CN102318738A - Development method for fenugreek seed meal-bifidobacterium synbiotics - Google Patents
Development method for fenugreek seed meal-bifidobacterium synbiotics Download PDFInfo
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Abstract
The invention discloses a development method for fenugreek seed meal-bifidobacterium synbiotics. The fenugreek seed meal-bifidobacterium synbiotics is divided into three layers, the capsule core is bifidobacterium, the capsule wall consists of sodium alginate, dried skim milk and calcium chloride, then, a traditional Chinese medicine granulator is adopted for covering the fenugreek seed meal at the outmost layer of bifidobacterium capsules, and fenugreek seed meal-bifidobacterium synbiotics particles are obtained. The fenugreek seed meal-bifidobacterium synbiotics developed by the method has the advantages that the average daily weight increase and the daily feed intake can be reduced, the feed conversion ratio is not obviously influenced, the intestinal mucosa villus height, the mucosa thickness and the villus height/crypt depth (V/C) value can be improved, the crypt depth is reduced, and the form development of the AA+ broiler intestinal tract tissue structure and the intestinal tract inner environment are improved.
Description
Technical field
The present invention relates to the method for a kind of medium-height grass dregs of a decoction dregs of rice and probio development symphysis unit, refer in particular to a kind of sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit method of production that can improve immunity of organisms.
Background technology
Now, along with the continuous enhancing of human living standard's raising and self health consciousness, people recognize the substantial connection of food security and human health strongly.Therefore, people not only require nutritiously when selecting animal products, and safety, health, health care etc. are brought up to critical role, wherein one of essence be exactly drug residue free (poisonous and harmful substances such as antibiotic, steroids and some heavy metal classes).Along with the internationalization in animal products market is further accelerated, carry out strict medicine inspection system between international trade, brought severe challenge also for China's animal husbandry.Develop nuisanceless green animal husbandry, strengthen the international competitiveness of China's animal products, become the vital task of herding authorities and manufacturing enterprise.The feed addictive that development does not contain antibiotic, hormone and heavy metal class poisonous and harmful substance has seemed particularly important.But this product must be a prerequisite with the green non-pollution, has the balance that after getting into the animal body intestines and stomach, can keep its inner little ecology; In the external characteristics of not destroying whole " the big ecosystem ".
Summary of the invention
The technical problem that the present invention will solve provides a kind of sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit method of production, and this method of production is processed probiotics symphysis unit with the Chinese medicine slag dregs of rice sophora alopecuroide dregs of rice and the combination of probio bifidobacterium bifidum.
The technical problem that the present invention will solve is realized by following scheme: the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit is divided into three layers: the capsule heart is a Bifidobacterium; Cyst wall is made up of sodium alginate, skimmed milk powder and calcium chloride; And then using the Chinese medicine granulator to encapsulate the sophora alopecuroide dregs of rice at the bifid bacterium microcapsule outermost layer, its viable count requires to reach 7.67 * 10 at least
7Cfu/g.Preparation symphysis unit at first will carry out the oxytolerant domestication to bifidobacterium bifidum; The domestication back is adopted colony counting method to measure its viable count and has been carried out the kind evaluation of bacterium; The sophora alopecuroide dregs of rice and the bifidobacterium bifidum after the oxytolerant domestication that selection accounts for total liquor capacity 4%-8% carry out the microcapsules preparation of symphysis unit for combination, and program is:
