CN100475952C - Preparation of feeding live bacterial acid, and dedicated bacterial strain - Google Patents
Preparation of feeding live bacterial acid, and dedicated bacterial strain Download PDFInfo
- Publication number
- CN100475952C CN100475952C CNB2007100650175A CN200710065017A CN100475952C CN 100475952 C CN100475952 C CN 100475952C CN B2007100650175 A CNB2007100650175 A CN B2007100650175A CN 200710065017 A CN200710065017 A CN 200710065017A CN 100475952 C CN100475952 C CN 100475952C
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- acid
- preparation
- live bacterial
- feeding live
- subtilis
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Abstract
This invention discloses a feed-use living bacterium acidic preparation and its special strain. The active component of the feed-use living bacterium acidic preparation is Bacillus subtilis subsp. Subtilis Gendone-01 (CGMCC No.1946). The feed-use living bacterium acidic preparation can be used for livestock as a substitute for antibiotics. And the livestock product does not have residual antibiotics, which is good for human health. Besides, the feed-use living bacterium acidic preparation can increase feed utility, reduce feed cost, sterilize in intestines, reduce diarrhea, and improve anti-stress ability and immunity.
Description
Technical field
The present invention relates to a kind of preparation of feeding live bacterial acid and special strain therefore thereof.
Background technology
Along with the raising of living standards of the people with to the reinforcement of environmental consciousness, requirement to livestock product quality and animal rearing hygienic condition is more and more higher, traditional feeding antibiotic additive progressively is under an embargo or limits use, in January, 2006, European Union has formally determined to forbid adding the microbiotic that promotes growth is used in animal and fowl fodder.Asia developed countries such as Japan, Korea S have also formulated the policies and regulations of corresponding restriction animal product antibiotic remains subsequently.
Seek antibiotic substitute, alleviating the negative impact that the microbiotic forbidding brings Production of Livestock and Poultry is the problem that various countries' livestock industry presses for solution.The lot of domestic and foreign report is thought, probiotics, souring agent, Chinese herbal feed additive, fodder enzyme preparation, functional oligosaccharide etc. can be used as antibiotic substitute products, but because breeding environment is poor, the souring agent additive capacity is not enough, probiotic bacterium vigor in enteron aisle is not high, often can not replace the promotes growth microbiotic fully in the actual production.Compound several substitute is sought the probiotics of high vigor, anti-adverse circumstance, utilizes the powerful sterilizing ability of its antibacterial peptide to substitute microbiotic shortcoming still at present.
Souring agent, active bacteria formulation can not substitute feeding antibiotic fully.Souring agent is remarkable at the effect of pig, especially to weanling pig, can remedy the gastric acid secretion defect of insufficient, the activator enzymic activity promotes nutrient digestion to absorb, and can be used as the first-selected product of antibiotic substitute, but product as an alternative, addition is essential wants big, and it is also bigger influenced by the surge capability of daily ration, can't add heavy dose of souring agent in the production practice.Probiotics is product as an alternative, its inferior position be the easy inactivation of active bacteria formulation, not anti-stomach sour environment, be difficult in gastrointestinal bacterial flora, be in competitive edge.
Owing to be subjected to the constraint of national conditions, the envrionment conditions of animal rearing is poor, China's its feeding of the overwhelming majority at present also be unable to do without microbiotic, antibiotic remains is than higher in the animal products, substantially can not export, seriously restrict the sound development of livestock industry, not only can improve the livestock and poultry quality so Development and Production can substitute antibiotic highly effective and safe fodder additives, and, realize that the livestock industry sustainable and healthy development all is highly significant for improving the ecological environment.
Summary of the invention
The purpose of this invention is to provide a kind of preparation of feeding live bacterial acid and special strain therefore thereof.
The special strain therefore of preparation of feeding live bacterial acid provided by the present invention is subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 02 15th, 2007 and (be called for short CGMCC, the address is: the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City), preservation registration number is CGMCC No.1946.
Subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01CGMCC No.1946 separates obtaining from Shunyi, Beijing plant soil.Concrete grammar is as follows: 12 parts of the earth of fetching earth from the different location of Shunyi, Beijing plant, each soil sample are all about the 2-3 gram.Join respectively in the 100ml stroke-physiological saline solution, vibration mixed 1 minute, left standstill then 5 minutes.Respectively get the 1ml supernatant liquor, join respectively among the 10ml aseptic culture fluid A that (every liter of nutrient solution A contains: glucose 5.0 grams, peptone 5.0 grams, extractum carnis 3.0 grams, yeast powder 1.0 grams, MgSO47H
2O 0.5 gram, MnSO4H
2O 0.005 gram, pH 7.0), (4 kinds of indicators are respectively: streptococcus aureus Staphylococcus aureusIVDC C56005 respectively to add 4 kinds of each 1ml of indicator nutrient solution simultaneously; Salmonella typhimurium Salmonella typhimurium IVDC C79-13; Intestinal bacteria K88E.coli IVDC C83901; E. coli k99 E.coli IVDC C83529).In 37 ℃ of mixed culture 48 hours (60rpm slowly runs), then nutrient solution is placed 80 ℃ of water-bath insulations 10 minutes, kill vegetative cell.10000 times of nutrient solution dilutions after will handling with stroke-physiological saline solution, get diluent 0.5ml and be uniformly coated on the solid medium B (adding 1.8% agar in nutrient solution A), 37 ℃ leave standstill cultivation 48 hours, select wherein maximum single bacterium colony, switching is in slant tube, 4 ℃ of preservations.Finishing screen is selected the good subtilis withered grass subspecies of proterties (Bacillus subtilis subsp.subtilis), laboratory called after: Gendone-01 (preservation registration number: CGMCC No.1946).
