CN103392911A - Feed-use high-activity lactobacillus solid preparation and preparation method thereof - Google Patents
Feed-use high-activity lactobacillus solid preparation and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a feed-use high-activity lactobacillus solid preparation and a preparation method thereof. The feed-use high-activity lactobacillus solid preparation comprises growing beneficial lactobacillus bacterium mud, a protection formula and a basic formula. The invention also provides the preparation method of the feed-use high-activity lactobacillus solid preparation, lactobacillus can be protected through synergistic effects of components in the protection formula and the basic formula, and the living bacterium survival rate in the whole process can be improved. The lactobacillus in the prepared feed-use high-activity lactobacillus solid preparation has improved high temperature and high humidity resistances, has a high survival rate in a simulated intestinal fluid, and a substantial improved animal application effect.
Description
Technical field
The present invention relates to a kind of additive for microbe feedstuff, particularly a kind of feeding high-activity lactic acid bacteria solid formulation and preparation method thereof, belong to the microorganism formulation field.
Background technology
Probio, as one of antibiotic substitute, is applied in feed additive field widely.In animal body, microorganism is never manufactured energy in the chyme of gastro-intestinal digestion, stimulates intestinal tract immune system, or essential vitamin is provided, and adjusts intestinal microecology and metabolic activity thereof.Although probiotics preparation progressively develops into the antibiotic substitute products that have application prospect most; but probio especially has the lactic acid bacteria of enteron aisle adhesive attraction; inactivation very easily in production, processing, storage and use procedure; resistance to hydrochloric acid in gastric juice, cholate etc. is poor; therefore, the probiotics preparation technology of the effective protection of research and development production overall process has become focus both domestic and external.
In prior art and patent, the effect of probiotics preparation (especially lactic acid bacteria) is subjected to various factors, as holding time of bacterium composition, feeding method, dosage, moisture, temperature, pH value and the bacteria preparation of animal species, physiological status, feed type, processing technology, preparation etc.The stability that how to keep microorganism is be engaged in lactobacillus preparation research and produce the most real and sixty-four dollar question that faces.
(1) activity problems of lactic acid bacteria.Transportation, use and the preservation process in easy inactivation, thereby reduce biological action, cause repeatability and the less stable of preparation result of use.
(2) stability problem in the feed process.To lactobacillus feed additive, must stand the test of 80 ℃ of high temperature in feed processing pelletization, so bacterial classification and preparation thereof seem particularly important to the stability of temperature.Different bacterial classifications is larger to the tolerance difference of high temperature, and general pelleting temperature is less on the bacillus impact, and is larger on impacts such as Bacillus acidi lactici, saccharomycete.In addition, the holding time of feed, the mineral matter in feed are (as heavy metal ion Cu
2+, Zn
2+, Mn
2+, Fe
2+Deng), choline etc. also can affect the vigor of probio.
(3) in animal body, lactobacillus preparation must pass through gastric environment, with a large amount of viable bacterias, arrives enteron aisle and is settled in its physiological function of competence exertion on intestinal mucosa.Yet because most of lactic acid bacteria is just dead because of hydrochloric acid in gastric juice and bile effect before entering enteron aisle, therefore the ability of survival propagation is lower in host, has affected to a great extent their prebiotic effect.In the survival rate of enteron aisle, also just become an important indicator of lactic acid bacteria effect.And any product standard of declaring to be added with probio is to contain at least 10
6-10
7The activated probio of cfu/g (FAO/WHO, 2001).
(4) pharmaceutical formulation is single, and versatility is not strong, thereby has limited same technique or the application of method in lactobacillus preparation prepared by non-designated bacterial classification.
Summary of the invention
First technical problem that the present invention will solve is to provide a kind of feeding high-activity lactic acid bacteria solid formulation, in said preparation, Lactobacillus Survival is high, the high-temp resisting high-humidity resisting ability improves, and survival rate is high in intestinal juice, alternative antibiotic, can significantly promote production performance and the health performance of meat chick, effectively reduce the grice diarrhoea rate.
