter sheet that contains this Lactobacterium acidophilum with Lactobacterium acidophilum of the present invention, the vigor that can preserve former bacterium of longer date can not go down, easy to carry, use simple.
No matter Lactobacterium acidophilum milk powder or Lactobacterium acidophilum starter sheet, its manufacture method has following three kinds:
1. the Lactobacterium acidophilum powder (can adopt the preparation of vacuum freeze-drying method) with pure culture mixes, packs, carries out aseptic technique with good matter milk powder composition in sterilisable chamber.The Lactobacterium acidophilum milk powder quality that this method is made is fine.
2. adopt the vacuum freeze-drying method.Be about to the skimming milk nutrient solution and the spissated milk elements mixing of process sterilization of the Lactobacterium acidophilum of pure culture, carry out vacuum freeze-drying again.The Lactobacterium acidophilum milk powder quality that this method is made is high.
3. spray method.Spraying drying behind skimming milk nutrient solution of the Lactobacterium acidophilum of pure culture soon and the spissated milk elements mixing of process sterilization.The content of the Lactobacterium acidophilum milk powder Central Plains bacterium that this method is made, more preceding dual mode is low.If but control properly, and still good goods can be obtained, and a complete set of equipment of producing common milk powder can be used, must not increase new installation in addition, invest less.
The product index of Lactobacterium acidophilum milk powder generally should reach following requirement.
Moisture (being no more than) 4%
Solubleness (being not less than) 98%
Impurity degree (being no more than) 2ppm
Lactobacterium acidophilum content (being not less than) 6,000,000/gram
Milk powder restores the back and is incubated setting times (being no more than) 10 hours at 40～42 ℃
Grumeleuse is evenly dense, the agalasisa sorting from, no bubble produce, flavor is pure and sour, do not have other peculiar smell microscopys does not find other assorted bacterium
The Lactobacterium acidophilum sour milk
Technical process is as follows:
Compound, homogeneous, sterilization, cooling add spices then, and small vessels can, cultivation, cooling obtain the coagulating type yogurt milk;
Compound, homogeneous, sterilization, cooling add starter then, add fermentor cultivation, stirring, cooling, add fruit material, spices, small vessels can again, obtain stir-type yoghurt;
The Lactobacterium acidophilum milky-drinks
A, liquid acid cow's milk type Lactobacterium acidophilum beverage
The fermentation compound is the skimming milk of strengthening with condensed skim milk or skimmed milk powder, and the material that adds promotions milk-acid bacteria growth such as glucose in case of necessity is configured.Compound heat-sterilization 5～10 minutes under 90～95 ℃ of temperature is cooled to 37 ℃; Add 2～3% Lactobacterium acidophilum starter then therein, cultivate down, make lactic acid degree reach 1.5～2.0% at 35～37 ℃.General incubation time with 24 hours with interior for well, at this moment the bacterium number reaches 108CFU/ml.Cultivate the back if improve the bacterium number, be advisable to fall culture temperature slightly.Compound after the cultivation should cool off immediately, maintains the temperature at below 10 ℃ till when using.
In addition, will be made into syrup, below 10～30 minutes postcooling to 10 of 80～90 ℃ of heat-sterilizations ℃ as dissolvings such as the granulated sugar of auxiliary material, stablizer, pigments.The a spot of water dissolution of stablizer stirs with machinery is powerful simultaneously, fully disperses the post-heating dissolving.
Fermented-milk is mixed with syrup, add citric acid in case of necessity and regulate acidity; After adding spices,, make fermenation raw liquid with the pressure homogeneous of 9.8～14.72 MPas; Add the dilution of a certain amount of water coolant, at last 3～5 ℃ goods can is refrigerated in container.
B, fruit juice type Lactobacterium acidophilum beverage
Press formula ratio with mixed dissolutions such as fruit juice, sucrose, pulp dark reddish brown elements, add stablizer in case of necessity, behind the citric acid, through 80～85 ℃, 10～15 minutes or 90～95 ℃, sterilization in 15 seconds postcooling to 3～5 ℃.Add the lactobacillus starter thorough mixing of cultivating in addition, can is back refrigeration in container.
