CN102899274B - Bacillus macerans from chicken, prepared fungicide and application thereof - Google Patents
Bacillus macerans from chicken, prepared fungicide and application thereof Download PDFInfo
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- CN102899274B CN102899274B CN201210430869.0A CN201210430869A CN102899274B CN 102899274 B CN102899274 B CN 102899274B CN 201210430869 A CN201210430869 A CN 201210430869A CN 102899274 B CN102899274 B CN 102899274B
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Abstract
The invention discloses a bacillus macerans from chicken, a prepared fungicide and an application thereof. A preserving number of the bacillus macerans from chicken is CGMCC NO.6573. In brooding stage, 0.15wt% bacillus macerans microbial agent is added into feeds, and from a breeding stage to the offering day, 0.1wt% bacillus macerans microbial agent is added into the feeds. According to the invention, a bacillus macerans is separated from the contents of the cecum of a 160-day stocked cock which is health and adult. After the manual gastrointestinal tract environment tolerance selection is carried out, the solid fermentation condition of the bacillus macerans is optimized and the bacillus macerans microbial agent with an enough viable count is produced. According to the invention, the bacillus macerans microbial agent is directly added into the feeds, so that the special 'chicken flavor' volatile flavor component and the content in the cage-rearing chicken are obviously increased.
Description
Technical field
The microbial inoculum of the screening and preparation thereof that the present invention relates to a strain chicken source Bacillus macerans is applied the improvement of chicken flavor, belongs to biological technical field.
Background technology
After the feed chicken being weary of few local flavor or stable breeding chicken, in recent years, people more and more pursue the mouthfeel under raising scattered, local flavor all more excellent put native chicken in a suitable place to breed.Backyard poultry improves a kind of feeding manner of income as peasant household.Put chicken in a suitable place to breed under the habitat expanded, enough free choice feeding behaviors increase, and not only reduce feed intake, its chicken flavor is also more subject to liking of human consumer.Be different from controllable cage conditions, chicken in the environment of putting in a suitable place to breed can contact free to various microorganism, what they pecked at behavior along with chicken entering enteron aisle surely grow, on the absorption of the formation of the intestinal microenvironment of host, stable, nutritive substance or improve feed utilisation transformation efficiency and have impact.
Microbiobacterial agent is added in animal diets, probiotic bacterium is that the disorder effect of intestinal microflora is obvious with the organism form of living to the change of prevention external environment, research shows that it can the growth of direct immune stimulatory organ, or SIgA biosynthesizing is increased, raising alimentary canal mucous membrane immunologic function.And the fungistatic effect of bacillus category has found that there is the effect promoting rabbit immunity of organism; In addition, the short chain unsaturated fatty acids that genus bacillus produces in growth and breeding process can reduce the pH value of enteron aisle, and effectively suppresses the growth of pathogenic bacteria; Or participate in the synthesis of multiple nutrients material (as VITAMIN, amino acid, organized enzyme), and participate in the metabolism of enteron aisle internal protein, fat, trace element, for animal metabolism provides energy.
Disclose the large quantity research of poultry local flavor, it is great that aliphatics aromatic compound is different from the contribution of other animal local flavors to chicken flavor.The matter interaction of the reaction of Maillard reaction, degradation of lipid and their generations can produce a large amount of volatilization flavor substances relevant with chicken flavor.Wherein, the product that in muscle, unsaturated fatty acids adds thermogenetic many lipid oxidations is as 2 anti-, and 4 trans-decadienals and γ-dodecalactone have lower taste threshold, extremely important to characteristic chicken flavor.
Microorganism plays positive effect in synthesis flavor compound.Such as Lactococcus lactis (Lactococcus lactis) or other milk-acid bacterias create the very important material of the local flavor of milk-product---diacetyl.And corynebacterium glutamicum (Corynebacterium glutamicum) can improve the content of Pyrazine material, and increase the local flavor of nut and baking in meat.Therefore, by putting to from 160 age in days health the strain Bacillus macerans separated in chicken caecum in a suitable place to breed, after the resistance test by simulation artificial human gastro intestinal tract environment, powdery microbiobacterial agent is prepared according to solid fermentation optimal condition, be added directly in chicken group feeding material, the application value improving (72 age in days) animal individual chicken flavor index when going on the market age in days is great.
