CN106148242B - A method of using chondroitin sulfate production Industry Waste bone mud as raw material solid fermenting and producing bacillus subtilis - Google Patents
A method of using chondroitin sulfate production Industry Waste bone mud as raw material solid fermenting and producing bacillus subtilis Download PDFInfo
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- CN106148242B CN106148242B CN201610753634.3A CN201610753634A CN106148242B CN 106148242 B CN106148242 B CN 106148242B CN 201610753634 A CN201610753634 A CN 201610753634A CN 106148242 B CN106148242 B CN 106148242B
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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
Abstract
Of the invention providing is a kind of using chondroitin sulfate production Industry Waste bone mud as the method for raw material solid fermenting and producing bacillus subtilis, to extract Industry Waste bone mud caused by chondroitin sulfate technique as matrix using animal cartilage, wheat bran, water are mixed as culture medium, bacillus subtilis is accessed after high-temperature sterilization and carries out solid fermentation, and bacillus subtilis is made through drying and crushing in fermentation material.Inexpensive leftover bits and pieces can be efficiently used using the method for the present invention to give up bone mud production high added value bacillus subtilis, reaches the purpose that turns waste into wealth, and simple process, low in cost, easy to operate.Chondroitin sulfate production Industry Waste bone mud is applied to fermenting and producing bacillus subtilis for the first time by the present invention, opens the new purposes of chondroitin sulfate production Industry Waste bone mud.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of using chondroitin sulfate production Industry Waste bone mud as raw material
The method of solid fermentation production bacillus subtilis.
Background technique
Chondroitin sulfate is to exist in cartilaginous tissue in the form of proteoglycan, chondroitin sulfate polysaccharide chain in proteoglycan
With core protein by being covalently keyed (cardohydrata-peptide linkage), and the aggregation of proteoglycan then passes through secondary key (hydrogen bond and sat linkage) shape
At.Have to take suitable effective ways to destroy polysaccharide chain and albumen it can be seen that extracting chondroitin sulfate from animal cartilaginous tissue
Covalent bond efficiently separates polysaccharide chain with albumen, and polysaccharide chondroitin sulfate could high efficiency extraction.Domestic production producer at present
Albumen dissociation is carried out frequently with proteolysis albumen+isoelectric point precipitates albumen method.The step production technology can generate largely
Protein precipitation belongs to chondroitin sulfate production waste, referred to as useless bone mud.Useless bone mud rich in nutrition content, suitable micro-organisms
Breeding, ordinary room temperature, which is placed, can occur corruption for 2~3 days, generate foul odour, and generate a large amount of waste liquids, cause very big environment
Pollution, is industrial production urgent problem.
Bacillus subtilis, The Ministry of Agriculture of the People's Republic of China, MOA announce No. 2045 " catalogue of feed additive varieties
(2013) " approved its can be used as beneficial microbe (probiotic) and be added in feed and use.There are many bacillus subtilis type,
It can produce the enzymes such as amylase, protease, lipase, cellulase, in alimentary canal together with the intracorporal digestive enzymes of animal
It plays a role, cellulose, the protein that a part cannot be digested and assimilated completely in degradation chicken, pannage improve feed conversion
Rate increases the cultivated animals speed of growth, and the protein that also can avoid these cannot be utilized by cultivated animals excretes, by micro- life
Object, which decomposes, generates poisonous and hazardous ammonia；The subtilin of generation, more Acarasiales during bacillus subtilis thalli growth simultaneously
Element, nystatin, gramicidins isoreactivity substance, opportunistic of these active materials to animal pathogen or autogenous infection
Bacterium has apparent inhibiting effect, can effectively reduce Animal diseases incidence.At this stage, with the increasing of health of people awareness of safety
By force, bacillus subtilis increases year by year as green, safe and efficient probiotic feed additive, demand.