Second technical problem that the present invention will solve is to provide a kind of preparation method of feeding high-activity lactic acid bacteria solid formulation, and this preparation method has following characteristics:
(1) has versatility, technique is simple, to common commercial lactic acid bacterium, comprise VREF, enterococcus faecalis, lactoenterococcus, bifidobacterium bifidum, lactobacillus acidophilus, Lactobacillus casei, lactobacillus lactis, Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus or bacillus bulgaricus, has applicability widely, and can be used for a large amount of production, simple to operate, cost of supplementary product is cheap;
(2) adopt " two-step method ", namely add basic recipe and protection formula, prepare microorganism formulation, lactic acid bacteria production, storage and use procedure are effectively protected, improved the viable bacteria survival rate in whole process;
(3) unique recipe ingredient, ratio and order of addition, make lactic acid bacteria, protective agent, the prebiotic factor and various auxiliary agent interact, cooperatively interact, to reach the purpose of effective assurance viable count.
For solving first technical problem, the present invention by the following technical solutions:
The invention provides a kind of feeding high-activity lactic acid bacteria solid formulation, it comprises probiotic lactobacillus bacterium mud, protection formula and basic recipe; Wherein,
described probiotic lactobacillus bacterium mud is VREF (Enterococcus faecium), enterococcus faecalis (Enterococcus faecalis), lactoenterococcus (Enterococcus lactis), bifidobacterium bifidum (Bifidobacterium bifidum), lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus casei (Lactobacillus casei), lactobacillus lactis (Lactobacillus lactis), Lactobacillus plantarum (Lactobacillus plantarum), Pediococcus acidilactici (Pedicoccus acidilacticii), the bacterium mud of one or more in Pediococcus pentosaceus (Pediococcus pentosaceus) or bacillus bulgaricus (Lactobacillus bulgaria), parts by weight are 1～5 part,
Above-mentioned these bacterial classifications all can obtain by the mode that business is bought.
Described protection formula comprises the component of following parts by weight: the food stage glycerine of 0.1～2 part, the sucrose of 0.4～2 part, the maltodextrin of 0.3～5 part, the plant extracts of the whey powder of 0.1～3 part and 0.06～1.5 part;
Described basic recipe comprises the component of following parts by weight: the prebiotic factor of 3～10 parts, the adhesive of 3～10 parts, the coating agent of 20.5～55 parts, the starch of 34～67 parts.
Further, plant extracts of the present invention is preferably one or more in eucommia leaf extract, dried orange peel extracts or licorice.
The described prebiotic factor is one or both in xylo-oligosaccharide or FOS.
Described adhesive is one or both in sodium carboxymethylcellulose or microcrystalline cellulose.
Described coating agent is one or more in polyacrylic resinⅡ, sodium alginate or calcium chloride; Preferably, described coating agent is polyacrylic resinⅡ or sodium alginate and two kinds of combinations of calcium chloride or polyacrylic resinⅡ, sodium alginate and three kinds of combinations of calcium chloride.
Described starch is one or more in cornstarch, wheat kind of starch or farina.
For solving second technical problem, the present invention by the following technical solutions:
A kind of preparation method of feeding high-activity lactic acid bacteria solid formulation, the method comprises the following steps:
(1) take one or more in VREF, enterococcus faecalis, lactoenterococcus, bifidobacterium bifidum, lactobacillus acidophilus, Lactobacillus casei, lactobacillus lactis, Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus or bacillus bulgaricus, ferment as bacterial classification, then zymotic fluid is centrifugal, make bacterium mud;
(2) by parts by weight, be that the above-mentioned bacterium mud of 1～5 part mixes with the various components in protection formula, then add water, dispersion stirs evenly, make bacterium liquid, wherein, described protection formula comprises the component of following parts by weight: the food stage glycerine of 0.1～2 part, the sucrose of 0.4～2 part, 0.3 the maltodextrin of～5 parts, the plant extracts of the whey powder of 0.1～3 part and 0.06～1.5 part; In this step, the purpose that adds water is in order to disperse bacterium mud, and according to routine operation, as long as amount of water guarantees to make bacterium mud to disperse, the weight ratio of the described water of ordinary circumstance and bacterium mud is (5-7): 1;
(3) the bacterium liquid that step (2) is made and the various component mix and blends in basic recipe, and in whipping process, add water, then carry out wet granulation, drying, obtain finished product, wherein, described amount of water is 40%～50% of basic recipe component gross weight; Described basic recipe comprises the component of following parts by weight: the prebiotic factor of 3～10 parts, the adhesive of 3～10 parts, the coating agent of 20.5～55 parts, the starch of 34～67 parts.