Summary of the invention
The object of this invention is to provide Bacillus macerans that is that a strain separates from chicken cecal content and that tested by simulation artificial human gastro intestinal tract environmental resistance, and be the solid fermentation condition optimizing of substratum with feedstuff raw material to this bacterial strain, prepare impregnation fiber crops bacillus living microbiobacterial agent and to the application improved on chicken flavor.
One strain chicken source Bacillus macerans, deposit number: CGMCC NO.6573.
Microbial inoculum prepared by described chicken source Bacillus macerans, preparation method comprises the following steps: (1) seed culture: by described chicken source Bacillus macerans bacterial strain, aseptically with inoculating articulating 1 ~ 2 ring in 40ml liquid seed culture medium, cultivate 48h with the rotating speed of 120 revs/min, 37 DEG C of constant-temperature tables, obtain primary seed solution; To inoculate articulating 2 ~ 3 ring in the liquid seed culture medium of same formula, cultivate 36 hours with the rotating speed of 120 revs/min, 37 DEG C of constant-temperature tables, obtain secondary seed solution;
(2) solid fermentation is cultivated: with the inoculum size of 3% ~ 10% mass ratio, secondary seed solution is inoculated in the solid medium of sterilizing in 121 DEG C, 30 minutes, be 8% ~ 24% after fermentation flask mixes according to material-water ratio 1 ︰ 0.9-1.5 g/ml and volume ratio, be placed on constant incubator, the temperature that controls environment at 35 ~ 42 DEG C, and is sealed with band 4 layers of sterile gauze; Often pat fermentation flask wall, make upper, middle and lower expect to limber up, mix, ferment 72 hours, fermentation ends;
(3) tunning dries: by tunning in clean enamel tray, add doubling dose fodder maize powder (crossing 40 mesh sieves) to mix, dividing scatters is positioned over ventilation natural air drying, when water ratio lower than 10% time terminate air drying process, collect product and pulverize, cross 40 mesh sieves, gained powder is the solid fermentation finished product of impregnation fiber crops bacillus living.
The formula of described solid medium, by mass percentage: 2% glucose, 1% peptone, 0.5% sodium-chlor, 0.5% extractum carnis, 1.7% agar powder, add aqua sterilisa to dissolving completely, regulate pH to 6.8 ~ 7, high pressure 121 DEG C of sterilizings cool for subsequent use after 30 minutes;
The formula of described liquid nutrient medium, by mass percentage: 2% glucose, 1% peptone, 0.5% sodium-chlor, 0.5% extractum carnis, add aqua sterilisa to dissolving completely, regulate pH to 6.8 ~ 7, high pressure 121 DEG C of sterilizings cool for subsequent use after 30 minutes;
Above-mentioned solid fermentation culture medium prescription and preparation method are: with percentages, and corn, dregs of beans, wheat bran were smashed 40 mesh sieves, according to corn 600g, dregs of beans 300g, wheat bran 50g, high pressure 115 DEG C of sterilizings 15 minutes, phosphoric acid buffer 1000ml is added, natural pH under aseptic condition after cooling.
The application of described microbial inoculum, the stage of brooding is added in feed by 0.15wt% Bacillus macerans microbiobacterial agent; The incubation stage is added in feed to listing age in days by 0.1wt% Bacillus macerans microbiobacterial agent.
The present invention from 160 ages in days grow up put cock in a suitable place to breed cecal content separate a strain Bacillus macerans, and the solid fermentation condition optimizing bacterial strain is to improve the content of living bacteria count, in every gram of solid fermentation finished product, Bacillus macerans viable count reaches 6.9 × 10
9cFU/mL; Added in fowl feed by this microbiobacterial agent, the cage bird group that feeds is to develop the better chicken of local flavor.