In conclusion one side Industry Waste bone mud contains the nutritional ingredients such as a large amount of protein, amino acid, faced in reality as
What makes full use of the problem of useless bone mud resource；Another aspect bacillus subtilis demand increases year by year, if the two is organic
Combine, develops that easily operated, composition is simple, convenient production, the fermentation of low-cost useless bone mud generate bacillus subtilis
Bacterium technology will have practical significance: both having solved the useless bone mud environmental pollution of waste, realized waste utilization, and can open up
The road of one more convenient, less expensive bacillus subtilis production.And currently with useless bone mud fermenting and producing bacillus subtilis
Bacterium has not been reported.
Summary of the invention
It is withered as raw material solid fermenting and producing using chondroitin sulfate production Industry Waste bone mud that the object of the present invention is to provide one kind
The method of careless bacillus can efficiently use the useless bone mud production high added value bacillus subtilis of inexpensive leftover bits and pieces using this method
Bacterium reaches the purpose that turns waste into wealth.
To achieve the above object, the technical solution used in the present invention is: a kind of produce Industry Waste bone with chondroitin sulfate
Mud is the method for raw material solid fermenting and producing bacillus subtilis, to be extracted produced by chondroitin sulfate technique using animal cartilage
Industry Waste bone mud be matrix, mixing wheat bran, water are used as culture medium, access bacillus subtilis progress solid hair after high-temperature sterilization
Bacillus subtilis is made through drying and crushing in ferment, fermentation material.
The useless bone mud, wheat bran, water mass ratio be 3~10: 10~30: 20~80；
It is 5 × 10 that the access bacillus subtilis, which connects bacterium number amount,5~5 × 106A/g culture medium；
The solid fermentation condition is 35~37 DEG C of temperature, and ferment 48~72h；
Preferably, useless bone mud used, wheat bran, water quality ratio are 3: 15: 30.
Preferably, it is 1 × 10 that bacillus subtilis used, which connects bacterium number amount,6A/g culture medium.
Preferably, solid fermentation condition used is solid fermentation 60h under the conditions of 35~37 DEG C.
Common chondroitin production technology is animal cartilage by cleaning and crush at present, and enzyme hydrolysis simultaneously combines isoelectric precipitation
Removing protein is separated, cleaner liquid is extracted using ethyl alcohol and obtained.Separation removing protein production technology can generate a large amount of protein precipitations, belong to
Chondroitin sulfate produces waste, referred to as useless bone mud.Useless bone mud rich in nutrition content, suitable micro-organisms breeding, ordinary room temperature are put
Corruption can be occurred for 2~3 days by setting, and generated foul odour, and generate a large amount of waste liquids, caused very big environmental pollution, be industrial production
Urgent problem.The present inventor has found the useless bone mud due to containing more polypeptide, amino acid, micro by practical studies
The nutritional ingredients such as element can be used for fermenting and producing bacillus subtilis.
Nitrogen content in the present inventor's Kjeldahl nitrogen determination useless bone mud, and measure acidity and host inorganic salt,
Be respectively as follows: total nitrogen >=12.1%, ammonia nitrogen >=2.3%, sodium chloride≤0.64%, bone slag≤3.5%, fats≤1.3%,
PH 6.5~7.0, chondroitin sulfate production Industry Waste bone mud can provide nitrogen for the culture of feeding bacillus subtilis as the result is shown
The nutrients such as source and inorganic salts, on this basis the research of the invention finds that only need to add wheat bran can meet as carbon source it is withered
Careless fermentation of bacillus production needs, and carries out any pretreatment without producing useless bone mud to chondroitin sulfate, and be suitable for institute
There is bacillus subtilis strain.
Chondroitin sulfate production Industry Waste bone mud is applied to fermenting and producing bacillus subtilis for the first time by the present invention, is opened
The new purposes of chondroitin sulfate production Industry Waste bone mud.
In addition, the feed addictive includes the bacillus subtilis powder the present invention also provides a kind of feed addictive
Body preparation.
