CN106190904B - Fly-maggot protein biology solid-state degradation prepares ammonia nitrogen feed process - Google Patents
Fly-maggot protein biology solid-state degradation prepares ammonia nitrogen feed process Download PDFInfo
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- CN106190904B CN106190904B CN201610564317.7A CN201610564317A CN106190904B CN 106190904 B CN106190904 B CN 106190904B CN 201610564317 A CN201610564317 A CN 201610564317A CN 106190904 B CN106190904 B CN 106190904B
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/075—Bacillus thuringiensis
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
Abstract
The technique for being prepared ammonia nitrogen feed using microbe in solid state degradation the present invention relates to a kind of fly-maggot protein is carried out fly-maggot protein biodegradation using serum anomaly bacillus thuringiensis KF228911 and prepares ammonia nitrogen feed.Amino nitrogen content is 2.162mg/g in raw material fly-maggot protein, and the technique that biological solid-state degradation fly-maggot protein prepares ammonia nitrogen feed is：Best degradation culture medium is 8 parts of Flyblow albumen powders, 1.5 parts of rice meals, 0.03 part of MgSO4, 5 parts of the water of pH5.5 stirred to culture medium complete wetting, 121 DEG C of high pressure sterilization 30min.Best degradation condition is that the bacteria suspension of bacillus thuringiensis KF228911 kinds age 60h is inoculated with 5% inoculum concentration, it is cultivated 60 hours in 34 DEG C of incubators after shaking up, the feed amino nitrogen content of production is 14.51mg/g feeds, amino nitrogen content improves nearly 7 times, the nutritive value of fly-maggot protein fodder is improved, and has vast potential for future development.
Description
Technical field
The technique for preparing ammonia nitrogen feed using microbe in solid state degradation the present invention relates to a kind of fly-maggot protein belongs to agricultural
Microorganisms technical field, suitable for the production of high-quality protein feed.
Background technology
In recent years, with the fast development of China's feed industry, there is lack of raw materials that problem seriously limits for protein feed
The development of Zhi Liao China feed industry.At present, China mainly by imported fish meal to solve the problems, such as the deficiency of protein resource, institute
Become the hot subject of scientific research to develop new protein resource.Soybean is good protein raw materials, but its higher valency
The development that lattice will undoubtedly limit feed industry.Therefore, it develops, seek with promoting novel, safe protein feed raw material as early as possible
It is current top priority.
Housefly is a kind of most commonly seen insect in area from all parts of the world, and quantity is huge, and fertility is strong, and reproduction speed is fast.
Fly maggot is the Larva Morpho. Logy of housefly, containing there are many aliphatic acid, amino acid, vitamin and minerals.In China, fly maggot can carry out
Breeding has higher protein content, can be developed.Flyblow albumen powder has been made at present.Albumen in fly maggot
Matter content is about 60.53%, and amino acid classes are complete, and 8 containing needed by human body kinds of essential amino acids and histidines, required
Amino acid content accounts for the 42.14% of total amino acid content, should well beyond the FAO/WHO good protein essential amino acids suggested
Account for the standard of total amino acid content 40%.Fly-maggot protein is more not a halfpenny the worse compared with other high-quality animal proteins, and nutritive value is very
To being higher than other animal proteins, therefore it is even more a kind of good protein resource.If but fly-maggot protein Direct-fed,
But there are fly-maggot protein nutritional utilization it is low the problem of, always affect the application prospect of this fly-maggot protein fodder.
Chinese patent application 2009100447955, a kind of " preparation method of fly maggot biological albumen powder and its in feedstuff for live pigs
In application " and Chinese patent application 2004100225764, " engineering fly biology high protein powder " disclose one kind and pass through culture
The method of fly maggot and expanding production fly-maggot protein.However, this method be only in that how to obtain Flyblow albumen powder and by its directly
For admixing feed, being even used as protein food, do not overcome the nutritional utilization present in Direct-fed fly-maggot protein low
The problem of.