(1), the bifidobacterium bifidum freeze-dried vaccine powder after the oxytolerant domestication is joined in the ratio of 3%-5% optimizes activation in the TPY fluid nutrient medium; Cultivate 20h-40h; It is the first generation of activation; With cultured first generation bacterium liquid piping and druming mixing; Therefrom draw the bacterium liquid that accounts for new optimization TPY fluid nutrient medium volume 5%; Receiving in the new optimization TPY fluid nutrient medium and cultivate 20h-40h, is the second generation of activation, with cultured second generation bacterium liquid piping and druming mixing; Therefrom draw the bacterium liquid that accounts for new optimization TPY fluid nutrient medium volume 5%; Receiving in the new optimization TPY fluid nutrient medium and cultivate 20h-40h, is the third generation of activation, optimizes TPY fluid nutrient medium formula rate to be: contain tryptone 0.5g-3g, soy peptone 0.5g-2g, glucose 0.5g-2g, lactose 0.5g-1.5g, dusty yeast 0.5g-2g, magnesium chloride 0.03g-0.08g, sodium chloride 0.5g-1g, sodium hydrogen phosphate 0.5g-1g, potassium dihydrogen phosphate 0.1g-0.2g, Tween-80 0.05g-0.16g, ferrous sulfate 0.001g-0.002g, L-cysteine hydrochloride 0.02g-0.1g, water 85-99g in every 100g optimization TPY fluid nutrient medium;
(2), with above-mentioned activated third generation bacterium liquid with 3000rpm-6000rpm/min, 2 ℃-8 ℃ the centrifugal 10min-30min of centrifuge; Collect bacterium mud; In collecting good bacterium mud, add emulsification 8min-15min in the skimmed milk power protective agent that concentration is 6%-10%; The addition of skimmed milk power is the long-pending 40%-60% of above-mentioned third generation bacteria liquid; Be that the sodium alginate of 4%-6% mixes with high pressure steam sterilization concentration then, be prepared into bacteria suspension, the addition of sodium alginate is the long-pending 40%-60% of above-mentioned third generation bacteria liquid; Make bifid bacterium microcapsule with laboratory self-control microcapsules maker through extrusion again, extruding splashes into the CaCl that concentration is 1%-3%
2In the solution, the stirring that does not stop with the speed of 120rpm-180rpm/min is simultaneously left standstill 20min-40min the bacteria suspension that splashes into is solidified;
(3), with the above-mentioned bacteria suspension that is cured with physiological saline cleaning and filtering 2 times-5 times, vacuum freeze drying 24h-72h gets bifid bacterium microcapsule;
(4), re-use the Chinese medicine granulator and encapsulate the sophora alopecuroide dregs of rice that concentration is 4%-8% at the bifid bacterium microcapsule outermost layer, obtain the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit granule, require its viable count to reach 7.67 * 10 at least
7Cfu/g, the sophora alopecuroide dregs of rice are 1000g-1200g: 100g with the mass ratio that has encapsulated the Bifidobacterium behind the microencapsulation.
Advantage of the present invention: the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit of this method development is divided into three layers: the capsule heart is a Bifidobacterium; Cyst wall is made up of sodium alginate, skimmed milk powder and calcium chloride, and then uses the Chinese medicine granulator to encapsulate the sophora alopecuroide dregs of rice at the bifid bacterium microcapsule outermost layer.The sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit can reduce average daily gain searched for food with day, and feedstuff-meat ratio is not had appreciable impact, can improve intestinal mucosa height of naps, mucous membrane thickness and V/C value (height of naps/crypts degree of depth), reduced the crypts degree of depth, improved AA
+The morphological development and the intestinal environment of meat chick intestinal tissue structure.
Description of drawings
Fig. 1 is this method of production process chart
The specific embodiment
Lift embodiment method of production be detailed:
One, the oxytolerant of bifidobacterium bifidum domestication
This experiment is to tame through increasing in the culture medium method of oxygen partial pressure gradually, passage and attenuation repeatedly under each oxygen partial pressure, and observation inoculates in the next oxygen partial pressure and cultivates after OD value and pH value tend towards stability.Experiment increases the oxygen partial pressure in the culture medium through following approach: the one, and culture environment cultivated to forward in 5% CO2gas incubator by anaerobism cultivate, forward cultivation common condition of culture under again to, shaken cultivation goes down to posterity 5 times in shaking table at last.The 2nd, when changing culture environment, under the certain condition of liquid amount, increase the volume of cultivating vessel gradually, promptly cultivate and convert the cultivation of different volumes triangular flask gradually into by test tube.And with spectrophotometer and pH meter mensuration bacterium liquid light absorption value (OD
600) and the pH value, in the time of in this two-value fluctuating range is in a stability range all the time, confirm naturalized strain at last.
Two, the kind of oxytolerant domestication back bifidobacterium bifidum is identified
(1) bacterial strain WY001 thalli morphology is observed: microscopy behind the employing Gram;
(2) bacterial strain WY001 biochemical identification: reference literature;
(3) bacterial strain WY001 molecular biology identification.