Further, described plant extracts is one or more in eucommia leaf extract, dried orange peel extracts or licorice, concrete preparation method is: 1～10 part of folium cortex eucommiae, dried orange peel or Radix Glycyrrhizae are dried to moisture≤10% under 40 ℃～55 ℃ conditions, be crushed to 60～80 orders, absolute ethyl alcohol extraction 1～3h, filter, and rotary evaporation is extracted thing, with adding 10～200 parts of water, obtain.
Further, the present invention is when carrying out strain fermentation, can use the culture medium that is suitable for growth well known in the prior art, for example, MRS culture medium, LB culture medium etc., the present invention provides a kind of formula of preferred culture medium at this: sucrose 1～4% (W/V), corn steep liquor 1～3% (W/V), yeast extract 0.4～2% (W/V), magnesium chloride 0.06～0.1% (W/V), manganese sulfate 0.04～0.2% (W/V), calcium carbonate 0.1～0.4% (W/V), sodium chloride 0.2～0.6% (W/V), water 100ml, pH7.0 ± 0.2.
" W/V " used in the present invention refers to w/v, i.e. g/ml or kg/L.
Further, the prebiotic factor described in said method is one or both in xylo-oligosaccharide or FOS; Described adhesive is one or both in sodium carboxymethylcellulose or microcrystalline cellulose; Described coating agent is one or more in polyacrylic resinⅡ, sodium alginate or calcium chloride, preferably, described coating agent is a kind of component of polyacrylic resinⅡ or sodium alginate and two kinds of combinations of calcium chloride or polyacrylic resinⅡ, sodium alginate and three kinds of combinations of calcium chloride.Described starch is one or more in cornstarch, wheat kind of starch or farina.
Further, in step (3), described drying is cyclone drying under 20 ℃～45 ℃ conditions.
Further, the present invention is when carrying out strain fermentation, and fermentation temperature is: 30 ℃～39 ℃.In sweat, can also control the amount of oxygen, generally at aerobic or micro-aerobic condition bottom fermentation, cultivate, when selecting micro-aerobic condition fermentation, the amount of oxygen is 0～0.5mg/L dissolved oxygen.
Beneficial effect of the present invention is:
1. feeding high-activity lactic acid bacteria solid formulation morphosis of the present invention is stable, in production, processing, storage, use procedure, effectively protects viable bacteria.With the lactobacillus preparation that only adds basic recipe, compare, said preparation after granulation, dry after the viable bacteria survival rate improve; Shelf life extension, store 6 months under normal temperature, and effectively the viable bacteria survival rate is more than 80%; Bacterium survival rate in simulated intestinal fluid all reaches more than 87%; Hot and humid tolerance improves 50%.
2. the animal applications effect significantly improves: feeding high-activity lactic acid bacteria solid formulation of the present invention improves meat chicken production performance, significantly improves daily gain, reduces feedstuff-meat ratio; Under the 10-100g/T addition, with the basal diet group, to compare, said preparation reduces feedstuff-meat ratio more than 9.5%; Improve the broiler health performance, intestinal villus is obviously elongated, eurythmy, and marshalling, evenly, on unit are, intestinal villus quantity is many for height; Antioxygenic property strengthens, and TAC and total superoxide dismutase level are improved (P<0.05), and in enteron aisle, Bifidobacterium and lactobacillus content significantly improve (P<0.05).
In addition, can also improve weaned piglets, improve daily gain, reduce the material anharmonic ratio; Under the 30-100g/T addition, with only adding the basic recipe group, to compare, said preparation improves daily gain more than 1.37%; Improve the piglet health performance, significantly reduce the grice diarrhoea rate in (P<0.05) more than 18%, can partly substitute zinc oxide and antibiotic.
The specific embodiment
The present invention will be further described below in conjunction with specific embodiment, but be not to limit the present invention.Experimental technique in following embodiment, if no special instructions, be conventional method.Test material used in following embodiment, if no special instructions, be the routine biochemistry reagent suppliers and buy and obtain.