The invention has the following advantages:
(1) present invention solves the problems, such as the Industry Waste bone sludge-polluted that chondroitin sulfate generates.Rough estimates national sulphur every year
Aching and limp ossein generates nitrogenous industrial wastewater and is up to hundreds of thousands ton, and such a large amount of waste causes the very big pollution of environment.The present invention
The completely new purposes for opening the useless bone mud, is used for the feeding bacillus subtilis of fermenting and producing, turns waste into wealth, and no longer generates and appoints
What waste or waste water, it is environmentally protective.It is not only that enterprise reduces the heavy burdens, but also can increase business economic income.
(2) present invention process is simple, easy to operate.The bone mud that gives up used in the present invention needs not move through pretreatment, directly addition wheat bran
Fermentation of bacillus subtilis production needs can be met.Entire process flow is simple, and process control condition is mild, equipment requirement
Lower, small investment is convenient for industrialization large-scale production application.
(3) the method for the present invention is low in cost, and bacillus subtilis price can be greatly reduced.Sulfuric acid is extracted using animal cartilage
The a large amount of trade wastes generated during chondroitin, wherein containing nutritional ingredients such as more protein, amino acid, in the past all
It is no economic value as waste discharge.And the wheat bran added is the byproduct obtained after wheat processing flour, economic value
Also not high, it is cheap and easily-available.And raw material needs not move through any processing, whole production cost is more cheap.Because currently, withered grass gemma
Culture medium raw material in bacillus active bacteria formulation production process generally has: beef extract, peptone, sodium chloride, starch, glucose,
Urea and other inorganic salts, it is clear that cheap more of the method for the present invention fermentation costs.Obvious the method for the present invention can be greatly reduced withered
Careless bacillus price.
(4) the effective viable bacteria of gained bacillus subtilis powder is high.Do not have to add the evaporation of solid content convection drying again after fermentation
Bacterium powder can be obtained after crushing.According to GB/T 26428-2010 " detection of bacillus subtilis in Application of Direct-fed Microbials " method
Detection Bacillus subtilis number reaches 6~18 × 1010A/g.It can be used for animal feed and microbial-bacterial fertilizer, promote green raw
The development of state agricultural, generates good economic benefit and social effect.
Detailed description of the invention
Fig. 1 is influence of the different incubation times to bacterial count of bacillus subtilis.
Fig. 2 is that difference connects influence of the bacterium amount to bacterial count of bacillus subtilis.
Specific embodiment
Technical solution of the present invention and its generated technical effect are carried out below with reference to specific test method and attached drawing
Further elucidated above, the following description is only intended to explain the invention, but the present invention is limited in any way, based on this
It is any made by invention introduction to transform or replace, it all belongs to the scope of protection of the present invention.
Method used in the present invention is conventional method in that art unless otherwise specified.It is used in following embodiments
Test material, reagent etc., be commercially available unless otherwise specified.
Useless bone mud used in following embodiment is with liquid and waste slag produced caused by animal cartilage extraction chondroitin sulfate technique
It is obtained after conventional filtration.
Embodiment 1: solid fermentation culture medium 100kg, stirring are prepared according to the mass ratio 3: 20: 60 of useless bone mud, wheat bran, water
After uniformly, 120 DEG C of high-temperature sterilization 20min, by 5 × 105A/g culture medium accesses bacillus subtilis, 35~37 DEG C of solid fermentations
48h, fermentation process in two stages: 1-8h uses static gas wave refrigerator, by 35~37 DEG C of temperature of sterile wind control after 8h, at interval of 6h
Open stirring 20rpm time 10min.It ferments after 48h, collects tunning.When 50~60 DEG C of dryings to moisture content is lower than 8%,
It crushed 80 meshes and feeding bacillus subtilis be made.According to GB/T 26428-2010 " withered grass gemma in Application of Direct-fed Microbials
The detection of bacillus " method detection Bacillus subtilis number > 6.0 × 1010A/g.