The result of morphological specificity, physio-biochemical characteristics and the 16SrDNA sequential analysis of comprehensive subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01CGMCC No.1946 is accredited as subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) with it.Concrete qualification result is as follows:
(1) morphological specificity of thalline
Gram-positive, cell shape are shaft-like, and cell dia forms gemma smaller or equal to 1 μ m, and gemma does not expand, and gemma is non-circular, does not form parasporal crystal.
(2) physio-biochemical characteristics
The physio-biochemical characteristics of grass genus bacillus withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01 CGMCCNo.1946 are as shown in table 1.
The physicochemical characteristics of table 1 subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01CGMCC No.1946
| Test subject | The result | Test subject | The result |
| Catalase oxydase anaerobic growth VP test VP＜pH6 VP＞pH7 methyl red test utilizes carbohydrate to produce sour grapes sugar wood sugar L-arabinose N.F,USP MANNITOL | + + - + + - + + + + + | Utilize the glucose aerogenesis to utilize the liquefaction of 50 ℃ of growths of Citrate trianion nitrate reduction pH5.7 growth 7%NaCl growth starch gelatin hydrolysate to decompose casein | - + + + + + + + + |
Annotate: "+" expression positive findings; "-" expression negative findings.
(3) 16SrDNA sequential analysis
The 16SrDNA sequencing result of subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01CGMCC No.1946 is shown in sequence in the sequence table 1.
Preparation of feeding live bacterial acid provided by the present invention, its activeconstituents comprise (being mainly) subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01 CGMCC No.1946.
In order to strengthen result of use, the activeconstituents of described preparation of feeding live bacterial acid also contains organic acid.
Described organic acid can be a kind of or its arbitrary combination in citric acid, fumaric acid, lactic acid (calcium lactate), dystopy acid, acetate, propionic acid, formic acid (calcium formiate), oxysuccinic acid, Sorbic Acid and succsinic acid etc. and its esters.
Described organic acid is preferably a kind of or its arbitrary combination in citric acid, fumaric acid, lactic acid, oxysuccinic acid and its esters.
In the described preparation of feeding live bacterial acid, the content of subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01 CGMCC No.1946 can be 5-50 * 10
8The CFU/g preparation of feeding live bacterial acid.
In the described preparation of feeding live bacterial acid, organic acid content can be the 400-800g/kg preparation of feeding live bacterial acid.
In the described preparation of feeding live bacterial acid, all the other compositions are the feed assistant agent.Existing feed all can be selected for use with assistant agent, and assistant agent can be a kind of or its arbitrary combination in hydrated SiO 2, whey powder and the lactose as described.
The present invention also provides the method for a kind of cultivation subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01 CGMCC No.1946.
The method of cultivation subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01 CGMCC No.1946 provided by the present invention, be in fermention medium, at 32-40 ℃, cultivated 33-39 hour, and collected and obtain subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01 CGMCC No.1946 thalline;
The pH of described fermention medium is 6.0-8.0, is made into by following material: Semen Maydis powder 2.5-3.5g; Soyflour 5.0-7.0g; Peptone 1.5-2.5g; Glucose 2.0-4.0g; Urea 1.5-2.5g; Magnesium sulfate heptahydrate 0.3-0.7g; Manganese sulfate monohydrate 0.3-0.7g; Potassium primary phosphate 0.8-1.2g adds water and is settled to 1000ml.
In order to obtain higher thalline output, described culture temperature is 35-38 ℃, and incubation time 35-37 hour, the pH of described fermention medium was 6.5-7.5, is made into by following material: Semen Maydis powder 3.0g; Soyflour 6.0g; Peptone 2.0g; Glucose 3.0g; Urea 2.0g; Magnesium sulfate heptahydrate 0.5g; Manganese sulfate monohydrate 0.5g; Potassium primary phosphate 1.0g adds water and is settled to 1000ml.
Subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01CGMCC No.1946 has high sterilizing power to multiple enteron aisle pathogeny bacterium such as intestinal bacteria, Salmonellas and Listeria monocytogeness, and fungicidal effectiveness reaches the close effect of microbiotic.
Conventional feeder acidulant exists that addition is not enough, acidizing effect is not obvious, effective many deficiencies such as sterilization when reality is used; The organic acid preparation of feeding live bacterial acid that contains of the present invention utilizes souring agent to diet pH value, gi tract pH value and oxidetic reduction, directly acts on body, and sterilization in advance reduces harmful microorganism in the enteron aisle; Utilize subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) the Gendone-01 CGMCC No.1946 that can effectively kill multiple enteron aisle pathogeny bacterium such as intestinal bacteria and Salmonellas again, make enteric pathogenic bacteria quantity descend nearly 100 times, probiotics quantity increases, clean enteron aisle.Thereby than using conventional souring agent more can improve gi tract acidity rapidly, and be that the inherent mechanism of environment reaches the purpose that reduces pH and improve digestive function in the balance gi tract microorganism.If in every gram preparation of feeding live bacterial acid of the present invention, the content of the active brood gemma of subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01 CGMCC No.1946 surpasses 2,500,000,000, and the product that adds the 1-3kg/ ton in swine feed just can replace microbiotic fully and not influence the production performance of animal.
