CN114262676A - Method for producing amino acid by efficiently degrading feather keratin with bacillus - Google Patents
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Abstract
The invention provides a method for producing amino acid by efficiently degrading feather keratin with bacillus and application of the method in degrading chicken feather meal. The technical scheme adopted by the invention is as follows: inoculating bacillus into a feather meal culture medium for culture, and adjusting a fermentation project to include two stages of aerobic fermentation and standing reaction to obtain a fermentation product, namely the amino acid aqueous solution. 10% (w/w) of feathers are degraded and converted in the 6d of the invention, the content of free amino acid in the product is as high as 39g/L, and simultaneously, the product contains a large amount of free polypeptide.
Description
Technical Field
The invention relates to a method for producing amino acid by efficiently degrading feather keratin with bacillus, belonging to the field of fermentation engineering.
Background
More than 90% of protein in the feathers exists in the form of keratin, which is a water-insoluble fibrous structural protein rich in threonine, arginine, cysteine and some hydrophobic amino acids and has high potential nutritional value. Keratin is ubiquitous in nature and is usually the ectoderm of animal skin, such as feathers, hair, scales, nails, claws, and the like. The keratin has disulfide bonds, hydrogen bonds, salt bonds and other crosslinking effects in and among molecular chains, so that the feather has strong degradation resistance and is difficult to hydrolyze by common protease (trypsin, pepsin, papain and the like).
The annual feather production waste of China is more than million tons, most of feathers are usually buried and burned, and serious resource waste, environmental pollution and even diseases are caused. Therefore, the feather waste is fully utilized, so that the environmental pollution can be reduced, and the resource recycling can be promoted.
The existing feather degradation methods mainly comprise a physical method (a high-temperature cooking method and a puffing method) and a chemical method (an acid-base hydrolysis method), and the methods have the advantages of high energy consumption, complex process and easy environmental pollution, and can damage partial amino acid to cause nutrient loss. The microbial method and the enzyme method are two emerging feather degradation methods, the process for degrading the waste feathers is mild, the environment is friendly, the amino acid structure is not damaged, and the produced amino acid product can be widely applied to the industries of fertilizer, medicine, feed and the like. However, the enzymatic degradation has the problems of complex process, high cost and the like, and the enzymatic degradation of feathers at the present stage can not meet the industrial requirements at all. The main problems of feather degradation by microorganisms include incomplete feather degradation and low amino acid yield, so that sample pretreatment and fermentation process optimization are urgently needed, and the amino acid conversion rate is further improved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for efficiently degrading feather keratin to produce amino acid by using aerobic microorganism bacillus licheniformis ZSZ6, so that the amino acid conversion rate is improved, and the fermentation period is shortened.
The technical scheme adopted by the invention is as follows:
the invention provides a method for efficiently degrading feather keratin by using bacillus, which comprises two stages of aerobic fermentation and standing reaction, preferably, Na with the weight of 0.5-1 percent of the total weight of the culture medium is added into the culture medium of the aerobic fermentation2SO3(ii) a Adding manganese ion-containing salt in an amount of 0.5-1% by weight based on the total weight of the system into the system for standing reaction.
The manganese ion-containing salt of the present invention can be any soluble manganese ion-containing salt, including, but not limited to, for example, MnSO4、MnCl2Or Mn (NO)3)2。
The bacillus used in the invention can be bacillus with feather degradation function known in the field, and the inventor finds that the degradation rate can be obviously improved through different bacillus strain experiments, and the method has wide application range. In a specific embodiment of the present invention, the strain used is bacillus licheniformis ZSZ6 in granted patent CN110183252B, which is preserved in the common microorganism center of the chinese committee for culture collection of microorganisms (CGMCC) with the preservation number: CGMCC No. 172458.
In some embodiments of the invention, the culture medium for aerobic fermentation comprises 90-120 g/L chicken feather, 0.8-1.2 g/L NaCl and KH2PO40.8 to 1.2g/L, K2HPO40.8-1.2 g/L, and the pH value is 6.8-7.4; in a specific example, the proportion of the culture medium for aerobic fermentation is 100g/L of chicken feather, 1g/L of NaCl and KH2PO4Is 1g/L, K2HPO41g/L and a pH value of 6.8.
In some embodiments of the present invention, the feather of the present invention is pretreated, specifically, the feather of chicken is washed and crushed.