Embodiment 1
(1) preparation of plant extracts
Respectively 1～10 part of folium cortex eucommiae, dried orange peel and Radix Glycyrrhizae are dried to moisture≤10% under 40 ℃～55 ℃ conditions, are crushed to 60～80 orders, 10～50 parts of absolute ethyl alcohols, extraction 1～3h, filter, and rotary evaporation is extracted thing (density 0.7～1.5g/cm
3), with adding 10～200 parts of water, obtain.
(2) preparation MRS culture medium and preferred culture medium
The preparation of MRS culture medium: get peptone 1% (W/V), glucose 2% (W/V), beef extract 1% (W/V), anhydrous sodium acetate 0.5% (W/V), yeast extract 0.5% (W/V), Tween 80 0.1ml, dipotassium hydrogen phosphate 0.2% (W/V), diammonium hydrogen citrate 0.2% (W/V), bitter salt 0.058% (W/V), Manganous sulfate monohydrate 0.025% (W/V), water 100ml, regulate pH to 7.0 ± 0.2; Adopt moist heat sterilization, temperature is 115 ℃, sterilization time 30min.
The preparation of preferred culture medium: sucrose 1～4% (W/V), corn steep liquor 1～3% (W/V), yeast extract 0.4～2% (W/V), magnesium chloride 0.06～0.1% (W/V), manganese sulfate 0.04～0.2% (W/V), calcium carbonate 0.1～0.4% (W/V), sodium chloride 0.2～0.6% (W/V), water 100ml, pH7.0 ± 0.2; Adopt moist heat sterilization, temperature is 115 ℃, sterilization time 30min.
(3) bacterium is clay standby
A. choose VREF (CGMCC2516), lactobacillus acidophilus (ATCC4356), respectively by bacterial classification under 35 ℃ of micro-aerobic conditions (being the 0.5mg/L dissolved oxygen), in the MRS culture medium, cultivate 8h, obtain VREF and lactobacillus acidophilus primary seed solution;
B. respectively the primary seed solution of two kinds of bacterium is cultivated by expansion in 2% inoculum concentration access seeding tank, under 35 ℃ of micro-aerobic conditions, in the MRS culture medium, cultivate 6h, obtain the secondary seed solution of two kinds of bacterium;
C. respectively the secondary seed solution of two kinds of bacterium is accessed in fermentation tank and carries out fermented and cultured by 5% inoculum concentration, use preferred culture medium to carry out fermented and cultured, under 38 ℃ of micro-aerobic conditions, cultivate 24h; Zymotic fluid, with the centrifugal bacterium mud that obtains two kinds of bacterium of tube centrifuge 10000rpm, can be mixed two kinds of bacterium mud according to arbitrary ratio as required, make Mixed Microbes mud, for example, the mass ratio of VREF bacterium mud and lactobacillus acidophilus bacterium mud is 1:1,1:2,1:3,2:1, or 3:1 etc.;
(4) formula of solid formulation:
Mixed Microbes mud 50g;
Protection formula: 5g food stage glycerine, 20g sucrose, 30g maltodextrin, 30g whey powder, 13g eucommia leaf extract, 1g dried orange peel extracts;
Basic recipe: 300g xylo-oligosaccharide, 20g sodium carboxymethylcellulose, 450g microcrystalline cellulose, 8g polyacrylic resinⅡ, 550g sodium alginate, 600g calcium chloride, 1700g cornstarch.
(5) formulation samples preparation
Portion adds 20 times standby (sample one) of sterilized water dilution, and portion only adds the basic recipe component to granulate by (sample two), and portion adds basic recipe and protects granulate (sample three) of filling a prescription.
A. only add the basic recipe component to granulate
Get the various component mix and blends in above-mentioned bacterium mud and basic recipe, and in whipping process, add water (described amount of water be basic recipe component gross weight 40%～50%), then wet granulation, throw circle, cyclone drying under 20 ℃～45 ℃ conditions, obtain finished product.
B. add basic recipe and protection formula to granulate
Get above-mentioned bacterium mud and with various components in the protection formula, mix immediately, then add water, disperse to stir evenly, standing 15-60min, make bacterium liquid standby;
Get the various component mix and blends in standby bacterium liquid obtained above and basic recipe, and in whipping process, add water (described amount of water be basic recipe component gross weight 40%～50%), then carry out wet granulation, throw circle, cyclone drying under 20 ℃～45 ℃ conditions, obtain finished product.Granulation, dry rear viable count are 1.0 * 10
10More than CFU/g.