Preparation of feeding live bacterial acid of the present invention can replace microbiotic, the grice diarrhoea rate descends significantly, feed conversion rate improves 2-5%, the growing-finishing pig feed conversion rate on average improves 2-6%, after omnidistance the use, the pork product antibiotic remains is reduced under the required standard, reaches export standard, is the green product innovation that substitutes feeding antibiotic.
The experimentation on animals result shows that use feeding microbial inoculum of the present invention has promoted hydrochloric acid in gastric juice and pepsic secretion significantly in the daily ration, trypsinase and diastatic activity have been improved, promoted the breeding of distal colon lactobacillus, suppressed the growth of harmful bacterium such as intestinal bacteria, and then improved the production performance of weanling pig significantly, reduced the generation of diarrhoea.
Preparation of feeding live bacterial acid of the present invention is applied to livestock and poultry, can substitute feeding antibiotic fully, and the animal products antibiotic-free is residual, guarantee human health, improve food utilization efficiency simultaneously, reduce feeding cost, play the enteron aisle sterilization, reduce diarrhoea, anti-stress and raise immunity effect.
The preparation of feeding live bacterial acid of the present invention of compatibility high reactivity gemma active bacteria formulation and souring agent can substitute feeding antibiotic, compare with common active bacteria formulation, the ability that genus bacillus in the preparation of feeding live bacterial acid of the present invention is killed the harmful bacterium of body is strong more than 10 times, the brood gemma excellent stability of genus bacillus, easy to use.Preparation of feeding live bacterial acid of the present invention substitutes feeding antibiotic from traditional part and rises to the feeding antibiotic that substitutes pig fully; Just can substitute microbiotic from rising to from the back 10 days beginnings of weaning or wean at alternative microbiotic of growing and fattening phase; The meat product antibiotic remains is reduced to below the standard code.Simultaneously, preparation of feeding live bacterial acid of the present invention does not influence the growth performance of animal, can effectively control grice diarrhoea.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
The cultivation of embodiment 1, subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01 CGMCC No.1946 and security detect
1, yeast culture
Consisting of of the seed culture medium that this embodiment is used: every liter of nutrient solution contains: glucose 5.0 grams, peptone 5.0 grams, extractum carnis 3.0 grams, yeast powder 1.0 grams, MgSO47H
2O 0.5 gram, MnSO4H
2O 0.005 gram, pH 7.0.
The composition of fermention medium (g/L): Semen Maydis powder 3.0; Soyflour 6.0; Peptone 2.0; Glucose 3.0; Urea 2.0; Magnesium sulfate heptahydrate 0.5; Manganese sulfate monohydrate 0.5; Potassium primary phosphate 1.0, pH6.5-7.5.
Gendone-01 CGMCC No.1946 encircles in seed culture medium with transfering loop picking 2 with activated subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis), behind the shaking table shaking culture 24h, get 2% (volume ratio) seed culture fluid and be forwarded in 5 tons of fermentor tanks that 3.6 tons of fermented liquids are housed and cultivated 36 hours.Wherein, culture temperature is 35-38 ℃; Ventilation: 0-6 hour, 0.2vvm; 6-10 hour, 0.4vvm; 10-18 hour, 0.6vvm; 18-36 hour, 0.4vvm.After the fermentation ends, mixing with nutrient solution with corn cob meal (particle diameter is 0.02-0.20mm), is 1: 1 (corn cob meal/nutrient solution) by weight proportion.In 50-60 ° of C air seasoning to water content is 10%, and packing is sealed then, obtains the feeding live bacterial agent.In this feeding microbial inoculum, brood gemma quantity is every gram 10,000,000,000 CFU, and the high 5-10 of viable bacteria content that produces than routine techniques doubly.
2, safety evaluation
Select the SD rat of 12 body weight basically identicals, initial body weight is 200 ± 20 grams, and male and female half and half are divided into 2 test group.Use 5ml subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01 CGMCC No.1946 gemma suspension (2-5 * 10 for one group
8Cfu/ml) be expelled in the rat abdominal cavity, another group with the 5ml stroke-physiological saline solution in contrast.Fasting is 6 hours before the injection, gavages the back and observes its toxicity symptom, and the time was 1 week.The result shows that all rats that gavage subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01 CGMCC No.1946 gemma suspension all do not have obvious clinical symptom, none death.Test group and control group do not have notable difference.Subtilis is widespread in nature as a kind of bacterium of generally recognized as safe (GRAS), can use safely.
The preparation of embodiment 2, preparation of feeding live bacterial acid
With the feeding live bacterial agent preparation preparation of feeding live bacterial acid of embodiment 1 preparation, its composition is as shown in table 2.Wherein the content of subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01 CGMCCNo.1946 is 1.4 * 10
9The CFU/g preparation of feeding live bacterial acid.