The present invention isolates a strain Bacillus macerans by the cecal content of putting cock from healthy adult 160 age in days in a suitable place to breed.After experience artificial human gastro intestinal tract environmental resistance is selected, optimize the solid fermentation condition of this bacterial strain and the Bacillus macerans microbiobacterial agent produced containing enough viable counts.Microorganism live bacteria preparation is added directly into feed by the present invention, has feature " chicken flavor " Volatile flavor components and content is significantly improved in chicken of raising in cages.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1:
(1) host selects: choose healthy 160 age in days cocks from raising chicken farmer family scattered; Except feeding except basal diet, animal individual is freely looked for food in the outdoors of abundance;
(2) strain separating and screening: be separated from raising chicken cecal content scattered; Getting 2g content to be placed in 80 DEG C of thermostat water baths after 10min, is directly applied on genus bacillus solid medium, cultivates 24 hours for 37 DEG C; By single bacterium colony renewed vaccination the highest for genus bacillus cultured on solid medium proportion on new genus bacillus solid medium, cultivate 24 hours for 37 DEG C;
(3) bacterial strain preservation: step (2) is screened the Bacillus macerans SCS3 bacterial strain obtained and is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 18th, 2012, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address Institute of Microorganism, Academia Sinica, deposit number: CGMCC NO.6573, Classification And Nomenclature: Bacillus macerans Bacillus macerans.
(4) artificial gastric juice resistance's test: by described to (3) bacterial strain, aseptically cultivate 2 hours in the simulated gastric fluid of pH2 ~ 4 according to serial dilutions, and carry out dilution plate counting calculating viable count, with 0 hour viable count for reference;
Simulated gastric fluid formula is: according to Chinese Pharmacopoeia 2010 editions, gets 16.4 milliliters, dilute hydrochloric acid, add water about 800 milliliters with stomach en-10 grams, after shaking up, thin up becomes 1000 milliliters and get final product, and puts into 4 DEG C of refrigerators for subsequent use;
(5) enumeration: carry out plate count with serial dilutions, result is: (3) described bacterial classification Bacillus macerans through artificial gastric juice resistance test after viable count in pH2 simulated gastric fluid, cultivate 0 hour after viable count be 2.12 × 10
6cFU/mL, after two hours, viable count is 2.34 × 10
6cFU/mL; In pH3 simulated gastric fluid, cultivate viable count after 0 hour is 3.1 × 10
6cFU/mL, after two hours, viable count is 1.3 × 10
6cFU/mL; In pH4 simulated gastric fluid, cultivate viable count after 0 hour is 3.3 × 10
6cFU/mL, after two hours, viable count is 2.45 × 10
6cFU/mL.
(6) artificial pancreatic juice resistance test: by described to (3) bacterial strain, aseptically cultivate 0 ~ 3 hour according in serial dilutions again simulated intestinal fluid, and carry out dilution plate counting calculating viable count, with 0 hour viable count for reference.
Artificial pancreas's liquid formula is: according to Chinese Pharmacopoeia 2010 editions, get potassium primary phosphate 6.8 grams, add water 500 milliliters and dissolve, with 0.4% sodium hydroxide readjustment pH to 6.8, every 100 milliliters of liquid add 1 gram of trypsinase, mixing, after filtering with the aseptic filter of 0.2 micron and get final product, put into 4 DEG C of refrigerators for subsequent use.
(7) enumeration: carry out plate count with serial dilutions, result is: above-mentioned steps (3) described bacterial classification Bacillus macerans viable count after artificial pancreatic juice resistance test is 2.6 × 10 after cultivating at 0 hour
7cFU/mL, cultivating after 2 hours is 1.52 × 10
7cFU/mL, cultivating after 3 hours is 3.6 × 10
7cFU/mL.
(8) artificial cholate solution resistance test: by described to (3) bacterial strain, aseptically cultivate 0 ~ 4 hour according in the artificial cholate solution of serial dilutions 0% ~ 0.3% 4 concentration gradient again, and carry out dilution plate counting calculating viable count, to cultivate the viable count of 0 hour under 0% concentration for reference.