The preparation of the culture medium for aerobic fermentation can be carried out according to a conventional method, for example, the culture medium is prepared according to a formula and then treated at 121 ℃ for 20min for sterilization. In some particularly preferred embodiments of the invention, 0.5% to 1% Na is added to the culture medium2SO3Then processing at 120-125 ℃ for 15-25 min, coolingAnd standing for 20-25 h after the temperature is reduced to room temperature.
The aerobic fermentation of the invention is to inoculate strain seed liquid into a culture medium, and the inoculation amount can be selected according to the conventional method, for example, the inoculation amount is 2-5%. Seed liquids may be prepared according to methods conventional in the art, for example: inoculating the bacterial liquid into a seed culture medium, and culturing at 37 ℃ and 180-260 rpm for 16-24 h. The seed culture medium comprises: 10g/L tryptone, 5g/L yeast powder, 10g/L NaCl, pH 7.0, sterilizing at 121 deg.C for 20 min. In some embodiments, the seed liquid for inoculation has a cell density of 107～1010cfu/ml。
In some embodiments of the invention, the fermentation temperature of the aerobic fermentation is 35-38 ℃, and the fermentation time is 24-48 h.
The dissolved oxygen of the aerobic fermentation can be controlled according to the conventional method in the field, for example, 20-50%.
In some embodiments of the invention, after the aerobic fermentation is finished, 0.1% -0.5% of manganese ion-containing salt is added and then the standing reaction is directly carried out.
In some embodiments of the invention, the standing reaction temperature is adjusted to be 37-60 ℃ for 72-96 h; the preferable reaction temperature is 40-55 ℃.
In some embodiments of the invention, the pH value of the aerobic fermentation is controlled to be 6.8-7.4, and the pH value of the standing reaction is controlled to be 8-9; the pH can be adjusted according to methods conventional in the art.
The invention also provides the amino acid polypeptide aqueous solution prepared by the invention.
The amino acid polypeptide aqueous solution prepared by the invention can be applied to preparation of organic fertilizers, feed processing, amino acid purification and the like.
The invention forms a novel amino acid aqueous solution production technology by optimizing reaction conditions. Compared with the prior art, the invention has the beneficial effects that:
(1) the invention pretreats the raw materials, in particular to add Na before fermentation2SO3Then standing for 1d, setting the fermentation reaction into two stages of aerobic fermentation and standing reaction,and further, manganese ion-containing salt is added in the standing reaction stage, so that the yield and the speed of amino acid after feather degradation are improved.
(2) Furthermore, the yield of amino acid is further improved by adjusting the temperature of each reaction stage, and the content of free amino acid in the product is remarkably improved by hydrolyzing for 5-6 d by using the method.
(3) The method has wide application range and can realize the improvement of the content of free amino acid in different bacillus fermentation products.
Drawings
FIG. 1 shows the yield of amino acids of Bacillus licheniformis ZSZ6 under different reaction conditions;
FIG. 2 shows the amino acid yield of Bacillus licheniformis ZSZ6 under different pretreatment conditions;
FIG. 3 shows the amino acid yield of Bacillus licheniformis ZSZ6 when different reagents are added during the standing period;
FIG. 4 shows the amino acid yield ratio of Bacillus licheniformis ZSZ6 with the addition of different manganese salts;
FIG. 5 shows the polypeptide yields before and after optimization of fermentation conditions for Bacillus licheniformis ZSZ 6;
FIG. 6 shows the application of the method to other Bacillus species.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the following embodiments, the detection method of free amino acids adopts an indetrione method (GB/TB 314-2002), and the polypeptide adopts a biuret method.
If not specifically stated, the strain used in the following examples is Bacillus licheniformis ZSZ6 in the granted patent CN110183252B, and the seed liquid is prepared specifically as follows: selecting Bacillus licheniformis ZSZ6 ring preserved on slant, inoculating into test tube containing seed culture medium, activating at 37 deg.C for 16h to obtain first-stage seed solution, inoculating the first-stage seed solution to 100ml seed culture according to 2% inoculum sizeCulturing at 37 deg.C for 16h to obtain secondary seed solution, i.e. seed solution for inoculation, with thallus density of about 2 x 109cfu/ml。
The seed culture medium used included: 10g/L tryptone, 5g/L yeast powder, 10g/L sodium chloride and pH value of 7.0, and sterilizing at 121 ℃ for 20 min.
If not otherwise stated, the feather meal medium used in the eutrophy stage in the following examples includes: chicken hair powder 100g/L, NaCl 1g/L, KH2PO41g/L，K2HPO41g/L and pH 6.8.