Chinese patent application 2015110036682, " a kind of preparation method of novel fermentation feed and worm ferment " disclose
Using the strain isolated and purified out from fly maggot metabolite, using Flyblow albumen powder, dregs of beans, roughage as primary raw material, into
The method of row fermentation production of fodder and worm ferment.However, the strain used in this method includes bacillus, saccharomycete, breast
A variety of strains such as sour bacterium, photosynthetic bacteria, nitrogen-fixing bacteria, complicated component influence the quality control of final fermentation results.Meanwhile the party
Method must carry out direct fermentation with large-scale feed, it is desirable that fairly large production scale, the fly maggot egg of gained feed product
Bai Hanliang, which has been fixed, to be difficult to adjust, and can not be realized and be carried out diversified adjustment for different poultry and livestock feeds, once extensive
It breaks down in fermentation production process or unexpected, then will lead to the discarded and waste of feedstuff, therefore this method is in the presence of certain
The defects of.
Chinese patent application 2012102464800, " the protein feed production technology produced with insect " disclose a kind of profit
With the method for insect production protein feed, including adult is milled into slurries, protease hydrolytic form pure worm slurry protein powder, and with
The microbiological feed of crushing is mixed, and obtains worm peptide protein biology feedstuff.However, this method mainly passes through expensive enzyme preparation
Barley worm is hydrolyzed, and microbiological feed cost used is excessively high (containing hyperglobulinemia：30%；Shrimp powder：10%；Fish meal：20%；
Yeast：20% etc.), if purchased protease preparation vigor is low, hydrolysis degree is difficult to meet production requirement, therefore does not overcome feeding
The problem of nutritional utilization present in hello fly-maggot protein is low.
The present inventor is on the basis of above, and research is using microbial degradation fly-maggot protein, and obtaining certain effect, (China is specially
Profit application is 2014107293361).In this study, the present inventor screens and obtains a kind of Su Yun of efficient degradation fly-maggot protein
Golden Bacillus strain, it is commonly micro- which is that on October 17th, 2014 is preserved in China Committee for Culture Collection of Microorganisms
Bio-Centers, preserving number are the bacterial strain of CGMCC NO.9789.The bacterial strain is Gram-negative bacillus, is serum anomaly
Property bacillus thuringiensis.Bacterial strain Sep-7S ribosomal RNA gene accession number is KF228911.Inventor's discovery,
Fly-maggot protein culture medium is carried out liquid fermentation using the bacterial strain, soluble Amino acid content can be generated as 1.744mg/mL
(being equivalent to amino nitrogen content 0.1348mg/mL), this imply that the bacterial strain can be used for the application of the feed processing of fly-maggot protein
In.
However, ammonia nitrogen 2.162mg/g is contained in raw material fly-maggot protein in this method, on after liquid degradation
It is 1.744mg/mL (being equivalent to amino nitrogen content 0.1348mg/mL) that soluble Amino acid content is obtained in clear liquid, causes amino
The loss of nitrogen, while the supernatant that this method is obtained, are directly incorporated into cross there are feed in feed and are at most difficult to mixing, and raise
Expected few then excessively moist, being easy to go mouldy is difficult to preserve, therefore causes certain difficulty for the production of biological feedstuff.
Therefore, a kind of technique that fly-maggot protein can be prepared to ammonia nitrogen feed using biological solid-state degradation is needed at present,
So as to develop a kind of novel, useful protein feed, to solve the problems, such as that current fly-maggot protein fodder nutrient utilization is low,
And the method for producing the biological feedstuff of the ammonia nitrogen enrichment of bioavailable in fly-maggot protein.
Invention content
The present invention uses bacillus thuringiensis bacterial strain (Bacillus thuringiensis serovar
Thuringiensis) China Committee for Culture Collection of Microorganisms's common micro-organisms is preserved on October 17th, 2014
Center, preserving number：CGMCC No.9789.
The present invention provides a kind of method of the biological feedstuff of ammonia nitrogen enrichment for producing bioavailable in fly-maggot protein, step
Suddenly include：
(1) activation of fly-maggot protein degradation bacteria strains, wherein the bacterial strain that it is CGMCC NO.9789 that the bacterial strain, which is preserving number,；
(2) the expansion culture of the degradation bacteria strains；
(3) the degradation culture medium of fly-maggot protein is prepared；
(4) degradation bacteria strains of activation are inoculated into degradation culture medium, solid or semisolid fermentation is carried out after mixing；
(5) solid or semisolid tunning is mixed and made into pre-mixing agent for raising directly as feed or by this product with carrier
Feed livestock and poultry fish；Wherein
In step (3), by the Flyblow albumen powder that parts by weight are 7-9 parts, 1-2 parts of rice meals, 0.02-0.04 parts of MgSO4With
And 4-6 parts of water (first adjusting pH 5.5), it stirs to culture medium complete wetting, in 121 DEG C of high pressure sterilization 30min, obtains solid or half
The degradation culture medium of solid；
In step (4), by the degradation bacteria strains bacteria suspension of activation be equivalent to degradation culture medium about 5% inoculum concentration (v/v or
V/m it) is inoculated with, after abundant mixing, is cultivated 60 hours in 34 DEG C of incubators.