The composition of table 2 preparation of feeding live bacterial acid
| Material name | Proportioning (kilogram) |
| Hydrated SiO 2 (auxiliary material) | 8 |
| The feeding live bacterial agent of embodiment 1 preparation | 14 |
| Anhydrous calcium lactate | 10 |
| Citric Acid, usp, Anhydrous Powder | 40 |
| Fumaric acid | 5 |
| Oxysuccinic acid | 3 |
| Lactose (as auxiliary material) | 20 |
| Add up to (kilogram) | 100 |
Its preparation method is as follows:
250 kilograms of every batch of turnout, take by weighing 100 kilograms of Citric Acid, usp, Anhydrous Powders, 25 kilograms of anhydrous calcium lactates, 12.5 kilograms of fumaric acid, 7.5 kilograms of oxysuccinic acid earlier, carry out thorough mixing for 20 kilograms with hydrated SiO 2, in case of necessity oarse-grained organic acid (fumaric acid etc.) is pulverized, required whole mistake 60 mesh sieves.Next step is with the organic acid that mixes (and its esters) and auxiliary material totally 165 kilograms of feeding live bacterial agent (10,000,000,000 CFU/g feeding live bacterial agent), 50 kilograms of lactose thorough mixing that prepare with 35 kilograms of embodiment 1.Sieve at last, be finished product after the check, weighing, packing.
Embodiment 3, experimentation on animals
Percentage composition in this experimentation on animals is the quality percentage composition.
1, experimental design
In this experimentation on animals, select 21 ages in days for use, mean body weight be 5.3 ± 0.1kg (Du Luoke * length is white * Da Bai) 72 of weanling pigs, be divided into 4 processing, each handles 18.First group is control group, the basal diet a of the table 3 of only feeding; Second group is the preparation of feeding live bacterial acid group, and daily ration b feeds; The 3rd group is the happy acid group that reaches, and daily ration c feeds; The 4th group is strong acid spirit group, and daily ration d feeds.Each handles 6 repetitions, and each repeats 3 pigs.Feeding period is 28 days.
Wherein, basal diet a is based on corn, swelling soya dreg, expanded soybean, and adds fish meal, whey powder and glucose on a small quantity.Composition and the trophic level of basal diet a see Table
On the basis of basal diet a, reduce the consumption of corn, add respectively in the daily ration embodiment 2 preparations preparation of feeding live bacterial acid, happyly reach acid (Spain Le Da company) or strong acid spirit (Zhuhai overflow the production of how sharp company), obtain daily ration b, daily ration c and daily ration d.
The composition of table 3 basal diet 1 and trophic level
The composition of daily ration b is compared with basal diet a, and the percentage composition that removes corn is 56.9%, and outside the preparation of feeding live bacterial acid of embodiment 2 preparations of interpolation 0.1%, other raw material is with basal diet a.
The composition of daily ration c is compared with basal diet a, and the percentage composition that removes corn is 56.75%, and the pleasure of interpolation 0.25% reaches outside the acid (Spain Le Da company), and other raw material is with basal diet a.
The composition of daily ration d is compared with basal diet a, and the percentage composition that removes corn is 56.8%, and outside the strong acid spirit of interpolation 0.2% (the excessive how sharp company in Zhuhai produces), other raw material is with basal diet a.
2, feeding and management
In semi-hermetic type child care house, each repeats feeding piglet in a hurdle for the examination swine rearing, and particulate material is fed, free choice feeding and drinking-water, and room temp remains on more than 22 ℃, health in the cleaning sterilization every day house, flushing water fountain.By normal immune programme for children immunity.Every day is by special messenger's observed and recorded test baby pig disease death condition.
3, data processing
Testing data adopts SPSS 10.0 statistical softwares to add up, and carries out variance analysis and Duncan ' s multiple comparisons.
4, index detects
(1) day weight gain, food consumption and feedstuff-meat ratio
After on-test, claim to survey initial weight, weigh weekly then 1 time, ramming material 1 time, the statistics food consumption, and calculate day weight gain and feedstuff-meat ratio.
(2) diarrhoea observation and diarrhoea exponential are determined
Observe the diarrhoea situation every day in 2 week of test, writes down diarrhoea pig's head number of times, and give different marks according to the situation of ight soil, statistics diarrhoea index, and concrete methods of marking sees Table 4.
Table 4 diarrhoea exponential standards of grading
| The ight soil outward appearance | Hard excrement | Even viscosity fecal | Soft, the part moulding | Half aqueous, paste shape ight soil | Watery stools |
| Scoring | 1 | 2 | 3 | 4 | 5 |
The day weight gain of different treatment, food consumption, feedstuff-meat ratio, diarrhoea observation and diarrhoea index result are as shown in table 5, compare with control group, add souring agent in the daily ration and all improved the day weight gain (P＜0.05) of wean back piglet in 4 weeks significantly, best with preparation of feeding live bacterial acid effect of the present invention.Adding preparation of feeding live bacterial acid of the present invention in the feed has the piglet of raising food consumption, improves the trend of feed efficiency, but difference not significantly (P＞0.05).Aspect diarrhoea exponential sum diarrhea rate, in the first two weeks after wean, add souring agent in the daily ration and all can improve piglet ight soil situation (P＜0.05) significantly, reduce the generation of diarrhoea, wherein best with preparation of feeding live bacterial acid effect of the present invention.