Embodiment 2: solid fermentation culture medium 100kg, stirring are prepared according to the mass ratio 8: 30: 80 of useless bone mud, wheat bran, water
After uniformly, 120 DEG C of high-temperature sterilization 20min, by 1 × 106A/g culture medium accesses bacillus subtilis, 35~37 DEG C of solid fermentations
60h.Fermentation process is in two stages, and: 1-8h uses static gas wave refrigerator, by 35~37 DEG C of temperature of sterile wind control after 8h, at interval of 6h
Open stirring 20rpm time 10min.It ferments after 60h, collects tunning.When 50~60 DEG C of dryings to moisture content is lower than 8%,
It crushed 80 meshes and feeding bacillus subtilis be made.According to GB/T 26428-2010 " withered grass gemma in Application of Direct-fed Microbials
The detection of bacillus " method detection Bacillus subtilis number > 1.0 × 1011A/g.
Embodiment 3: solid fermentation culture medium 100kg, stirring are prepared according to the mass ratio 3: 10: 20 of useless bone mud, wheat bran, water
After uniformly, 120 DEG C of high-temperature sterilization 20min, by 5 × 106A/g culture medium accesses bacillus subtilis, 35~37 DEG C of solid fermentations
72h.Fermentation process is in two stages, and: 1-8h uses static gas wave refrigerator, by 35~37 DEG C of temperature of sterile wind control after 8h, at interval of 6h
Open stirring 20rpm time 10min.It ferments after 72h, collects tunning.When 50~60 DEG C of dryings to moisture content is lower than 8%,
It crushed 80 meshes and feeding bacillus subtilis be made.According to GB/T 26428-2010 " withered grass gemma in Application of Direct-fed Microbials
The detection of bacillus " method detection Bacillus subtilis number > 8 × 1010A/g.
Embodiment 4: solid fermentation culture medium 100kg is prepared according to the mass ratio 10: 25: 60 of useless bone mud, wheat bran, water, is stirred
After mixing uniformly, 120 DEG C of high-temperature sterilization 20min, by 5 × 105A/g culture medium accesses bacillus subtilis, 35~37 DEG C of solid hairs
Ferment 66h.Fermentation process is in two stages, and: 1-8h uses static gas wave refrigerator, by 35~37 DEG C of temperature of sterile wind control after 8h, at interval of
6h opens stirring 20rpm time 10min.It ferments after 66h, collects tunning.50~60 DEG C of dryings to moisture content is lower than 8%
When, it crushed 80 meshes and feeding bacillus subtilis be made.According to GB/T 26428-2010 " withered grass in Application of Direct-fed Microbials
The detection of bacillus " method detection Bacillus subtilis number > 9 × 1010A/g.
Embodiment 5: solid fermentation culture medium 100kg, stirring are prepared according to the mass ratio 3: 15: 30 of useless bone mud, wheat bran, water
After uniformly, 120 DEG C of high-temperature sterilization 20min, by 1 × 106A/g culture medium accesses bacillus subtilis, 35~37 DEG C of solid fermentations
60h. fermentation process is in two stages, and: 1-8h uses static gas wave refrigerator, by 35~37 DEG C of temperature of sterile wind control after 8h, at interval of 6h
Open stirring 20rpm time 10min.It ferments after 60h, collects tunning.When 50~60 DEG C of dryings to moisture content is lower than 8%,
It crushed 80 meshes and feeding bacillus subtilis be made.According to GB/T 26428-2010 " withered grass gemma in Application of Direct-fed Microbials
The detection of bacillus " method detection Bacillus subtilis number > 1.8 × 1011A/g.