(6) Performance Detection of the prepared lactobacillus preparation of the present embodiment
A. by product in the middle of the processing of the sample two in (5) and sample three (after granulating, after drying) sampling, detect sample viable count and survival rate, in Table 1; B. 3 samples in (5) are placed in to simulated intestinal fluid (Chinese Pharmacopoeia formula) 2h, detect sample viable count and survival rate, in Table 2; C. the sample in (5) is placed in to 85 ℃, 60% humidity environment 2min, detects sample viable count and survival rate, in Table 3; D. by 3 sample sealing preservations in (5), 20 ℃ of normal temperature storage, detect sample viable count and survival rate, in Table 4.
Table 1
Annotate: the bacterium unit of number: CFU value/g.Lower same.
Table 2
Table 3
| Sample | The 0h original value | 2min | Survival rate % after 2min |
| Sample one | 1.21×10 10 | 7.1×10 6 | -- |
| Sample two | 1.17×10 10 | 1.15×10 9 | 9.8 |
| Sample three | 1.01×10 10 | 6.16×10 9 | 61.01 |
Table 4
By on can draw: the product contrast shows in the middle of a. production process: after granulating, after dry, with the lactobacillus preparation viable bacteria survival rate that " two-step method " made, than the preparation that only adds basic recipe, improve respectively 6.05% and 20.01%; B. simulated intestinal fluid tolerance experiment shows: in the lactobacillus preparation that utilizes " two-step method " to make, effectively the viable bacteria survival rate is significantly higher than respectively the bacterium liquid that does not add any auxiliary material protection, the preparation that only adds basic recipe is 16.42% and 8.69%; C. hot and humid tolerance experiment shows: utilize in the lactobacillus preparation that " two-step method " make effectively the viable bacteria survival rate to be significantly higher than the preparation that only adds basic recipe and surpass 50%; D.6 month storing experiment shows: utilize in the lactobacillus preparation that " two-step method " make effectively the viable bacteria survival rate more than 80%, far above the bacterium liquid that does not add any auxiliary material protection, only add the preparation of basic recipe.
Embodiment 2
(1) prepare the preparation of LB culture medium and preferred culture medium
The preparation of LB culture medium: get tryptone 1% (W/V), yeast extract 0.5% (W/V), sodium chloride 1% (W/V), water 100ml, pH7.0 ± 0.2; Adopt moist heat sterilization, 121 ℃, 20min.
The preparation of preferred culture medium: sucrose 1～4% (W/V), corn steep liquor 1～3% (W/V), yeast extract 0.4～2% (W/V), magnesium chloride 0.06～0.1% (W/V), manganese sulfate 0.04～0.2% (W/V), calcium carbonate 0.1～0.4% (W/V), sodium chloride 0.2～0.6% (W/V), water 100ml, pH7.0 ± 0.2; Adopt moist heat sterilization, temperature is 115 ℃, sterilization time 30min.
(2) bacterium is clay standby
A. choose enterococcus faecalis (ATCC19433), bacterial classification, under 35 ℃ of conditions, is cultivated to 8h in the LB culture medium, obtain primary seed solution;
B. primary seed solution is cultivated by expansion in 2% inoculum concentration access seeding tank, under 37 ℃ of micro-aerobic conditions, in the LB culture medium, cultivate 6h, obtain secondary seed solution;
C. secondary seed solution is accessed in fermentation tank and carries out fermented and cultured by 5% inoculum concentration, use preferred culture medium to carry out fermented and cultured, under 38 ℃ of micro-aerobic conditions, cultivate 24h; By zymotic fluid with the centrifugal bacterium mud that makes of desk centrifuge 6000rpm.
(3) formula of solid formulation
Bacterium mud 100g;
The protection formula is: 25g food stage glycerine, 45g sucrose, 30g maltodextrin, 28g whey powder, 8g eucommia leaf extract, 1.5g dried orange peel extracts, 4.5g licorice; The preparation method of described plant extracts is with embodiment 1;
Basic recipe is: 325g xylo-oligosaccharide, 1.0g sodium carboxymethylcellulose, 334g microcrystalline cellulose, 18g polyacrylic resinⅡ, 1050g sodium alginate, 1200g sodium chloride, 6700g cornstarch;
(4) formulation samples preparation
Portion only adds the granulation of basic recipe component, and portion adds basic recipe, and (granulation, the rear viable count of drying are 1.0 * 10 with protecting formula to granulate
10More than CFU/g).Concrete preparation method is with embodiment 1.