This experiment is through identifying the kind of the bifidobacterium bifidum after the oxytolerant domestication; At last; According to the above 16SrDNA sequence cluster analysis result that obtains; Combine thalli morphology microscopy and biochemical test to identify again, the result shows the change that the kind attribute of bacterium does not take place after this bacterium is through the oxytolerant domestication, has strengthened the oxytolerant characteristic of normal existence under aerobic environment on the contrary.Therefore, bacterial strain WY001 is accredited as bifidobacterium bifidum.
Three, the preparation of the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit
1), at first bifidobacterium bifidum is carried out oxytolerant domestication, the domestication back adopts colony counting method to measure the kind attribute that its viable count also carried out bacterium to identify.
2), select for use the sophora alopecuroide dregs of rice (2%, 4%, 6%, 8%) of variable concentrations to detect it to the active influence of Bifidobacterium, confirm that finally the sophora alopecuroide dregs of rice under 6% concentration are the strongest to the active facilitation of Bifidobacterium.
3), Bifidobacterium is inserted to contain to cultivate in the 6% sophora alopecuroide dregs of rice fluid nutrient medium process liquid symphysis unit; Then with its respectively with chicken colibacillosis and white diarrhea Salmonella at external Mixed culture 24h; Measure the sophora alopecuroide dregs of rice and bifidobacterium bifidum symphysis unit to chicken colibacillosis and white diarrhea Salmonella body outer suppressioning test, result of the test shows: along with its also enhancing thereupon of inhibitory action to two kinds of pathogens of increase of symphysis unit adding proportion.
4), the preparation of bifidobacterium bifidum microencapsulation: through several kinds of Microencapsulation Method screenings commonly used are shown that it is better that extrusion encapsulates the bifidobacterium bifidum effect.Bifidobacterium bifidum freeze-dried vaccine powder after the 0.15g oxytolerant domestication is joined activation in the optimization TPY fluid nutrient medium of 5g; Cultivate 34h; It is the first generation of activation; With cultured bacterium liquid piping and druming mixing; And the bacterium liquid of therefrom drawing 0.25g joins in the new optimization TPY fluid nutrient medium of 5g; Cultivation 34h is the second generation, and with second generation bacterium liquid piping and druming mixing, the bacterium liquid of therefrom drawing 0.25g joins 5g and newly optimizes in the TPY fluid nutrient medium; Cultivating 34h is the 3rd generation of activation, and every 5g newly optimizes in the TPY fluid nutrient medium and contains: tryptone 0.05g, soy peptone 0.025g, glucose 0.05g, lactose 0.05g, dusty yeast 0.025g, magnesium chloride 0.0025g, sodium chloride 0.025g, sodium hydrogen phosphate 0.04495g, potassium dihydrogen phosphate 0.0075g, Tween-80 0.0075g, ferrous sulfate 0.00005g, L-cysteine hydrochloride 0.0025g, water 4.71g.Optimizing the TPY culture medium prescription is: contain tryptone 1g, soy peptone 0.5g, glucose 1g, lactose 1g, dusty yeast 0.5g, magnesium chloride 0.05g, sodium chloride 0.5g, sodium hydrogen phosphate 0.899g, potassium dihydrogen phosphate 0.15g, Tween-80 0.15g, ferrous sulfate 0.001g, L-cysteine hydrochloride 0.05g, water 94.2g in every 100g culture medium.With activation three generations's bacterium liquid with 4000rpm/min, 4 ℃ the centrifugal 20min of centrifuge; Collect bacterium mud, adding concentration is 8% skimmed milk power protective agent in collecting good bacterium mud, emulsification 10min; The addition of skimmed milk power be above-mentioned third generation bacteria liquid long-pending 50%; Be that 5% sodium alginate mixes with high pressure steam sterilization concentration then, be prepared into bacteria suspension, the addition of sodium alginate be above-mentioned third generation bacteria liquid long-pending 50%; Make bifid bacterium microcapsule with laboratory self-control microcapsules maker through extrusion again, it is 2% CaCl that extruding splashes into concentration
2In the solution, the stirring that does not stop with the speed of 150rpm/min is simultaneously left standstill 30min the bacteria suspension that splashes into is solidified; With physiological saline cleaning and filtering 3 times, vacuum freeze drying 72h gets microcapsules with the bacteria suspension that is cured; Bifidobacterium bifidum behind the microencapsulation, its acid resistance and storage-stable are enhanced, and have the insoluble characteristic of enteric stomach.