The addition of starter, it is different to press bacterial classification, bacterium number, vigor etc., and common 5% with interior enough.If the addition of starter is too much, then produce the casein precipitation, reduce commodity value.
The Lactobacterium acidophilum ice cream
Method with routine obtains ice cream, adds living lactobacillus acidophilus bacterium powder of the present invention or Lactobacterium acidophilum active-fermented broth of the present invention before packing, promptly obtains the Lactobacterium acidophilum ice cream after the stirring.
The composition of raw material sees the following form with becoming score value:
| Material name | Material composition value (%) | |||
| Fat | Non-fat solid | The sweet taste degree | Total solid matters | |
| Skimming milk | ????8.5 | ????8.5 | ||
| Cow's milk | ????3.3 | ????8.5 | ????11.5 | |
| Give birth to rare cream | ????40.0 | ????5.1 | ????45.1 | |
| Cream | ????82.0 | ????1.0 | ????83.0 | |
| Full fat concentrated milk | ????8.0 | ????21.5 | ????43.0 | ????72.5 |
| Skimmed condensed milk | ????30.0 | ????42.0 | ????72.0 | |
| Skimmed milk powder | ????97.0 | |||
| Granulated sugar | ????100.0 | ????100.0 | ||
| Caramel powder | ????19.0 | ????96.0 | ||
| Emulsion stabilizer | ????100.0 | |||
[description of drawings]
Fig. 1 is the processing process (is example with the Radix Dauci Sativae) of garden spgarden stuff;
Fig. 2 is fermentation of Lactobacterium acidophilum Radix Dauci Sativae juice and technology of the package;
Fig. 3 is the manufacturing process flow diagram of Lactobacterium acidophilum sour milk;
Fig. 4 is a liquid acid cow's milk type Lactobacterium acidophilum drink manufacturing process flow sheet;
Fig. 5 is the technological process of production figure of Lactobacterium acidophilum ice cream.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
Acidproof bile tolerance experiment
A. experimental program
1. bacterial strain to be measured is activated 1 time from the inclined-plane, cultivate 18hr for 37 ℃ and take out standby;
2. with sterile saline thalline is washed from the inclined-plane, use 3.0 opacity tubes to carry out than turbid;
3. acidproof: get in the phosphate buffered saline buffer that the 0.1ml bacteria suspension joins 2mlPH3.0,37 ℃ of anaerobism are cultivated 3hr;
Acidproof bile tolerance: get in the phosphate buffered saline buffer that the 0.1ml bacteria suspension joins 2mlPH3.0, after 37 ℃ of anaerobism are cultivated 1hr, get 1ml and join in 0.15% the cholate solution, 37 ℃ of anaerobism are cultivated 3hr;
4. suitably carry out plate count after the dilution.
B. experimental result
Acidproof:
| Bacterial strain | Survival rate (%) |
| Lactobacterium acidophilum (SY02) | ????27.78 |
| Lactobacillus paracasei (SY13) | ????0.018 |
| Lactobacillus paracasei (Sy33) | ????0.176 |
| Bifidus longum bb (Sy31) | ????1.197 |
| Lactobacterium acidophilum (Sy32) | ????4.64 |
Acidproof bile tolerance:
| Bacterial strain | Survival rate (%) |
| Lactobacterium acidophilum (SY02) | ????86.67 |
| Lactobacillus paracasei (SY13) | ????0.685 |
| Lactobacillus paracasei (Sy33) | ????3.50 |
| Bifidus longum bb (Sy31) | ????42.63 |
| Lactobacterium acidophilum (Sy32) | ????61.06 |
Embodiment 2
The decreasing cholesterol experiment
A. experimental program
Chromatographic condition:
Instrument model: VISTA6000GC
Detector: flame ionization detector
Chromatographic column: 2m*I.D.2mm 5%0V-17 on 80/100 order GCQ glass column, or the instrument with identical performance and the chromatographic column of other company's production.