Table 5 preparation of feeding live bacterial acid group is to the influence of weanling pig production performance and diarrhoea situation
Annotate: letter shows the result of variance analysis, with the average tool significant difference on α=0.05 level that indicates different letters in the delegation.
Behind the early weaning piglet, because psychological stress, environmental stress and nutrition stress, probioticss such as original enteron aisle dominant microflora one lactobacillus are owing to lack substrate, the breeding survival rate reduces significantly, harmful microorganisms such as intestinal bacteria take this opportunity to breed in a large number, intestinal microbial balance faces a severe challenge, and gastric acid secretion this moment deficiency, pepsin activity is lower, the nutritive ingredient that is not digested in the stomach enters the enteron aisle back segment, for condition has been created in the breeding of pathogenic bacteria, trophism diarrhea and pathogenicity bo diarrhoea very easily appear in piglet.
Add the pH value that souring agent can reduce gastric content in the daily ration, improve pepsin activity and then promote digesting and assimilating of nutritive substance, reduce the generation of grice diarrhoea, this is the basic model that conventional souring agent is taked.If on this basis, external source is added probiotic bacterium, suppresses the growth of pathogenic micro-organism, will further improve the effect of souring agent.Also to be preparation of feeding live bacterial acid of the present invention be better than the major cause of other treatment group to the improvement of production performance of piglet and diarrhoea situation for this.
(3) feed pH and sour bonding force
The index that often will consider when the sour bonding force of feed is the high-grade suckling piglet feed of preparation, the sour bonding force of feedstuff raw material and feed can regularly detect in feed with good conditionsi producer, it is generally acknowledged that the sour bonding force of pig starter feed is advisable with about 30.In the research in early days, in order to reach so sour bonding force, often add a large amount of mineral acids or organic acid, though can play the oxidetic effect that reduces feed, but owing to need to add lot of organic acids or mineral acid, improved feed cost and feed processing machinery is had a negative impact, adding a large amount of organic acids in therefore producing at present, to reduce the oxidetic way of daily ration inadvisable.
Get the 20g feed, add the 30ml deionized water, the formation suspension that stirs, and measure its pH value with acidometer.The acid bonding force then is defined as the mmole number of the hydrochloric acid that consumes when the 100g feed is titrated to pH=4.0.Measurement result is as shown in table 6, shows to add pH value and the sour bonding force that souring agent all can reduce feed, and the effect that reaches acid with pleasure is best, and the strong acid spirit is taken second place, and the amplitude that preparation of feeding live bacterial acid of the present invention reduces is minimum.This mainly is to contain a large amount of mineral acids because pleasure reaches acid, and the pH value is lower, due to usage quantity is big.
Table 6 pair feed pH value and oxidetic influence
| Control group | The preparation of feeding live bacterial acid group | The happy acid group that reaches | Strong acid spirit group | |
| Feed pH value | 6.41 | 6.22 | 6.12 | 6.18 |
| Acid bonding force (mmol) | 41 | 38 | 34 | 37 |
(4) hydrochloric acid in gastric juice, enzyme are lived and the colon microbioassay
Carry out slaughter experiment after raising for 2 weeks, each repeats to get 1 piglet that body condition is medium, and each treatment group is got 6 piglets, raises injection of heart narcotic after back 1 hour morning, after waiting to lose consciousness, and the heart sacrificed by exsanguination.Cut the abdominal cavity open, take out internal organ, isolate stomach, duodenum, jejunum and ileum, get the partial content thing, measure the pH value.
The mensuration of a, gastric acid secretion
In fact hydrochloric acid in gastric juice be exactly hydrochloric acid, by measuring the chlorine ion concentration of gastric content chlorine ion concentration feed resource, can get the chlorine ion concentration of stomachial secretion.Concrete grammar is, gets the gastric content supernatant liquor, uses AgNO
3[the Cl of titration measuring gastric content and feed resource
-], can get [the Cl in the hydrochloric acid in gastric juice
-].
Preparation of feeding live bacterial acid of the present invention and different souring agents to piglet gastrointestinal content pH value and gastric acid secretion to influence the result as shown in table 7, compare with control group, use preparation of feeding live bacterial acid of the present invention in the daily ration, happyly reach the pH value (P＜0.05) that acid and strong acid spirit all can reduce gastric content significantly, wherein best with preparation of feeding live bacterial acid effect of the present invention; Add the pH value (P＜0.05) that preparation of feeding live bacterial acid of the present invention has reduced duodenum, jejunum and ileal contents significantly in the daily ration, promoted gastric mucosa Cl significantly
-Secretion (P＜0.05), illustrate that preparation of feeding live bacterial acid of the present invention can promote the secretion of hydrochloric acid in gastric juice.Compare with preparation of feeding live bacterial acid of the present invention, use in the daily ration happyly to reach acid and strong acid spirit all not to intestinal contents pH value, the secretion of hydrochloric acid in gastric juice generation influences significantly.