(5) Performance Detection of the prepared lactobacillus preparation of the present embodiment: to broiler growth and healthy action effect.
Animal used as test: 120 public broiler chicken of AA;
Feeding and management: whole feeding period amounts to 42d, is divided into two stage: 1-21d and is early stage, and 22-42d is the later stage.Employing is raised in cages (each cage is exactly a repetition), and whole experimental period carries out immunity, sterilization routinely, whole day 24h illumination, and full phase free choice feeding and drinking-water, temperature automatically controlled control is wet.Before brood time, the 3d room temperature is controlled at 33 ℃, descends weekly 3 ℃ until 24 ℃ of constant temperature later; Humidity 1-21 day remains on 65-70%, and 22-42 keeps 60-65%; 1-42d continues 24h illumination and ventilation.
Packet design:
120 meat chicks are divided into to 4 groups at random, every group of 6 repetitions, 5 of every repetitions.Raise in SPF room, pilot scale base, computer is controlled humiture.
A group: the basal diet of added with antibiotic of feeding not+only add basic recipe lactobacillus preparation
B group: the not lactobacillus preparation 1.00 * 10 of the basal diet of added with antibiotic+" two-step method " protection of feeding
10The CFU/kg feed.
Experimental result is as table 5-6.
The impact of the lactobacillus preparation that table 5 " two-step method " is made on the broiler growth performance
The different lowercase alphabet differentials of colleague shoulder mark different significantly (P<0.05), identical lowercase or without letter representation difference not significantly (P > 0.05).Following table is same.
Table 6 TAC (T-AOC)
| ? | The A group | The B group |
| 21 ages in days | 11.11±0.84 a | 15.54±1.19 c |
| 42 ages in days | 9.49±0.93 bc | 10.89±1.28 c |
The impact of the lactobacillus preparation that table 7 " two-step method " is made on meat chick Morphological Indices of Duodenum Villus structure
The impact of the lactobacillus preparation that table 8 " two-step method " is made on meat chick gut flora
Table 5-table 8 shows, with the group that only adds the basic recipe preparation, compares, and the lactobacillus preparation group broiler chicken feedstuff-meat ratio that " two-step method " made is remarkable reduces by 9.66%; TAC significantly improves 14.75%-39.87%; Unit are intestinal villus quantity is many, and intestines suede height, the crypts degree of depth all have significant change; In ileum, Bifidobacterium, lactobacillus quantity significantly increase.
Embodiment 3
(1) prepare culture medium
Preparation LB culture medium: get tryptone 1% (W/V),, yeast extract 0.5% (W/V), sodium chloride 1% (W/V), water 100ml, pH7.0 ± 0.2; Adopt moist heat sterilization, 121 ℃, 20min.
Preparation preferred culture medium: sucrose 1～4% (W/V), corn steep liquor 1～3% (W/V), yeast extract 0.4～2% (W/V), magnesium chloride 0.06～0.1% (W/V), manganese sulfate 0.04～0.2% (W/V), calcium carbonate 0.1～0.4% (W/V), sodium chloride 0.2～0.6% (W/V), water 100ml, pH7.0 ± 0.2; Sterilizing methods: moist heat sterilization, 115 ℃, 30min.