Column temperature: 285 ℃
Detector temperature: 325 ℃
Sampler temperature: 325 ℃
Nitrogen flow rate: 20ml/min
Sampling volume: 2 μ l
(1) measuring method of cholesterol level in the medium supernatant
With the bacterial suspension inoculation of 1.0ml in the test tube that 10ml MRS liquid nutrient medium is housed, do contrast with the 1ml sterile saline simultaneously;
2. add 1.0ml cholesterol solution (1.6);
3.37 ℃ cultivation is after 10 hours, centrifugal 10 minutes of 4000*g collects supernatant;
4. saponification: in a tool plug test tube, add the 0.1ml supernatant, 0.3ml 33%KOH, 3ml 95% ethanol, 60 ℃ of water-baths are 15 minutes behind the mixing, reduce to room temperature then rapidly;
5. extraction: add the 5ml normal hexane, 3ml distilled water mixes concussion 1 minute.It is to be measured in the volumetric flask to shift 1ml normal hexane layer.
(2) reduce cholesterol level experiment in the cultured milk prod
1. bacteria suspension 1ml is inoculated in the 100ml homogeneous raw dairy;
2. in 37 ℃ of incubators, cultivated 4-6 hour;
3. treat curdling back taking-up admittedly, put into refrigerator and cooled and hide standby;
4. sample treatment and detection method:
Accurately take by weighing 5g sample (newborn 50ml) in saponification flask, add 25ml 50%KOH solution and 25ml 95% ethanol then, connect prolong, water-bath backflow saponification 30 minutes.
The cooling saponification liquor all moves to saponification liquor in the 250ml separating funnel then to room temperature rapidly, adds the 50ml sherwood oil, and jolting extracted after 2 minutes, static layering; Emit water in another separating funnel, use the 50ml sherwood oil more respectively, aqueous phase extracted is twice as stated above; Merge organic phase, be washed till neutrality with distilled water after, use anhydrous sodium sulfate drying again.
On rotatory evaporator,, use 1ml normal hexane constant volume at last with the sherwood oil evaporate to dryness.
It is standby that the normal hexane of extraction back gained is partly drawn 1ml, detects with gas-chromatography then.
B. experimental result
In the substratum:
| Bacterial strain | Cholesterol decreasing ratio (%) |
| Lactobacterium acidophilum (SY02) | ????30.06 |
| Lactobacillus paracasei (SY13) | ????43.69 |
| Lactobacillus paracasei (Sy33) | ????48.85 |
| Bifidus longum bb (Sy31) | ????61.88 |
| Lactobacterium acidophilum (Sy32) | ????57.37 |
In the fermented-milk:
| Bacterial strain | Cholesterol decreasing ratio (%) |
| Lactobacterium acidophilum (SY02) | ????25.37 |
| Lactobacillus paracasei (SY13) | ????53.63 |
| Lactobacillus paracasei (Sy33) | ????59.96 |
| Bifidus longum bb (Sy31) | ????68.65 |
| Lactobacterium acidophilum (Sy32) | ????26.77 |
Embodiment 3
Adsorption experiment
A. experimental program
1. bacterial strain to be measured is activated 1 time from the inclined-plane, cultivate 18hr for 37 ℃ and take out standby;
2. with sterile saline thalline is washed from the inclined-plane;
3. clean 2 times with PBS, use the centrifugal 10min of 3000rpm at every turn.Be made into 1*10 at last
8The bacteria suspension of CFU/ml;
4. in 12 well culture plates of slide with cover, (cultivate individual layer HT-29 cell) and added the 0.5ml bacteria suspension, and added cell culture fluid.(culture condition is 10%CO to 37 ℃ of cultivation 60min
2, 90% air or 5%CO
2, 95% air);
5. remove nutrient solution, and clean 3-4 time seasoning with PBS;
6. with methyl alcohol, acetone or other immobilization of reagents;
7. gramstaining;
8. observe the absorption situation down at opticmicroscope (1000 times, oily mirror);
The counting and take pictures.