Table 7 preparation of feeding live bacterial acid is to the influence of gastrointestinal content pH value and gastric acid secretion
| Control group | The preparation of feeding live bacterial acid group | The happy acid group that reaches | Strong acid spirit group | SEM | The P-value | |
| Gastric content pH value | 5.13 b | 4.52 a | 4.57 a | 4.69 a | 0.08 | 0.027 |
| Duodenum content pH value | 5.95 b | 5.57 a | 5.82 ab | 5.92 b | 0.11 | 0.042 |
| The terminal content pH of jejunum value | 6.78 b | 6.35 a | 6.71 ab | 6.75 b | 0.13 | 0.028 |
| Terminal ileum content pH value | 7.04 b | 6.32 a | 6.98 b | 6.96 b | 0.15 | 0.015 |
| Total secretion Cl -(mmol) | 18.5 a | 67.8 b | 20.6 a | 19.3 a | 1.1 | 0.031 |
| Gastric mucosa secretion Cl -(mmol/g) | 4.8 a | 17.2 b | 5.3 a | 5.1 a | 0.3 | 0.018 |
Annotate: letter shows the result of variance analysis, with the average tool significant difference on α=0.05 level that indicates different letters in the delegation.
The acidity of gastric content is common decision of product acid power by the secretion of the character of feed, acidity, hydrochloric acid in gastric juice and intestinal microflora itself.Result from this test, although the sour bonding force of its daily ration of preparation of feeding live bacterial acid treatment group of the present invention will be higher than two other treatment group of using souring agent, but from gastric content pH value, duodenum, jejunum and ileum pH value, there is not difference between three treatment group, gi tract pH value with preparation of feeding live bacterial acid treatment group of the present invention is minimum, this may promote the secretion of hydrochloric acid in gastric juice with viable bacteria acid, and the breeding that has improved gi tract acid-producing bacteria group is relevant.
B, to the influence of digestive enzyme activity
Determination of peptic activity adopts An Senfa, and determination of tryptic activity adopts the method in Wei Erte-bamboo of executing, and amylase activity is measured the F.I.P method that adopts.Aforesaid method see for details " measuring method of enzyme " (Qian Jiayuan, " measuring method of enzyme ", Beijing: China Light Industry Press, 1992:21-67,210-234,256-270,324-332).Get the gastric content supernatant liquor, press An Senfa measured for total pepsin activity, promptly total stomach en-=stomach en-+propepsin; Regulating gastric content supernatant liquor pH=8.0, make stomach en-inactivation wherein, is the propepsin activity by what An Senfa measured again, deducts the propepsin activity from total enzyme activity, promptly gets pepsin activity.
Preparation of feeding live bacterial acid of the present invention and different souring agents to digestive enzyme activity to influence the result as shown in table 8, compare with control group, add preparation of feeding live bacterial acid of the present invention in the daily ration and improved gastric content protease activities (P＜0.01), improved the conversion ratio (P＜0.01) of propepsin.Add and happyly to reach the conversion that acid and strong acid spirit have all promoted propepsin, do not influence but the stomach en-enzymic activity produced significantly.Preparation of feeding live bacterial acid of the present invention and Le Da acid have improved trypsinase and diastatic activity (P＜0.01), and the strong acid spirit does not produce influence significantly.Souring agent does not all produce influence significantly to the propepsin activity.
Table 8 preparation of feeding live bacterial acid of the present invention is to the influence of digestive enzyme activity
Annotate: letter shows the result of variance analysis, with the average tool significant difference on α=0.05 level that indicates different letters in the delegation.
Stomach en-and gastric acid secretion have certain synchronism.From the result of this test, also confirmed this point.Preparation of feeding live bacterial acid of the present invention has also promoted propepsin to pepsic conversion in gastric acid secretion, makes the activity of preparation of feeding live bacterial acid treatment group Pepsin of the present invention be higher than other treatment group significantly.Pleasure reaches acid and the strong acid spirit also has the effect that promotes that stomach en-transforms to stomach en-, but effect does not have preparation of feeding live bacterial acid of the present invention so obvious.Its reason has two.The one, preparation of feeding live bacterial acid of the present invention has promoted pepsic secretion, the 2nd, and preparation of feeding live bacterial acid of the present invention itself contains the pepsic flora of generation, has produced certain stomach en-at stomach.
About the influence of souring agent to the small intestine digestive enzyme activity, this experimental result shows that preparation of feeding live bacterial acid of the present invention and Le Da acid all can improve trypsinase and diastatic activity significantly, and is best with preparation of feeding live bacterial acid effect of the present invention.The strong acid spirit also has the trend that improves trypsinase and amylase activity, but difference is not remarkable.
Preparation of feeding live bacterial acid of the present invention and Le Da acid improve the machine-processed not clear of trypsinase and amylase activity, may be relevant with the secretion that promotes stomach en-and gastric juice.On the one hand, the protein in the food by pepsin hydrolysis after, enter into the secretion that can stimulate the enteron aisle digestive ferment behind the duodenum; On the other hand, after chyme enters duodenum, acid (H wherein
+) can stimulate the intestinal mucosa cell to produce secretin, the pancreatic secretion that digestive ferment is rich in promotion makes trypsinase and amylase activity improve.