Reference examples 6: solid fermentation culture medium 100kg, stirring are prepared according to the mass ratio 0: 15: 30 of useless bone mud, wheat bran, water
After uniformly, 120 DEG C of high-temperature sterilization 20min, by 1 × 106A/g culture medium accesses bacillus subtilis, 35~37 DEG C of solid fermentations
48h, fermentation process in two stages: 1-8h uses static gas wave refrigerator, by 35~37 DEG C of temperature of sterile wind control after 8h, at interval of 6h
Open stirring 20rpm time 10min.It ferments after 48h, collects tunning.When 50~60 DEG C of dryings to moisture content is lower than 8%,
It crushed 80 meshes and feeding bacillus subtilis be made.According to GB/T 26428-2010 " withered grass gemma in Application of Direct-fed Microbials
The detection of bacillus " method detection Bacillus subtilis number > 5 × 108A/g.
It is compared as the result is shown by embodiment 1-5 and reference examples 6: withered only with wheat bran, water by fermentation bacillus subtilis
Careless bacillus number reaches 5 × 108A/g, after adding useless bone mud, bacillus subtilis bacterium number is increased to 6~18 × 1010A/g, mentions
It is 120~360 times high.
1. experiments of single factor of experimental example investigates the useless bone mud ratio of wheat bran, material-water ratio, incubation time, connects the factors pair such as bacterium amount
Fermentation produces the influence of bacillus subtilis
(1) as bacillus subtilis carbon nitrogen source, carbon-nitrogen ratio grows to pass weight to bacillus subtilis for wheat bran, useless bone mud
It wants, influences thallus output and quality.According to the method provided by the present invention, using single factor design, by useless bone mud: wheat bran ratio
It is respectively set to 2:1,1:1,1:3,1:5,1:7,1:10,1:15, carries out bacillus subtilis culture, experimental result is shown in Table 1.
The different useless bone mud of table 1: the bacillus subtilis bacterium number that the fermentation of wheat bran ratio obtains
As it can be seen from table 1 useless bone mud: when wheat bran=1:1~1:10, bacterial count of bacillus subtilis is up to 1010The order of magnitude
More than, especially useless bone mud: when wheat bran=1:5, bacterial count of bacillus subtilis reaches highest, and 1.8 × 1011, therefore useless bone mud: wheat bran
The method of the present invention culture bacillus optimum proportioning when=1:5.
(2) solid state fermentation maximum feature is exactly no free water, and the variation of substrate moisture content is to microorganism growth metabolism ability
It can have an important influence on.According to the method provided by the present invention, using single factor design, by material-water ratio example be respectively set to 2:1,
1.5:1,1:1,3:5,1:2,1:4,1:6, carry out bacillus subtilis culture, and experimental result is shown in Table 2
The different material-water ratio examples of table 2 influence bacterial count of bacillus subtilis
From table 2 it can be seen that bacterial count of bacillus subtilis reaches highest when material water=3:5,1.3 × 1011, therefore expect water
The method of the present invention culture bacillus optimum proportioning when than=3:5.
(3) in microculture, fermentation time is an important indicator, the time up to influencing thalli growth, also with production
Cost, production energy consumption are directly related.According to the method provided by the present invention, using single factor design, incubation time is set respectively
Be set to 12h, for 24 hours, 36h, 48h, 60h, 72h, 84h, carry out bacillus subtilis culture, experimental result is shown in Fig. 1.
It will be seen from figure 1 that incubation time 60h, bacterial count of bacillus subtilis reaches highest, therefore the method for the present invention culture
Bacillus optimum incubation time is 60h.
(4) it how much related to fermentation period initially connects bacterium amount, according to the method provided by the present invention, is set using experiment of single factor
Meter, will connect bacterium amount and is respectively set to 1 × 105、3×105、5×105、1×106、3×106、5×106A/g culture medium carries out withered
Careless bacillus culture, experimental result is shown in Fig. 2
Figure it is seen that connecing bacterium amount 1 × 106A/g culture medium, bacterial count of bacillus subtilis reach highest, are this hair
Bright method culture bacillus optimum connects bacterium amount.