Artificial cholate solution formula is: according to Chinese Pharmacopoeia 2010 editions, taking 1.5 grams of pig cholate joins in the triangular flask of 100 milliliter of 0.5% sterilizing sodium chloride solution, add 0.1 gram of trypsinase again, shake up, adjust pH to 7.0, namely be prepared into the cholate solution containing cholate 1.5%, put into 4 DEG C of refrigerators for subsequent use;
(9) enumeration: carry out plate count with serial dilutions, result is: (3) described bacterial classification Bacillus macerans viable count after artificial cholate solution resistance test is 7.8 × 10 after 0 hour in the artificial cholate water culture of 0.1% concentration
7cFU/mL, cultivating after 4 hours is 4.6 × 10
7cFU/mL; Viable count is 5.0 × 10 after the artificial cholate liquid of 0.2% concentration cultivates 0 hour
7cFU/mL, cultivating after 4 hours is 1.4 × 10
7cFU/mL; Cultivating 0 hour viable count at the artificial cholate liquid of 0.3% concentration is 5.0 × 10
7cFU/mL, cultivating after 4 hours is 2.9 × 10
6cFU/mL.
(10) Bacillus macerans seed culture: (genus bacillus solid medium is by percentage composition by described to (3) Bacillus macerans SCS3 slant activation, 2% glucose, 1% peptone, 0.5% sodium-chlor, 0.5% extractum carnis, 1.7% agar powder, add aqua sterilisa to dissolving completely, regulate pH to 6.8 ~ 7, high pressure 121 DEG C of sterilizings cooled after 30 minutes, were placed in 37 ± 2 DEG C of biochemical cultivation cases and cultivated 18h; Aseptically with inoculating articulating 1 ~ 2 ring in 40ml liquid seed culture medium, cultivating 48h with the rotating speed of 120 revs/min, 37 DEG C of constant-temperature tables, obtaining primary seed solution; To inoculate articulating 2 ~ 3 ring in the liquid seed culture medium of same formula, cultivate 36 hours with the rotating speed of 120 revs/min, 37 DEG C of constant-temperature tables, obtain secondary seed solution.
Enumeration: carry out plate count with serial dilutions, secondary seed solution viable bacteria content is 6.0 × 10
9the fermented liquid of CFU/mL.
Aforesaid liquid ferment-seeded substratum and preparation method are: by percentage composition, 2% glucose, 1% peptone, 0.5% sodium-chlor, 0.5% extractum carnis, and add aqua sterilisa to dissolving completely, regulate pH to 6.8 ~ 7, high pressure 121 DEG C of sterilizings cool for subsequent use after 30 minutes.
Embodiment 2:
(1) bacterial classification is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 18th, 2012, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address Institute of Microorganism, Academia Sinica, deposit number: CGMCC NO.6573, Classification And Nomenclature: Bacillus macerans Bacillus macerans.
(2) seed culture: by step (1) described bacterial strain, aseptically with inoculating articulating 1 ~ 2 ring in 40ml liquid seed culture medium, cultivating 48h with the rotating speed of 120 revs/min, 37 DEG C of constant-temperature tables, obtaining primary seed solution;
(3) to inoculate articulating 2 ~ 3 ring in the liquid seed culture medium of same formula, cultivate 36 hours with the rotating speed of 120 revs/min, 37 DEG C of constant-temperature tables, obtain secondary seed solution.
(4) solid fermentation is cultivated:
By step (1) described Bacillus macerans with the inoculum size of 5% mass ratio, secondary seed solution is inoculated in the solid medium of sterilizing in 121 DEG C, 30 minutes, be 1:1.1 g/ml and volume ratio according to material-water ratio be 16% access fermentation flask, mixing is placed in biochemical cultivation case, and with band 4 layers of sterile gauze bottleneck, it is 42 DEG C that temperature controls; Often pat fermentation flask wall, substratum limbered up, loading and unloading mixing, ferment 72 hours, fermentation ends;
(5) tunning dries:
By step (4) tunning in clean enamel tray, add doubling dose fodder maize powder (crossing 40 mesh sieves) to mix, dividing scatters is positioned over ventilation natural air drying, when water ratio lower than 10% time terminate air drying process, collect product and pulverize, cross 40 mesh sieves, gained powder is the solid fermentation finished product containing Bacillus macerans.