Example 1 comparison of different reaction conditions
Inoculating the secondary seed liquid of the bacillus licheniformis ZSZ6 into a feather meal culture medium according to the inoculation amount of 5%, wherein the shaking initial fermentation conditions are as follows: 5% inoculum size, at 37 degrees C, 180rpm conditions were shake culture.
Setting of reaction conditions experimental group: performing shake culture on the CK control group at 37 ℃ and 180rpm for 6 d; and the other fermentation conditions are that the degradation mode is adjusted after shaking culture is carried out for 2d under the conditions of 37 ℃ and 180rpm, the reaction is continued for 4d under the conditions of standing at 37 ℃, standing at 45 ℃ and standing at 55 ℃, and then the yield of the amino acid is measured. As shown in FIG. 1, the yield of amino acid was increased to various degrees in the stage of adjustment to the standing after 2d, wherein the yield of amino acid was about 20.93g/L when the solution was adjusted to the standing at 37 ℃ and about 25.87g/L when the solution was adjusted to the standing at 55 ℃.
Example 2 comparison of different pretreatment conditions for feather meal
Adding 1% DTT, mercaptoethanol, and Na into feather powder culture medium2SO3(CK is standing only, no reagent is added), treating at 121 ℃ for 20min, cooling to room temperature, and standing for 1 d. Then, after shaking-culturing at 37 ℃ and 180rpm for 2 days, the mixture was allowed to stand at 55 ℃ for 4 days. The results are shown in FIG. 2, where DTT and mercaptoethanol treatment under the assay conditions failed to improve amino acid production, using 1% Na2SO3The amino acid yield can be further remarkably improved when the pretreatment is carried out for 1d, and is about 29.42 g/L.
Example 3 comparison of the addition of different reagents during the resting phase
Adding 1% Na2SO3Treating at 121 deg.C for 20min, cooling to room temperature, standing for 1d, shake culturing at 37 deg.C and 180rpm for 2d, and adding 0.1% MgSO4、FeSO4、FeCl3、MnSO4、CuSO4Then, the mixture was allowed to stand at 55 ℃ for 4 days. The measurement results are shown in FIG. 3 (CK represents no Na addition before leaving standing)2SO3No reagent is added after standing), the MnSO of the invention is added4The yield of amino acid is obviously improved to 35.41 g/L. In addition, it was determined that the addition of other manganese ion-containing salts included MnCl2And Mn (NO)3)2The effect of time on the amino acid production of the strain is shown in FIG. 4, which is a graph of the amino acid production and MnSO4Approximate (ordinate is its amino acid production and addition of MnSO)4The ratio of the production of amino acids), namely, Mn is added in the standing stage2+It is helpful to improve the yield of amino acid.
Example 4 the effect of this method on polypeptide production was as follows:
the method of the embodiment comprises the following steps: performing shake culture on the CK control group at 37 ℃ and 180rpm for 6 d; the treatment group was 1% Na2SO3The chicken feather powder culture medium after 1d of pretreatment is subjected to shake cultivation at 37 ℃ and 180rpm for 2d, and then 0.1% MnSO is added4And continuously standing for 4d at 55 ℃, and respectively determining the polypeptide content in the products.
As shown in FIG. 5, the method of this example can improve the polypeptide yield by 45.91g/L, which is 73.31% higher than conventional treatment.
EXAMPLE 5 use of other Bacillus species
The method described in example 4 was used to replace the strains with three other Bacillus species C1, C2, C3, i.e., Bacillus species C1, C2, C3 were subjected to shake cultivation at 37 deg.C and 180rpm for 2d and then allowed to stand at 55 deg.C, and the polypeptide content in the product was determined. Bacillus strains C1, C2 and C3 are selected from the unit as strains with feather degradation ability, and can be obtained by the skilled person. As shown in FIG. 5, at 5d, the fermentation method greatly increased the amino acid production of other bacilli, and finally increased the amino acid production by 36.40%, 38.73%, and 56.07%, respectively.
Example 650L fermentation of Bacillus licheniformis ZSZ6 to produce amino acids in a fermenter
(1) Soaking two groups of 3kg chicken feather powder in advance, wherein the first group is not added with Na2SO3And the second group added 0.5% of Na2SO3And standing overnight.
(2) Inoculating the first-stage seed solution of Bacillus licheniformis ZSZ6 into LB liquid culture medium at a ratio of 2%, and culturing at 37 deg.C and 180rpm for 16 hr to obtain second-stage seed solution.