Claims (6)
1. it is a kind of using chondroitin sulfate production Industry Waste bone mud as the method for raw material solid fermenting and producing bacillus subtilis, it is special
Sign is that, to extract the bone mud that gives up caused by chondroitin sulfate technique as matrix using animal cartilage, mixing wheat bran, water are as culture
Base, access bacillus subtilis carries out solid fermentation after high-temperature sterilization, and bacillus subtilis is made through drying and crushing in fermentation material；
The useless bone mud, wheat bran, water mass ratio be 3~10: 10~30: 20~80；
It is 5 × 10 that the access bacillus subtilis, which connects bacterium number amount,5~5 × 106A/g culture medium；
The solid fermentation, condition are 35~37 DEG C of temperature, and ferment 48~72h.
2. as described in claim 1 using chondroitin sulfate production Industry Waste bone mud as raw material solid fermenting and producing bacillus subtilis
The method of bacterium, characterized in that useless bone mud used, wheat bran, water quality ratio are 3: 15: 30.
3. as described in claim 1 using chondroitin sulfate production Industry Waste bone mud as raw material solid fermenting and producing bacillus subtilis
The method of bacterium, characterized in that it is 1 × 10 that the bacillus subtilis, which connects bacterium number amount,6A/g culture medium.
4. as described in claim 1 using chondroitin sulfate production Industry Waste bone mud as raw material solid fermenting and producing bacillus subtilis
The method of bacterium, characterized in that the solid fermentation process in two stages: 1-8h uses static gas wave refrigerator, passes through sterile air control after 8h
35~37 DEG C of temperature processed, stirring 20rpm, time 10min are opened at interval of 6h.
5. as described in claim 1 using chondroitin sulfate production Industry Waste bone mud as raw material solid fermenting and producing bacillus subtilis
The method of bacterium, characterized in that the fermentation material is lower than 8% through 50~60 DEG C of dryings to moisture content, crushed 80 meshes and feeding is made
With bacillus subtilis powder body preparation.
6. a kind of feed addictive, characterized in that include the feeding bacillus subtilis powder body preparation described in claim 5.
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Citations (5)
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|---|---|---|---|---|
| CN101624230A (en) * | 2008-07-11 | 2010-01-13 | 烟台海岸带可持续发展研究所 | Method of processing waste water and recycling protein |
| CN102220270A (en) * | 2011-05-18 | 2011-10-19 | 江南大学 | Screening method for producing chondroitin sulfate bacterial strain and application of bacterial strain fermentation method in production of chondroitin sulfate |
| CN102337312A (en) * | 2011-10-19 | 2012-02-01 | 江南大学 | Method for increasing yield of chondroitin sulfate produced by fermentation method |
| CN102352334A (en) * | 2011-10-21 | 2012-02-15 | 山东省农业科学院高新技术研究中心 | Method for producing viable bacillus subtilis by way of solid fermentation |
| CN104446813A (en) * | 2014-12-11 | 2015-03-25 | 砀山县春源肥业有限公司 | Bioorganic fertilizer and preparation method thereof |
-
2016
- 2016-08-29 CN CN201610753634.3A patent/CN106148242B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101624230A (en) * | 2008-07-11 | 2010-01-13 | 烟台海岸带可持续发展研究所 | Method of processing waste water and recycling protein |
| CN102220270A (en) * | 2011-05-18 | 2011-10-19 | 江南大学 | Screening method for producing chondroitin sulfate bacterial strain and application of bacterial strain fermentation method in production of chondroitin sulfate |
| CN102337312A (en) * | 2011-10-19 | 2012-02-01 | 江南大学 | Method for increasing yield of chondroitin sulfate produced by fermentation method |
| CN102352334A (en) * | 2011-10-21 | 2012-02-15 | 山东省农业科学院高新技术研究中心 | Method for producing viable bacillus subtilis by way of solid fermentation |
| CN104446813A (en) * | 2014-12-11 | 2015-03-25 | 砀山县春源肥业有限公司 | Bioorganic fertilizer and preparation method thereof |
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