(6) enumeration: utilize the solid fermentation finished product of serial dilutions to impregnation fiber crops bacillus living to carry out enumeration, result is: the living bacteria count of Bacillus macerans microbiobacterial agent is 6.9 × 10
9/ gram fermentation finished product.
Aforesaid liquid ferment-seeded substratum and preparation method are: by percentage composition, 2% glucose, 1% peptone, 0.5% sodium-chlor, 0.5% extractum carnis, and add aqua sterilisa to dissolving completely, regulate pH to 6.8 ~ 7, high pressure 121 DEG C of sterilizings cool for subsequent use after 30 minutes.
Above-mentioned solid fermentation substratum and preparation method are: with percentages, and corn, dregs of beans, wheat bran were smashed 40 mesh sieves, according to corn 600g, dregs of beans 320g, wheat bran 50g, high pressure 115 DEG C of sterilizings 15 minutes, phosphoric acid buffer is added, natural pH by 1 ︰ 1.1 g/ml under aseptic condition after cooling.
Embodiment 3:
The animal experiment result of use of leavened prod of the present invention.
This example carries out in Sichuan Agricultural University's poultry breeding field, selects healthy 1 Japanese instar chickling 60 (cock), is divided into 2 process at random, each process 2 repetition, each repetition 15 chick, are respectively blank group and Bacillus macerans fermentation group of the present invention, 72 days trial periods;
Bacillus macerans fermentation group of the present invention: the organism of fermentation microbial inoculum of impregnation fiber crops bacillus living of the present invention, viable count>=10
9cFU/ gram, brood the stage (1 age in days ~ 42 age in days) is added in feed by 0.15wt% Bacillus macerans microbiobacterial agent; The incubation stage is added in feed to listing age in days (43 age in days ~ 72 age in days) by 0.1wt% Bacillus macerans microbiobacterial agent;
Butcher the collection with chicken breast: when reaching 72 age in days, extract 6 healthy chickens (each repetition 3) at random out from blank group and Bacillus macerans fermentation group of the present invention; After bloodletting, removing feather, unification rounds the left chest muscle meat of block; The chicken breast of each individuality gets 4 grams, puts into-20 DEG C of preservations after pack after rubbing, mark; Whole sampling process of butchering completed in 1 hour.
Chicken breast local flavor prepares before measuring: chicken breast sample is positioned over 4 DEG C of refrigerators, cured overnight; In the repeating groups of tape label, the chicken breast of two individualities is mixed into one and measures sample, and namely two test group have 4 and measure sample.
Chicken breast local flavor measures: adopt volatile matter in headspace solid-phase microextraction technology extraction chicken breast, and analyze volatile component and content in chicken breast with Gas chromatographyMass spectrometry;
Chicken breast local flavor results and analysis:
The comparison of volatile flavor component in table 1. Bacillus macerans fermentation of the present invention group and blank group chicken breast
Note:
*represent that the content of this volatile flavor component exists significant difference (P < 0.05) between Bacillus macerans fermentation group of the present invention and blank group;
*represent that the content of this volatile flavor component exists pole significant difference (P < 0.01) between Bacillus macerans fermentation group of the present invention and blank group.
Table 1 shows, by adding Bacillus macerans solid fermentation microbiobacterial agent in daily ration, the chicken flavor component of Bacillus macerans fermentation group of the present invention significantly improves, be in particular in volatile component trans-2-nonenal, anti-2, anti-4-nonadienal and 2-n-heptyl furans are significantly higher than blank group, they present strong chicken, chicken soup or roast chicken fragrance, and with grease fragrance, directly can enhance characteristic " chicken flavor " local flavor; And the octanal in Bacillus macerans fermentation group of the present invention, trans-2-octenal, trans-2-hexenoic aldehyde and trans 2-heptenic aldehyde composition are significantly higher than blank group equally, the flavor characteristic that they have jointly is grease fragrance, has contribution equally to the typical flavor of chicken breast; In addition, the alcohols material of high-content comprises 1-OCOL and n-Octanol obtains from Bacillus macerans fermentation group of the present invention detection, and the mushroom tone presented, blue or green perfume (or spice) improve the performance of the overall local flavor organoleptic feature of chicken breast.