(2) bacterium is clay standby
A. choose lactobacillus lactis (ATCC12315), bacillus bulgaricus (ATCC11842) and lactoenterococcus (ATCC19435), respectively by bacterial classification under 35 ℃ of micro-aerobic conditions (being the 0.3mg/L dissolved oxygen), in the LB culture medium, cultivate 8h, obtain the primary seed solution of three kinds of bacterium;
B. respectively the primary seed solution of three kinds of bacterium is cultivated by expansion in 2% inoculum concentration access seeding tank, under 37 ℃ of micro-aerobic conditions, in the LB culture medium, cultivate 6h, obtain the secondary seed solution of three kinds of bacterium;
C. respectively the secondary seed solution of three kinds of bacterium is accessed in fermentation tank and carries out fermented and cultured by 5% inoculum concentration, use preferred culture medium to carry out fermented and cultured, under 38 ℃ of micro-aerobic conditions, cultivate 24h; By zymotic fluid with the centrifugal bacterium mud that obtains of desk centrifuge 6000rpm.Can three kinds of bacterium mud be mixed according to arbitrary ratio as required, make Mixed Microbes mud, for example, the mass ratio of lactobacillus lactis bacterium mud, bacillus bulgaricus bacterium mud and lactoenterococcus bacterium mud is 1:1:1,1:2:1,1:1:3,2:1:1 etc.;
(3) formula of solid formulation
Mixed Microbes mud 50g
Protection formula: 10g food stage glycerine, 23g sucrose, 33g maltodextrin, 26g whey powder, 8g eucommia leaf extract, 1g dried orange peel extracts, 4g licorice; The preparation method of plant extracts is referring to embodiment 1;
Basic recipe: 450g xylo-oligosaccharide, 18g sodium carboxymethylcellulose, 320g microcrystalline cellulose, 10g polyacrylic resinⅡ, 1250g sodium alginate, 1550g calcium chloride, 3250g cornstarch.
(4) formulation samples preparation
Portion only adds the basic recipe component to granulate by (sample one), and portion adds basic recipe and protection formula to granulate (sample two) concrete preparation method with embodiment 1.
(5) Performance Detection of the lactobacillus preparation for preparing of the present embodiment
Experiment purpose: contrast only adds lactobacillus preparation that basic recipe and " two-step method " make to grice diarrhoea, daily gain.
Experimental animal: 102 of weanling pigs;
Duration of trial: test from wean 35 days experimental periods;
Packet design:
Weanling pig is divided into to 2 groups (two groups, one group, sample and samples), every group of 3 repetitions, 17 of every repetitions at random.
Diarrhea rate=diarrhoea piglet head/total head of test piglet
Diarrhoea piglet head=diarrhoea piglet head time (the 35th age in days)+diarrhoea piglet head time (the 36th age in days)+... + diarrhoea piglet head time (the 63rd age in days); The total head of a piglet=piglet number (the 35th age in days)+piglet number (the 36th age in days)+... + piglet number (the 63rd age in days).
Scours index: Scours index has reflected the order of severity of diarrhoea, and this index is higher, and representative diarrhoea is more serious.
Scours index=stools scored sum/total number of confession examination pig.Standards of grading see the following form.
Diarrhoea situation standards of grading
| The diarrhoea degree | The ight soil outward appearance | Scoring |
| Normally | Bar shaped or granular | 0 |
| Slightly | Soft fecal energy is shaped | 1 |
| Moderate | Thick shape, shapeless, liquid dung is without segregation phenomenon | 2 |
| Seriously | Aqueous, shapeless, liquid dung has segregation phenomenon, and mucus just or just dense | 3 |
| Pollute | The anus of pig and have the excrement thing to hang sample on every side | 4 |
| Red and swollen | The anus redness of pig | 5 |
To the testing result of two groups of samples as shown in table 9, table 10:
Table 9 piglet daily gain relatively
| Process | Average daily gain, g/ days |
| One group, sample | 464.13±30.1 |
| Two groups, sample | 470.5±15.3 |
Table 10 grice diarrhoea rate relatively
| Process | Diarrhea rate (%) | Scours index (%) | Death rate (%) |
| One group, sample | 2.48 b | 0.865 b | 1.6 |
| Two groups, sample | 2.01 a | 0.801 a | 0 |
As from the foregoing, with the preparation that only adds basic recipe, compare, the piglet daily gain of the lactobacillus preparation group that utilization " two-step method " is made improves 1.37%; Diarrhea rate significantly reduces (P<0.05), with control group, compares, and diarrhea rate descends 18.95%.
In addition, the ruddy degree of test group piglet skin and hair color brightness, in disorder degree are greatly improved.