(3) After filter sterilization and air digestion of the fermentation tank, the feather culture medium containing chicken feather meal was added to the fermentation tank and water was added to 22L for actual digestion. After cooling to room temperature, adding the secondary seed liquid according to the proportion of 5% for fermentation.
The first group of fermentation conditions is that the pH is controlled to be 7, the stirring speed is 180rpm, the fermentation temperature is 37 ℃, the dissolved oxygen DO is 20-40%, and the total time is 6 days.
The second set of adjusted fermentation conditions: the first stage is a thallus growth stage (within 48 h), the initial pH is 7, the stirring speed is 180rpm, the fermentation temperature is 37 ℃, and the dissolved oxygen DO is controlled to be 20-50%; the second stage is a stage of degrading chicken feather powder to produce amino acid (after 48 h), adjusting pH to 8, heating to 55 deg.C, adding 0.1% MnSO4And stopping stirring and ventilating, and stirring once every 12h for fermentation for 4 d.
The experimental results are as follows: under the condition of a 50L fermentation tank, the first group can produce about 18g/L of amino acid at the sixth day, and the second group can reach about 39g/L at the fifth day, so that the method can still greatly improve the yield of the amino acid even under the complex condition of the amplification treatment.
Finally, it should also be noted that the above list is only a specific implementation example of the present invention. It is obvious that the invention is not limited to the above embodiment examples, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (10)
1. A method for degrading feather keratin by using bacillus,the method is characterized by comprising two stages of aerobic fermentation and standing reaction, preferably, Na with the weight of 0.5-1 percent of the total weight of the culture medium is added into the culture medium of the aerobic fermentation2SO3(ii) a Adding manganese ion-containing salt with the weight portion of 0.5-1% of the total weight of the system into the system for standing reaction; more preferably, the manganese ion-containing salt is selected from MnSO4、MnCl2Or Mn (NO)3)2。
2. The method for degrading feather keratin by using bacillus as claimed in claim 1, wherein the ratio of the culture medium for aerobic fermentation is 90-120 g/L chicken feather, 0.8-1.2 g/L NaCl and KH2PO40.8 to 1.2g/L, K2HPO40.8-1.2 g/L, and the pH value is 6.8-7.4; preferably 100g/L of chicken feather, 1g/L of NaCl and KH2PO4Is 1g/L, K2HPO41g/L and a pH value of 6.8.
3. The method for degrading feather keratin by using Bacillus of claim 1, wherein 0.5% -1% of Na is added to the culture medium2SO3Then treating at 120-125 ℃ for 15-25 min, cooling to room temperature and standing for 20-25 h.
4. The method for degrading feather keratin by using bacillus as claimed in claim 2, wherein the feather is added to the culture medium after being pulverized.
5. The method for degrading feather keratin by using bacillus as claimed in claim 1, wherein the aerobic fermentation is to inoculate strain seed liquid into a culture medium; the preferred inoculation amount is 2 to 5 percent; preferably, the strain seed solution is Bacillus licheniformis ZSZ6 seed solution, and more preferably the seed solution has a thallus density of 107～1010cfu/ml。
6. The method for degrading feather keratin by using bacillus as claimed in claim 1, wherein the fermentation temperature of the aerobic fermentation is 35-38 ℃ and the fermentation time is 24-48 h; preferably, the dissolved oxygen is controlled to be 20-50%.
7. The method for degrading feather keratin by using bacillus as claimed in claim 1, wherein 0.1% -0.5% of manganese ion-containing salt is added after the aerobic fermentation is finished, and then the mixture is directly subjected to a standing reaction.
8. The method for degrading feather keratin by using bacillus as claimed in claim 1, wherein the reaction temperature of the standing reaction is 37-60 ℃ and the reaction time is 72-96 h; the preferable reaction temperature is 40-55 ℃.
9. The method for degrading feather keratin by using bacillus as claimed in claim 1, wherein the pH value is controlled to 6.8-7.4 by aerobic fermentation, and the pH value is controlled to 8-9 by standing reaction.
10. An aqueous solution of an amino acid polypeptide obtained by the method of any one of claims 1 to 9.
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| CN115055038A (en) * | 2022-06-02 | 2022-09-16 | 广东省科学院生物与医学工程研究所 | Method for preparing degradable air purification material by hydrolyzing waste feather through microbiological method |
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