5), re-use the Chinese medicine granulator and encapsulate the sophora alopecuroide dregs of rice at the bifid bacterium microcapsule outermost layer, obtain asking its sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit granule.Want viable count to reach 7.67 * 10 at least
7Cfu/g.The sophora alopecuroide dregs of rice are 1200g: 100g with the mass ratio that has encapsulated the Bifidobacterium behind the microencapsulation.Symphysis unit in order to last alternatives formulation is the peroral dosage form granule.
Four, the application of the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit on AA+ meat chick is raised
The microencapsulation bifidobacterium bifidum that obtains and the sophora alopecuroide dregs of rice are made an addition to the AA+ fryer of feeding in the meat chick fodder, with 420 AA of 1 age in days
+The meat chick is divided into 6 groups at random by body weight, and the I group is the blank group, the basal diet of feeding (not adding antibiotic basal diet); The II group is added on basal diet and is added 0.02g bifidobacterium bifidum freeze-dried vaccine powder in every 100g feed; III group basal diet adds the sophora alopecuroide dregs of rice that add 2g in every 100g feed; IV, V, VI group are added the symphysis unit (the sophora alopecuroide dregs of rice+bifidobacterium bifidum microcapsules) that adds 0.5g, 1.0g, 2.0g in every 100g feed respectively on basal diet, experimental period is 42 days.
The sophora alopecuroide dregs of rice-bifidobacterium bifidum symphysis unit is to AA+ meat chick Immune Effects.
Randomly drawing ten respectively at every group of 7,14,21,28,35,42 age in days cuts open extremely.
1), measures thymus index, index and spleen index, bursa of farbricius index, cecal tonsil index, NDHI antibody titer, NK cell killing activity in the blood, the concentration of interferon-and serum total cholesterol content in the serum.The result shows:
1. AA can improve in the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit
+Meat chick immune organ index (particularly aspect bursa of farbricius index, cecal tonsil index).
2. there is raising AA in the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit
+The trend of meat chick NDHI antibody titer.
3. AA can improve in the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit
+Meat chick NK cytoactive.
4. interferon-concentration in the serum can improve in the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit
5. the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit has the function that improves total cholesterol level in the serum.
6. make a general survey of the whole test result, the additive capacity of the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit can improve AA
+Meat chick immunologic function, the effect sophora alopecuroide dregs of rice or the Bifidobacterium group that is superior to feeding separately respectively, better to add the 1.0g sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit effect in every 100g feed especially, be suitable for production practices.
The sophora alopecuroide dregs of rice-bifidobacterium bifidum symphysis unit is to AA
+The influence of meat chick production performance and intestinal environment.
2), measure alkaline phosphatase, amylase, lipase and tryptic activity in the small intestine; Volatile fat acid content in the caecum; PH value in jejunum and the ileum; Make each intestinal segment histotomy of small intestine, measure intestinal mucosa height of naps, the crypts degree of depth and mucous membrane thickness.The result shows:
1. the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit can reduce average daily gain and day search for food, and feedstuff-meat ratio is not had appreciable impact.
2. intestinal mucosa height of naps, mucous membrane thickness and V/C value (height of naps/crypts degree of depth) can improve in the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit, reduce the crypts degree of depth, improve AA
+The morphological development of the intestinal tissue structure of meat chick.
3. alkaline phosphatase, amylase, lipase and tryptic activity in the enteron aisle can improve in the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit.
4. the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit does not have appreciable impact to volatile fatty acid in the enteron aisle.
5. the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit does not have appreciable impact to pH value in the enteron aisle.
6. under this test feeding manner, there is reduction AA in the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit
+The trend of meat chick production performance, but can improve its intestinal environment.