B. experimental result
| Bacterial strain | Total plate count units' (individual) of 10 cells absorption at random |
| Lactobacterium acidophilum (SY02) | 266 |
| Lactobacillus paracasei (SY13) | 152 |
| Lactobacillus paracasei (Sy33) | 88 |
| Bifidus longum bb (Sy31) | 65 |
| Lactobacterium acidophilum (Sy32) | 48 |
Embodiment 4
Bacteriostatic experiment
A. experimental program
(1) flat band method
1. pathogenic bacterium are activated 2 times with nutrition nutrient agar substratum, get 0.2ml respectively and carry out the flat board coating.
Place 30min, make its surface drying for 37 ℃.
2. in each flat board, place 4 Oxford cups, add PH3.5 lactic acid, Lactobacterium acidophilum culture supernatant, Lactobacterium acidophilum bacteria suspension and the blank MRS liquid nutrient medium of 0.1ml respectively.
3. cultivate or, measure inhibition zone diameter.
(2) liquid culture method
1. in MRS liquid nutrient medium (8ml), add pathogenic bacterium bacteria suspension 1ml, survey original bacterium number;
2. add Lactobacterium acidophilum bacteria suspension 1ml, contrast (using the MRS liquid nutrient medium) is set;
3. mixed culture 24-48hr;
4. survey the pathogenic bacterium bacterium and count CFU/ml (flat band method);
5. enumeration.
B. experimental result
Flat band method:
| Bacterial strain | Inhibition zone diameter (cm) | |||
| The white dysentery Salmonellas | Shigella dysenteriae | Streptococcus aureus | Dust Xi Shi intestinal bacteria | |
| Lactobacterium acidophilum (SY02) | ????1.546 | ????1.756 | ????1.092 | ????0.962 |
| Lactobacillus paracasei (SY13) | ????1.448 | ????1.574 | ????- | ????0.960 |
| Lactobacillus paracasei (Sy33) | ????1.450 | ????1.772 | ????- | ????0.844 |
| Bifidus longum bb (Sy31) | ????1.570 | ????1.478 | ????1.142 | ????0.904 |
| Lactobacterium acidophilum (Sy32) | ????1.540 | ????1.530 | ????0.992 | ????0.870 |
The liquid nutrient medium method:
| Lactobacterium acidophilum (SY02) | Lactobacillus paracasei (SY13) | Lactobacillus paracasei (Sy33) | Bifidus longum bb (Sy31) | Lactobacterium acidophilum (Sy32) | |
| Original bacterium number (* 10 5) | ???35 | ???51 | ???52 | ???110 | ???48 |
| Original bacterium number | Whole bacterium number | |||||
| Lactobacterium acidophilum (SY02) | Lactobacillus paracasei (SY13) | Lactobacillus paracasei (Sy33) | Bifidus longum bb (Sy31) | Lactobacterium acidophilum (Sy32) | ||
| The sramana | 10×10 5 | ????＜10 3 | ????＜10 3 | ????＜10 3 | ????＜10 3 | ????＜10 3 |
| Will is congratulated | 13×10 6 | ????＜10 3 | ????＜10 3 | ????＜10 3 | ????＜10 3 | ????＜10 3 |
| The gold Portugal | ??39×10 7 | ????38×10 7 | ????＜10 3 | ????14×10 7 | ????26×10 7 | ????14×10 7 |
| Large intestine | ??13×10 7 | ????25×10 3 | ????＜10 3 | ????＜10 3 | ????＜10 3 | ????＜10 3 |
Embodiment 5
Suppress the helicobacter pylori experiment
A. experimental program (flat band method)
1. prepare helicobacter pylori bacteria suspension (10
9CFU/ml), concentration 10
9CFU/ml Lactobacterium acidophilum bacteria suspension; The MRS liquid nutrient medium; The nutrient solution supernatant; Lactic acid solution (providing for oneself);
2. prepare the helicobacter pylori flat board;
3. add 0.2ml Helicobacter pylori bacteria suspension on the culture medium flat plate, evenly with the coating of trilateral glass stick.Put into incubator and cultivate 30min in advance;
4. put into 4 Oxford cups, add bacteria suspension, bacteria culture fluid supernatant, PH3.5 lactic acid solution (sterilization), MRS liquid nutrient medium successively;
5. incubator is cultivated 24-48hr;
6. survey inhibition zone diameter (use vernier callipers);
7. record data.