In one embodiment, the activation in step (1) includes：In 100mL water, 0.3g beef extracts, 1.0g are added in
Peptone, 0.5g NaCl, 2.0g agar adjust pH value 7.0, and 121 DEG C of sterilizing 30min, cool down bevel after packing.Inoculation
After degradation bacteria strains, cultivated for 24 hours in 37 DEG C of incubators.
In another embodiment, the expansion culture in step (2) includes：By 0.3 portion of beef extract, 1.0 parts of peptones,
0.5 part of sodium chloride, 100 parts of water after heating for dissolving, adjust pH value 7.0 and in 121 DEG C of high pressure sterilization 30min, obtain seed culture
Base.Then, degradation bacteria strains are inoculated with, and in 37 DEG C, the rotating speed culture of 150r/min 60 hours, obtain the bacterium in exponential phase
Suspension.
In any of the above-described embodiment, in the degradation culture medium, preferably described Flyblow albumen powder is 8 parts, 1.5 parts
Rice meal, 0.03 part of MgSO4 and 5 part of water (first adjusting pH 5.5).
In any of the above-described embodiment, the amino nitrogen content for measuring tunning is further included：Take a small amount of tunning with
After distilled water oscillation mixing 30min, centrifuged 10 minutes in 4000r/min, centrifugation sample liquid can be used for surveying ammonia in every gram of tunning
Base nitrogen content.In a preferred embodiment, amino nitrogen content is 13.56-14.51mg/g in the tunning.
In any of the above-described embodiment, the Flyblow albumen powder is after fly maggot is dried, and crushes to sieve with 100 mesh sieve and obtain
It obtains or purchased from commercial commercially available Flyblow albumen powder.
In any of the above-described embodiment, the feed is the feed for feeding poultry or aquatic livestock.
According to the tunning prepared by any of the above method, it is used to prepare the feed of feeding poultry or aquatic livestock.
Advantageous effect
1st, the deficiency of the fly-maggot protein degradation bacteria fermentation culture medium before the present invention is directed to reduces the use of Flyblow albumen powder
Amount, and cheap and a small amount of rice meal is selected to substitute expensive glucose, prepare solid or semisolid culture medium.It is being inoculated with
In the case of identical degradation bacteria strains, the required reaction volume of the present invention is relatively smaller, and tunning is solid-state or semisolid,
It is easily dried and crushes, and be mixed into feed, the defects of degradation cultured products before avoiding.
2nd, the present invention has adjusted the pH of degradation culture medium, and specific acid water (pH 5.5) has been selected to replace pH7.0.It is real
Verify the bright enrichment for being conducive to ammonia nitrogen in tunning.
3rd, it is solvable in 2.162mg/g and existing liquid fermentation production with amino nitrogen content in raw material fly-maggot protein
Acidic amino acid content is compared for 1.744mg/mL, and the present invention avoids ammonia nitrogen in raw material by the production method before improvement
Loss, and the amino nitrogen content (14.51mg/g) in significant enrichment tunning, so as to be conducive to improve the biology in feed
Available amino acid content：The ammonia nitrogen of 2.162mg/g is only the nitrogen that can be directly absorbed and utilized in raw material, and the present invention passes through
The protein that cannot directly utilize in fly-maggot protein is also become to the ammonia nitrogen that can be directly absorbed and utilized using biodegradation,
The utilization rate of nitrogen in fly-maggot protein is improved, avoids nitrogen with the waste caused by excrement discharge.
4th, the method for the present invention so that amino nitrogen content improves nearly 7 times in fly-maggot protein tunning, fly-maggot protein fodder
Nutritive value be improved, have vast potential for future development.
Specific embodiment
The present invention will be described further in conjunction with specific embodiments, these examples be for illustration purposes only rather than
Limit the scope of the invention.