This test illustrates, from 160 ages in days put in a suitable place to breed optimize chicken caecum and the solid fermentation microbiobacterial agent of impregnation fiber crops bacillus living that formed of preparation, to the chicken flavor Be very effective improving chicken under cage conditions.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
Claims (2)
1. the microbial inoculum prepared of chicken source Bacillus macerans, is characterized in that, described chicken source Bacillus macerans, deposit number: CGMCC NO.6573; Described bacterial preparation process comprises the following steps: (1) seed culture: by described chicken source Bacillus macerans bacterial strain, aseptically with inoculating articulating 1 ~ 2 ring in 40ml liquid seed culture medium, cultivate 48h with the rotating speed of 120 revs/min, 37 DEG C of constant-temperature tables, obtain primary seed solution; To inoculate articulating 2 ~ 3 ring in the liquid seed culture medium of same formula, cultivate 36 hours with the rotating speed of 120 revs/min, 37 DEG C of constant-temperature tables, obtain secondary seed solution; (2) solid fermentation is cultivated: with the inoculum size of 3% ~ 10% mass ratio, secondary seed solution is inoculated in the solid medium of sterilizing in 121 DEG C, 30 minutes, be 8% ~ 24% after fermentation flask mixes according to material-water ratio 1 ︰ 0.9-1.5g/ml and volume ratio, be placed on constant incubator, the temperature that controls environment at 35 ~ 42 DEG C, and is sealed with band 4 layers of sterile gauze; Often pat fermentation flask wall, make upper, middle and lower expect to limber up, mix, ferment 72 hours, fermentation ends;
(3) tunning dries: by tunning in clean enamel tray, add doubling dose fodder maize powder to mix, dividing scatters is positioned over ventilation natural air drying, when water ratio lower than 10% time terminate air drying process, collect product and pulverize, cross 40 mesh sieves, gained powder is the solid fermentation finished product of impregnation fiber crops bacillus living;
The formula of described liquid nutrient medium, by mass percentage: 2% glucose, 1% peptone, 0.5% sodium-chlor, 0.5% extractum carnis, add aqua sterilisa to dissolving completely, regulate pH to 6.8 ~ 7, high pressure 121 DEG C of sterilizings cool for subsequent use after 30 minutes;
Above-mentioned solid fermentation culture medium prescription and preparation method are: with percentages, and corn, dregs of beans, wheat bran were smashed 40 mesh sieves, according to corn 600g, dregs of beans 300g, wheat bran 50g, high pressure 115 DEG C of sterilizings 15 minutes, phosphoric acid buffer 1000ml is added, natural pH under aseptic condition after cooling.
2. the application of microbial inoculum according to claim 1, it is characterized in that, the stage of brooding is added in feed by 0.15wt% Bacillus macerans microbiobacterial agent; The incubation stage is added in feed to listing age in days by 0.1wt% Bacillus macerans microbiobacterial agent.
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| CN101851123A (en) * | 2010-05-26 | 2010-10-06 | 王金玲 | High-concentration compound microorganism bacterial fertilizer and production method thereof |
| CN102703563A (en) * | 2012-06-26 | 2012-10-03 | 太仓市周氏化学品有限公司 | Method for producing ethanol by adopting mixed culture organism by means of glycerol fermentation |
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Effective date of registration: 20200119 Address after: Ya'an City, Sichuan Province, 625000 new Yucheng Kang Road No. 46 Co-patentee after: WANYUAN HENGKANG AGRICULTURAL DEVELOPMENT CO.,LTD. Patentee after: SICHUAN AGRICULTURAL University Address before: 611130 No. 211 Huimin Road, Wenjiang District, Sichuan, Chengdu Patentee before: Sichuan Agricultural University |
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