Claims (10)
1. a feeding high-activity lactic acid bacteria solid formulation, is characterized in that, comprises probiotic lactobacillus bacterium mud, protection formula and basic recipe; Wherein,
Described probiotic lactobacillus bacterium mud is one or more the bacterium mud in VREF, enterococcus faecalis, lactoenterococcus, bifidobacterium bifidum, lactobacillus acidophilus, Lactobacillus casei, lactobacillus lactis, Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus or bacillus bulgaricus, and parts by weight are 1～5 part;
Described protection formula comprises the component of following parts by weight: the food stage glycerine of 0.1～2 part, the sucrose of 0.4～2 part, the maltodextrin of 0.3～5 part, the plant extracts of the whey powder of 0.1～3 part and 0.06～1.5 part;
Described basic recipe comprises the component of following parts by weight: the prebiotic factor of 3～10 parts, the adhesive of 3～10 parts, the coating agent of 20.5～55 parts, the starch of 34～67 parts.
2. feeding high-activity lactic acid bacteria solid formulation according to claim 1, is characterized in that, described plant extracts is one or more in eucommia leaf extract, dried orange peel extracts or licorice.
3. feeding high-activity lactic acid bacteria solid formulation according to claim 1, is characterized in that, the described prebiotic factor is one or both in xylo-oligosaccharide or FOS; Described adhesive is one or both in sodium carboxymethylcellulose or microcrystalline cellulose; Described coating agent is one or more in polyacrylic resinⅡ, sodium alginate or calcium chloride; Described starch is one or more in cornstarch, wheat kind of starch or farina.
4. the preparation method of a feeding high-activity lactic acid bacteria solid formulation, is characterized in that, the method comprises the following steps:
(1) take one or more in VREF, enterococcus faecalis, lactoenterococcus, bifidobacterium bifidum, lactobacillus acidophilus, Lactobacillus casei, lactobacillus lactis, Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus or bacillus bulgaricus, ferment as bacterial classification, then zymotic fluid is centrifugal, make bacterium mud;
(2) by parts by weight, be that the above-mentioned bacterium mud of 1～5 part mixes with the various components in protection formula, then add water, dispersion stirs evenly, make bacterium liquid, wherein, described protection formula comprises the component of following parts by weight: the food stage glycerine of 0.1～2 part, the sucrose of 0.4～2 part, 0.3 the maltodextrin of～5 parts, the plant extracts of the whey powder of 0.1～3 part and 0.06～1.5 part;
(3) the bacterium liquid that step (2) is made and the various component mix and blends in basic recipe, and in whipping process, add water, then carry out wet granulation, drying, obtain finished product, wherein, amount of water is 40%～50% of basic recipe component gross weight; Described basic recipe comprises the component of following parts by weight: the prebiotic factor of 3～10 parts, the adhesive of 3～10 parts, the coating agent of 20.5～55 parts, the starch of 34～67 parts.
5. preparation method according to claim 4, is characterized in that, described plant extracts is one or more in eucommia leaf extract, dried orange peel extracts or licorice.
6. preparation method according to claim 5, it is characterized in that, the preparation method of described eucommia leaf extract, dried orange peel extracts or licorice is: 1～10 part of folium cortex eucommiae, dried orange peel or Radix Glycyrrhizae are dried to moisture≤10% under 40 ℃～55 ℃ conditions, be crushed to 60～80 orders, absolute ethyl alcohol extraction 1～3h, filter, and rotary evaporation is extracted thing, with adding 10～200 parts of water, obtain.
7. preparation method according to claim 4, it is characterized in that, the culture medium prescription that uses during fermentation is: sucrose 1～4% (W/V), corn steep liquor 1～3% (W/V), yeast extract 0.4～2% (W/V), magnesium chloride 0.06～0.1% (W/V), manganese sulfate 0.04～0.2% (W/V), calcium carbonate 0.1～0.4% (W/V), sodium chloride 0.2～0.6% (W/V), water 100ml, pH7.0 ± 0.2.
8. preparation method according to claim 4, is characterized in that, the described prebiotic factor is one or both in xylo-oligosaccharide or FOS; Described adhesive is one or both in sodium carboxymethylcellulose or microcrystalline cellulose; Described coating agent is one or more in polyacrylic resinⅡ, sodium alginate or calcium chloride; Described starch is one or more in cornstarch, wheat kind of starch or farina.
9. preparation method according to claim 4, is characterized in that, in step (3), described drying is cyclone drying under 20 ℃～45 ℃ conditions.
10. preparation method according to claim 4, is characterized in that, in step (1), the temperature during fermentation is: 30 ℃～39 ℃.
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