1 sample amino nitrogen content of embodiment measures
1. ammonia nitrogen Specification Curve of Increasing
(1) prepared by ammonia nitrogen standard solution (14 μ g/mL)：Accurately weigh the tyrosine to dry to constant weight in 105 DEG C of baking ovens
0.1812g after being dissolved with 1mol/mL hydrochloric acid solutions 60mL, adds in distilled water 40mL, is settled to 0.1mol/mL hydrochloric acid solutions
1000mL obtains the ammonia nitrogen standard solution of 14 μ g/mL.
(2) ammonia nitrogen Specification Curve of Increasing：6 scale test tube numbers are taken, 14 μ g/mL ammonia nitrogen marks are separately added by table 1
Quasi- solution, without ammonia distilled water, ninhydrin and 0.1% ascorbic acid, be put into boiling water bath after mixing and heat 15min, often
Shake, the red for making to be formed during heating gradually oxidation by air and when fading to bluish violet, with 60% ethyl alcohol constant volume to 20mL,
Then mixing measures OD under 570nm wavelength570Value.
The drafting of 1 ammonia nitrogen standard curve of table
With OD570It is worth for abscissa, amino nitrogen content y (μ g/mL) is ordinate, draws standard curve, obtains its linear regression
Equation.
2. sample amino nitrogen content measures
Liquid 0.2mL is sampled, supplement 1.8mL is without ammonia distilled water, and 2mL TCA, 20min is kept the temperature in 40 DEG C of water-baths makes albuminous degeneration
Precipitation centrifuges 10min.
Take four 20mL scale test tubes, No. 0 control plus 2.0mL distilled water, No. 1,2,3 measure pipe plus 0.1mL centrifugates and
1.9mL is without ammonia distilled water, then is separately added into 3mL ninhydrins, and 0.1mLVc, boiling water bath 15min shake primary, use per 3min
60% ethyl alcohol is settled to 20mL.OD values are then surveyed at 570nm, equation of linear regression is brought into and calculates sample liquid amino nitrogen content y (μ
g/mL).Amino nitrogen content：Y (mg/g)=y (μ g/mL)/1000 × extension rate/dry solid weight (g)
Flyblow albumen powder sample after fly maggot dries pulverizing measures its amino nitrogen content as 2.162mg/ by above method
g。
The biodegradable fly-maggot protein of embodiment 2 prepares the tunning of enrichment ammonia nitrogen
1. according to table 2, fly-maggot protein biodegradation culture medium is prepared respectively：
2. the activation of fly-maggot protein degradation bacteria KF228911：In 100mL water, addition 0.3g beef extracts, 1.0g peptones,
0.5g NaCl, 2.0g agar adjusts pH value 7.0, and 121 DEG C of sterilizing 30min bevels, are inoculated with KF228911 bacterial strains after packing,
It is cultivated for 24 hours in 37 DEG C of incubators.
3.KF228911 the expansion culture of bacterial strain：
(1) formula of seed culture medium is：0.3g beef extracts, 1.0g peptones, 0.5g sodium chloride, 100mL water, heating are molten
Xie Hou adjusts 7.0,121 DEG C of high pressure sterilization 30min of pH value.
(2) by KF228911 inoculations in the sterile 250mL conical flasks equipped with 100mL seed culture mediums, temperature
Under the conditions of 37 DEG C, with the rotating speed culture of 150r/min 60 hours on shaking table, the bacteria suspension in exponential phase is obtained.
4. the best degradation technique of biological solid-state：The most preferably biodegradable culture medium of fly-maggot protein after sterilizing, Su Yun gold gemma
The bacteria suspension of bacillus KF228911 kinds age 60h carries out being seeded to fly-maggot protein degradation culture medium with 5% inoculum concentration (0.5mL)
In, it is cultivated 60 hours in 34 DEG C of incubators after shaking up, obtains ammonia nitrogen tunning.Tunning adds 95mL distilled water to vibrate
30min is extracted, 4000r/min is centrifuged 10 minutes, and centrifugation sample liquid can be used for surveying amino nitrogen content in every gram of tunning.
According to the method for embodiment 1, the amino nitrogen content in every gram of tunning is measured, the results are shown in Table 2.
It is prepared by the biodegradable culture medium of table 2
The table 2 of supplement is used for：1. illustrate that the range of each ingredient of culture medium of the present invention is reasonable；2. water pH is for hair
Obvious results fruit is critically important.Microorganism normal growth needs certain acid-base value, and acid-base value is usually represented with pH value.PH value is to micro- life
The growth of object and metabolite formation have many influences, and requirement of different types of microorganism to pH value is different.Serum becomes
It is 5.5 that special-shaped bacillus thuringiensis, which needs KF228911 the most suitable growthes pH value, and protease, albumen are generated in optimal pH conditioned growth
Fly-maggot protein is degraded generate ammonia nitrogen rapidly by enzyme.