The mensuration of c, colon microorganism
Get the part colonic contents, laboratory culture intestinal bacteria and lactobacillus are sent in 4 ℃ of preservations in 4 hours.Intestinal bacteria are adopted the aerobic cultivation counting of eosin methylene blue agar, and lactobacillus adopts lactobacillus culture medium culturing counting.Preparation of feeding live bacterial acid of the present invention and different souring agents to colon microorganism species quantity to influence the result as shown in table 9, add the quantity (P＜0.01) that preparation of feeding live bacterial acid of the present invention has improved the distal colon lactobacillus significantly in the daily ration, reduced colibacillary quantity (P＜0.01).Use happy reach acid and strong acid spirit then to the colon lactobacillus, the coliform count volume production is given birth to significantly influences.
Table 9 preparation of feeding live bacterial acid of the present invention is to the influence of colon microorganism
| Control group | The preparation of feeding live bacterial acid group | The happy acid group that reaches | Strong acid spirit group | SEM | The P-value | |
| Lactobacillus (10 9CFU/g) | 2.54 a | 175.3 b | 9.25 a | 6.42 a | 4.21 | ＜0.01 |
| Intestinal bacteria (10 7CFU/g) | 8.1 b | 1.8 a | 5.2 ab | 6.5 b | 1.5 | ＜0.01 |
Annotate: letter shows the result of variance analysis, with the average tool significant difference on α=0.05 level that indicates different letters in the delegation.
From the result of this test, add the breeding that preparation of feeding live bacterial acid of the present invention has promoted the distal colon lactobacillus significantly in the daily ration, suppressed the growth of harmful bacterium such as intestinal bacteria.This mainly is that lower pH value environment can promote the growth of probioticss such as lactobacillus because the pH value of enteron aisle can have influence on the quantity and the structure of its inner microorganism species.In addition, owing to added probiotic bacterium in the viable bacteria acid, when breeding, the probiotic bacterium of this part external source also can play certain restraining effect in enteron aisle to the growth of harmful bacterium such as intestinal bacteria.
This experimentation on animals result shows around the wean back, use souring agent all to improve the day weight gain (P＜0.05) of weanling pig significantly in the daily ration, reduced diarrhoea index (P＜0.05) and diarrhea rate, wherein best with preparation of feeding live bacterial acid effect of the present invention.Use preparation of feeding live bacterial acid of the present invention and Le Da acid all to promote hydrochloric acid in gastric juice and pepsic secretion (P＜0.05), trypsinase and diastatic activity (P＜0.05) in the duodenum content have been improved, increase the quantity of lactobacillus significantly, reduced intestinal bacteria quantity (P＜0.05).The strong acid spirit has the trend that increases duodenum lactobacillus quantity, but difference is not remarkable, and hydrochloric acid in gastric juice and pepsinia are not then produced influence significantly.
Preparation of feeding live bacterial acid of the present invention and benefit poultry precious (probiotics product, the U.S. is refined to come company to produce) and the happy performance index comparing result that reaches acid (composite souring agent, the production of Spain Le Da company) are as shown in table 10.
The performance index contrast of the preparation of feeding live bacterial acid of table 10 invention and probiotics and souring agent
| Index | The viable bacteria acid supplement | Benefit poultry precious (probiotics) | Find pleasure in and reach acid (composite souring agent) |
| Fungicidal effectiveness (relative value) | 100 times | 10 times | 10 times |
| Substitute feeding antibiotic | Substitute fully/part is alternative | Part substitutes | Part substitutes |
| Can withstand high temperatures | Energy | Can/can not | Energy |
| Addition, kg/t | 1-3 | 1-3 | 2-5 |
| The meat antibiotic remains | Do not have | Lack/have | Lack/have |
| Diarrhea rate reduces degree | 50-70％ | 10-20％ | 5-20％ |
| Save cost, a unit/pig | 15 | 8 | 5 |
Sequence table
<160>1
<210>1
<211>1427
<212>DNA
＜213〉subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis)
<400>1
tccccttcgg cggctggctc cataaaggtt acctcaccga cttcgggtgt tacaaactct 60
cgtggtgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc 120
cgcgattact agcgattcca gcttcacgca gtcgagttgc agactgcgat ccgaactgag 180
aacagatttg tgggattggc ttaacctcgc ggtttcgctg ccctttgttc tgtccattgt 240
agcacgtgtg tagcccaggt cataaggggc atgatgattt gacgtcatcc ccaccttcct 300
ccggtttgtc accggcagtc accttagagt gcccaactga atgctggcaa ctaagatcaa 360
gggttgcgct cgttgcggga cttaacccaa catctcacga cacgagctga cgacaaccat 420
gcaccacctg tcactctgcc cccgaagggg acgtcctatc tctaggattg tcagaggatg 480
tcaagacctg gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc caccgcttgt 540
gcgggccccc gtcaattcct ttgagtttca gtcttgcgac cgtactcccc aggcggagtg 600
cttaatgcgt tagctgcagc actaaggggc ggaaaccccc taacacttag cactcatcgt 660
ttacggcgtg gactaccagg gtatctaatc ctgttcgctc cccacgcttt cgctcctcag 720
cgtcagttac agaccagaga gtcgccttcg ccactggtgt tcctccacat ctctacgcat 780
ttcaccgcta cacgtggaat tccactctcc tcttctgcac tcaagttccc cagtttccaa 840
tgaccctccc cggttgagcc gggggctttc acatcagact taagaaaccg cctgcgagcc 900
ctttacgccc aataattccg gacaacgctt gccacctacg tattaccgcg gctgctggca 960
cgtagttagc cgtggctttc tggttaggta ccgtcaaggt gccgccctat ttgaacggca 1020
cttgttcttc cctaacaaca gagctttacg atccgaaaac cttcatcact cacgcggcgt 1080
tgctccgtca gactttcgtc cattgcggaa gattccctac tgctgcctcc cgtaggagtc 1140
tgggccgtgt ctcagtccca gtgtggccga tcaccctctc aggtcggcta cgcatcgtcg 1200
ccttggtgag ccgttacctc accaactagc taatgcgccg cgggtccatc tgtaagtggt 1260
agccgaagcc accttttatg tctgaaccat gcggttcaga caaccatccg gtattagccc 1320
cggtttcccg gagttatccc agtcttacag gcaggttacc cacgtgttac tcacccgtcc 1380
gccgctaaca tcagggagca agctcccatc tgtccgctcg acttgca 1427
Claims (10)
1, subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01CGMCC No.1946.