3), measure Bifidobacterium, Bacillus acidi lactici and colibacillary quantity in the chicken caecum.The result shows: the interior Bifidobacterium of AA+ meat chick enteron aisle and the quantity of Bacillus acidi lactici can improve in the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit, reduce colibacillary quantity simultaneously.
Claims (1)
1. the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit method of production is characterized in that:
(1), the bifidobacterium bifidum freeze-dried vaccine powder after the oxytolerant domestication is joined in the ratio of 3%-5% optimizes activation in the TPY fluid nutrient medium; Cultivate 20h-40h; It is the first generation of activation; With cultured first generation bacterium liquid piping and druming mixing; Therefrom draw the bacterium liquid that accounts for new optimization TPY fluid nutrient medium volume 5%; Receiving in the new optimization TPY fluid nutrient medium and cultivate 20h-40h, is the second generation of activation, with cultured second generation bacterium liquid piping and druming mixing; Therefrom draw the bacterium liquid that accounts for new optimization TPY fluid nutrient medium volume 5%; Receiving in the new optimization TPY fluid nutrient medium and cultivate 20h-40h, is the third generation of activation, optimizes TPY fluid nutrient medium formula rate to be: contain tryptone 0.5g-3g, soy peptone 0.5g-2g, glucose 0.5g-2g, lactose 0.5g-1.5g, dusty yeast 0.5g-2g, magnesium chloride 0.03g-0.08g, sodium chloride 0.5g-1g, sodium hydrogen phosphate 0.5g-1g, potassium dihydrogen phosphate 0.1g-0.2g, Tween-80 0.05g-0.16g, ferrous sulfate 0.001g-0.002g, L-cysteine hydrochloride 0.02g-0.1g, water 85-99g in every 100g optimization TPY fluid nutrient medium;
(2), with above-mentioned activated third generation bacterium liquid with 3000rpm-6000rpm/min, 2 ℃-8 ℃ the centrifugal 10min-30min of centrifuge; Collect bacterium mud; In collecting good bacterium mud, add emulsification 8min-15min in the skimmed milk power protective agent that concentration is 6%-10%; The addition of skimmed milk power is the long-pending 40%-60% of above-mentioned third generation bacteria liquid; Be that the sodium alginate of 4%-6% mixes with high pressure steam sterilization concentration then, be prepared into bacteria suspension, the addition of sodium alginate is the long-pending 40%-60% of above-mentioned third generation bacteria liquid; Make bifid bacterium microcapsule with laboratory self-control microcapsules maker through extrusion again, extruding splashes into the CaCl that concentration is 1%-3%
2In the solution, the stirring that does not stop with the speed of 120rpm-180rpm/min is simultaneously left standstill 20min-40min the bacteria suspension that splashes into is solidified;
(3), with the above-mentioned bacteria suspension that is cured with physiological saline cleaning and filtering 2 times-5 times, vacuum freeze drying 24h-72h gets bifid bacterium microcapsule;
(4), re-use the Chinese medicine granulator and encapsulate the sophora alopecuroide dregs of rice that concentration is 4%-8% at the bifid bacterium microcapsule outermost layer; Obtain the sophora alopecuroide dregs of rice-Bifidobacterium symphysis unit granule, the sophora alopecuroide dregs of rice are 1000g-1200g: 100g with the mass ratio that has encapsulated the Bifidobacterium behind the microencapsulation.
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Cited By (2)
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| CN104381617A (en) * | 2014-12-14 | 2015-03-04 | 河南联合英伟饲料有限公司 | Medicine core feed for preventing viral diseases of chickens and preparation method of medicine core feed |
| CN111534566A (en) * | 2020-06-17 | 2020-08-14 | 江南大学 | Gradient diluent and application thereof in bifidobacterium counting |
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2011
- 2011-05-17 CN CN201110136924A patent/CN102318738A/en active Pending
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| CN104381617A (en) * | 2014-12-14 | 2015-03-04 | 河南联合英伟饲料有限公司 | Medicine core feed for preventing viral diseases of chickens and preparation method of medicine core feed |
| CN111534566A (en) * | 2020-06-17 | 2020-08-14 | 江南大学 | Gradient diluent and application thereof in bifidobacterium counting |
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Application publication date: 20120118 |