Embodiment 3：Prepare the feed of enrichment ammonia nitrogen
Above-mentioned fermented and cultured product after drying and crushing, can be fed directly as feed for livestock and poultry fish；Or by this product with
Carrier is mixed and made into pre-mixing agent for feeding livestock and poultry fish.
Claims (5)
1. a kind of method of the biological feedstuff of ammonia nitrogen enrichment for producing bioavailable in fly-maggot protein, step include：
（1）The activation of fly-maggot protein degradation bacteria strains, wherein the bacterial strain that it is CGMCC NO.9789 that the bacterial strain, which is preserving number,；
（2）The expansion culture of the degradation bacteria strains；
（3）Prepare the degradation culture medium of fly-maggot protein：
（4）The degradation bacteria strains of activation are inoculated into degradation culture medium, solid or semisolid fermentation is carried out after mixing, is obtained
Amino nitrogen content is 13.56- 14.51mg/g in tunning；
（5）Solid or semisolid tunning is mixed directly as feed or by it with carrier pre-mixing agent for feed poultry
Fowl or aquatic livestock；Wherein,
Step（3）In, by the Flyblow albumen powder that parts by weight are 8 parts, 1.5 parts of rice meals, 0.03 part of MgSO4And 5 parts be first adjusted to pH
5.5 water is stirred to culture medium complete wetting, in 121 DEG C of 30 min of high pressure sterilization, obtains the degradation training of solid or semisolid
Support base；
Step（4）In, by the degradation bacteria strains bacteria suspension of activation be equivalent to the inoculum concentration of degradation culture medium about 5% v/v or v/m into
Row is inoculated with, and after abundant mixing, is cultivated 60 hours in 34 DEG C of incubators.
2. the method for claim 1 wherein steps（1）In activation include：First in 100 mL water, 0.3 g beef extracts of addition,
1.0 g peptones, 0.5 g NaCl, 2.0 g agar adjust pH to 7.0, and 121 DEG C of 30 min of sterilizing after packing, cooling is made
Inclined-plane；Then degradation bacteria strains are inoculated with, and 24 h are cultivated in 37 DEG C of incubators.
3. the method for claim 1 wherein steps（2）In expansion culture include：By 0.3 portion of beef extract, 1.0 parts of peptones,
0.5 part of sodium chloride, 100 parts of water after heating for dissolving, adjust pH to 7.0, and in 121 DEG C of 30 min of high pressure sterilization, obtain seed
Culture medium；Then, degradation bacteria strains are inoculated with, and in 37 DEG C, the rotating speed culture of 150 r/min 60 hours, are obtained in logarithmic growth
The bacteria suspension of phase.
4. the method for claim 1 wherein steps（4）The middle amino nitrogen content for measuring tunning includes：Take a small amount of fermentation production
It after object mixes 30 min with distilled water oscillation, is centrifuged 10 minutes in 4000 r/min, centrifugation sample liquid is used to survey every gram of fermentation production
Amino nitrogen content in object.
5. Claims 1-4 any one of them method, wherein the Flyblow albumen powder is after fly maggot is dried, to crush
Sieve with 100 mesh sieve acquisition or purchased from commercial commercially available Flyblow albumen powder.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102823718A (en) * | 2012-09-17 | 2012-12-19 | 青岛中仁药业有限公司 | Method for preparing probiotics through liquid and solid double fermentation |
| CN104450571A (en) * | 2014-12-04 | 2015-03-25 | 湖南农业大学 | Bacillus thuringiensis strain for efficient degradation of fly larvae protein |
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2016
- 2016-07-17 CN CN201610564317.7A patent/CN106190904B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102823718A (en) * | 2012-09-17 | 2012-12-19 | 青岛中仁药业有限公司 | Method for preparing probiotics through liquid and solid double fermentation |
| CN104450571A (en) * | 2014-12-04 | 2015-03-25 | 湖南农业大学 | Bacillus thuringiensis strain for efficient degradation of fly larvae protein |
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| Title |
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| Bt固态发酵条件的研究进展;姚伟芳等;《现代农药》;20060630;第5卷(第3期);4-6 * |
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