B. experimental result
| Bacterial strain | Inhibition zone diameter cm |
| Lactobacterium acidophilum (SY02) | 1.70 |
| Lactobacillus paracasei (SY13) | 1.60 |
| Lactobacillus paracasei (Sy33) | 1.65 |
| Bifidus longum bb (Sy31) | 1.70 |
| Lactobacterium acidophilum (Sy32) | 1.60 |
Embodiment 6
The Lactobacterium acidophilum bacterial classification is cultivated with liquid nutrient medium, collected and the washing thalline, add protective material, drying is prepared into active bacterium powder; Make the capsule material with gelatin, make end clothing, carry out outer dressing with beeswax again, with No. 0 capsulation with PVP.Obtain the Lactobacterium acidophilum capsule.
Embodiment 7
Get 50 kilograms in Radix Dauci Sativae, obtain aseptic garden spgarden stuff through sorting, cleaning, peeling, cutting, blanching, the making beating of squeezing the juice, batching, homogeneous, sterilization steps.
Pass through following steps then: with Lactobacterium acidophilum actication of culture, strain expanded culture, be inoculated into sterilization vegetables juice, fermentation, that sampling detects is qualified, cool, sterile filling, the packing of product, finished product, obtain the Lactobacterium acidophilum fermented fruits and vegetables juice, wherein, the qualified condition one of described sampling detection is that pH value reaches 4.2～4.5; The 2nd, viable count reaches 10
8More than the CFU/ml.
Embodiment 8
The Lactobacterium acidophilum bacterial classification is cultivated with liquid nutrient medium, collected and the washing thalline, add protective material, add starch then, milk powder is mixed into suspension or lyophilize becomes the bacterium powder; With conventional capsule material wiring solution-forming of the prior art, after sterilizing, mix with the bacterium powder; Then the mixture of bacterium powder and capsule material is squeezed into and carefully drips or spray in the 0.3mol/L calcium chloride solution of sterilization, slowly stir and solidify, collect microcapsule then, promptly get the Lactobacterium acidophilum microcapsule that wet three times with the damping fluid washing.
Embodiment 9
The skimming milk of strengthening with condensed skim milk or skimmed milk powder with compound heat-sterilization 60 minutes under 95 ℃ of temperature, is cooled to 37 ℃; Add 3% Lactobacterium acidophilum starter then therein, cultivate down, make acidity reach 1.5% at 35 ℃, incubation time 24 hours, at this moment the bacterium number is minimum reaches 10
8CFU/ml.Obtain the Lactobacterium acidophilum milky-drinks.
The foregoing description explanation product of the present invention has the better and more function than prior art really.At acidproof bile tolerance, suppress bacterium etc. and have special advantage aspect a lot.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, not departing from the these modifications or improvements basically of spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a lactobacillus acidophilus strains is characterized in that by the preserving number of China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation being: CGMCC No.1084.
2. the capsular preparation method of Lactobacterium acidophilum is characterized in that: the described Lactobacterium acidophilum bacterial classification of claim 1 is cultivated with liquid nutrient medium, collected and the washing thalline, add protective material, pack into after the drying and select capsule for use 0～No. 5.
3. the capsular preparation method of Lactobacterium acidophilum as claimed in claim 2, it is characterized in that: capsule is made end clothing with PVP, and CAP or beeswax carry out outer dressing again, and select for use gas impermeable material as wrapping material, and pack with vacuum or inflated with nitrogen.
4. the preparation method of a Lactobacterium acidophilum tablet is characterized in that: with Lactobacterium acidophilum bacterium powder and auxiliary material or the mixed Lactobacterium acidophilum tablet that is prepared into of gas-producing disintegrant.
5. the preparation method of a Lactobacterium acidophilum fermented fruits and vegetables juice, it is characterized in that: vegetables are processed into vegetables juice with ordinary method, pass through following steps then: with Lactobacterium acidophilum actication of culture, strain expanded culture, be inoculated into sterilization vegetables juice, fermentation, that sampling detects is qualified, cool, sterile filling, the packing of product, finished product, obtain the Lactobacterium acidophilum fermented fruits and vegetables juice, wherein, the qualified condition one of described sampling detection is that pH value reaches 4.2～4.5; The 2nd, viable count reaches 10
8More than the CFU/ml.