2, a kind of preparation of feeding live bacterial acid, its activeconstituents comprise subtilis withered grass subspecies Gendone-01CGMCC No.1946.
3, preparation of feeding live bacterial acid according to claim 2 is characterized in that: the activeconstituents of described preparation of feeding live bacterial acid also contains organic acid.
4, preparation of feeding live bacterial acid according to claim 3 is characterized in that: described organic acid is a kind of or its arbitrary combination in citric acid, fumaric acid, lactic acid, dystopy acid, acetate, propionic acid, formic acid, oxysuccinic acid, Sorbic Acid and succsinic acid and its esters.
5, preparation of feeding live bacterial acid according to claim 4 is characterized in that: described organic acid is a kind of or its arbitrary combination in citric acid, fumaric acid, lactic acid, oxysuccinic acid and its esters.
6, according to arbitrary described preparation of feeding live bacterial acid among the claim 2-5, it is characterized in that: in the described preparation of feeding live bacterial acid, the content of subtilis withered grass subspecies Gendone-01 CGMCC No.1946 is 5-50 * 10
8CFU/g.
7, according to arbitrary described preparation of feeding live bacterial acid among the claim 3-5, it is characterized in that: in the described preparation of feeding live bacterial acid, organic acid content is 400-800g/kg.
8, according to arbitrary described preparation of feeding live bacterial acid among the claim 2-5, it is characterized in that: in the described preparation of feeding live bacterial acid, also contain assistant agent.
9, a kind of method of cultivating subtilis withered grass subspecies Gendone-01 CGMCC No.1946, be in fermention medium, at 32-40 ℃, cultivated 33-39 hour, and collected and obtain subtilis withered grass subspecies (Bacillus subtilis subsp.subtilis) Gendone-01 CGMCC No.1946 thalline;
The pH of described fermention medium is 6.0-8.0, is made into by following material: Semen Maydis powder 2.5-3.5g; Soyflour 5.0-7.0g; Peptone 1.5-2.5g; Glucose 2.0-4.0g; Urea 1.5-2.5g; Magnesium sulfate heptahydrate 0.3-0.7g; Manganese sulfate monohydrate 0.3-0.7g; Potassium primary phosphate 0.8-1.2g adds water and is settled to 1000ml.
10, method according to claim 9 is characterized in that: described culture temperature is 35-38 ℃, and incubation time 35-37 hour, the pH of described fermention medium was 6.5-7.5, is made into by following material: Semen Maydis powder 3.0g; Soyflour 6.0g; Peptone 2.0g; Glucose 3.0g; Urea 2.0g; Magnesium sulfate heptahydrate 0.5g; Manganese sulfate monohydrate 0.5g; Potassium primary phosphate 1.0g adds water and is settled to 1000ml.
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| CN102613401A (en) * | 2012-02-29 | 2012-08-01 | 北京和利美生物科技有限公司 | 4% of premix for growing pig and daily ration prepared by using premix |
| CN105695373B (en) * | 2016-04-18 | 2019-05-24 | 广州市佰沃生物科技有限公司 | The fermentation process of bafillus natto |
| CN106561999A (en) * | 2016-11-01 | 2017-04-19 | 浙江海洋大学 | Method for preparing feeding peptide by using enzymolysis tuna dark meat |
| CN115873748B (en) * | 2022-08-18 | 2023-07-07 | 浙江省农业科学院 | Bacillus subtilis, bacillus subtilis ferment and application |
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Non-Patent Citations (2)
| Title |
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| 枯草芽孢杆菌粉剂作为鸡饲料添加剂的研究. 郑永利，雷秋波，徐润林，林继球.中山大学学报（自然科学版），第37卷第2期. 1998 |
| 枯草芽孢杆菌粉剂作为鸡饲料添加剂的研究. 郑永利，雷秋波，徐润林，林继球.中山大学学报（自然科学版），第37卷第2期. 1998 * |
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