6. the preparation method of Lactobacterium acidophilum microcapsule is characterized in that: the Lactobacterium acidophilum bacterial classification is cultivated with liquid nutrient medium, collected and the washing thalline, add then that starch, milk powder or other protective materials are mixed into suspension or lyophilize becomes the bacterium powder; With conventional capsule material wiring solution-forming of the prior art, after sterilizing, mix with the bacterium powder; Then the mixture of bacterium powder and capsule material is squeezed into and carefully drips or spray in the curing agent solution, slowly stir and solidify, collect microcapsule then, promptly get the Lactobacterium acidophilum microcapsule that wet three times with the damping fluid washing; Wherein, solidifying agent adopts the 0.1～0.3mol/L calcium chloride solution after sterilizing.
7. the preparation method of a Lactobacterium acidophilum milk powder is characterized in that: the Lactobacterium acidophilum powder of the present invention of pure culture is mixed, packs, carries out aseptic technique with good matter milk powder composition in sterilisable chamber.
8. the preparation method of Lactobacterium acidophilum milk powder as claimed in claim 7, it is characterized in that: with the skimming milk nutrient solution of the Lactobacterium acidophilum of pure culture with mixed through the spissated cow's milk of sterilization, carry out vacuum freeze-drying then, obtain Lactobacterium acidophilum milk powder.
9. the preparation method of a Lactobacterium acidophilum yogurt is characterized in that: after preparing the yogurt starter and mix by a certain percentage with the starter of the present invention's preparation with ordinary method, with common process production solidifiability and stirred yogurt.
10. the preparation method of a Lactobacterium acidophilum milky-drinks is characterized in that: the skimming milk of strengthening with condensed skim milk or skimmed milk powder with compound heat-sterilization 5～10 minutes under 90～95 ℃ of temperature, is cooled to 37 ℃; Add 2～3% Lactobacterium acidophilum starter then therein, cultivate down at 35～37 ℃, make lactic acid degree reach 1.5～2.0%, incubation time is lower than 24 hours, so or with ordinary method makes milky-drinks, and wherein, the bacterium number is minimum to reach 10
8CFU/ml.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1940060B (en) * | 2005-09-28 | 2010-05-05 | 曾浩洋 | Acidophilous milk and its use |
| CN101139558B (en) * | 2006-09-07 | 2010-05-12 | 内蒙古农业大学 | Lactobacillus acidophilus and its application for improvement of blood lipid metabolism and immunological regulation |
| CN101144062B (en) * | 2006-09-15 | 2011-11-16 | 内蒙古农业大学 | Lactobacillus casei strain and application for products thereof in bird immunity |
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| CN103013972A (en) * | 2012-12-19 | 2013-04-03 | 江苏绿科生物技术有限公司 | Preparation method of lactobacillus casei activity-improving probiotic fermenting agent |
| CN103385296A (en) * | 2013-06-14 | 2013-11-13 | 扬州市扬大康源乳业有限公司 | Preparation method for disturbing yoghurt |
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| CN1940060B (en) * | 2005-09-28 | 2010-05-05 | 曾浩洋 | Acidophilous milk and its use |
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| CN103695442B (en) * | 2013-12-13 | 2016-01-20 | 上海市农业科学院 | A kind of NFSAI gene coming from Lactobacterium acidophilum and its preparation method and application |
| CN103695442A (en) * | 2013-12-13 | 2014-04-02 | 上海市农业科学院 | NFSAI gene from lactobacillus acidophilus and preparation method and application thereof |
| CN105400726A (en) * | 2015-12-21 | 2016-03-16 | 南昌大学 | Human-derived lactobacillus acidophilus resisting gastrointestinal tract stress and application thereof |
| CN106350468A (en) * | 2016-09-08 | 2017-01-25 | 济南康多宝生物技术有限公司 | Novel lactobacillus acidophilus |
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