WO2020143205A1 - 一种利用废弃羽毛生产氨基酸液肥的生物制方法 - Google Patents

一种利用废弃羽毛生产氨基酸液肥的生物制方法 Download PDF

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WO2020143205A1
WO2020143205A1 PCT/CN2019/096125 CN2019096125W WO2020143205A1 WO 2020143205 A1 WO2020143205 A1 WO 2020143205A1 CN 2019096125 W CN2019096125 W CN 2019096125W WO 2020143205 A1 WO2020143205 A1 WO 2020143205A1
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amino acid
enzymolysis
enzyme
feather
protease
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PCT/CN2019/096125
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English (en)
French (fr)
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朱红惠
周莲
谢小林
陈美标
姚青
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广东省微生物研究所（广东省微生物分析检测中心）
广东博沃特生物科技有限公司
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Priority to US17/762,751 priority Critical patent/US11667586B2/en
Publication of WO2020143205A1 publication Critical patent/WO2020143205A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05C—NITROGENOUS FERTILISERS
    • C05C11/00—Other nitrogenous fertilisers
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F1/00—Fertilisers made from animal corpses, or parts thereof
    • C05F1/005—Fertilisers made from animal corpses, or parts thereof from meat-wastes or from other wastes of animal origin, e.g. skins, hair, hoofs, feathers, blood
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/10—Fertilisers containing plant vitamins or hormones
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G5/00—Fertilisers characterised by their form
    • C05G5/20—Liquid fertilisers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/20—Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses

Definitions

  • the invention belongs to the technical field of fertilizer processing, and specifically discloses a method for preparing amino acid liquid fertilizer by stepwise compound enzymatic hydrolysis of waste feathers using keratinase, neutral protease, alkaline protease, amino acid peptidase and acid protease.
  • Feather is the waste left over from poultry treatment. Worldwide, millions of tons of waste feathers are produced every year, and the degree of resource utilization is less than 20%. Except for a small amount of warm clothing filling and animal feed, these feathers are mostly treated as waste, not only causing waste of resources, but also traditional incineration, landfill and other treatment methods will cause serious pollution to the environment. Therefore, the management of feather waste has become an urgent problem for poultry slaughter enterprises.
  • feather waste is a rich natural protein resource. Its crude protein content is as high as 85% to 90%. Except for the content of lysine and methionine in feather protein, the composition of other animal essential amino acids is slightly higher than that of fish meal. This means that the use of protein hydrolysis to produce amino acid-related products has significant potential economic benefits.
  • the protein in poultry feathers is mainly composed of keratin, keratin contains more cystine, and a large number of disulfide bonds are formed between them, plus hydrogen bonds and intermolecular forces are difficult for ordinary proteases (such as alkali Sex protease, trypsin, pepsin, papain, etc.) hydrolysis.
  • proteases such as alkali Sex protease, trypsin, pepsin, papain, etc.
  • Traditional methods such as high-temperature and high-pressure cooking hydrolysis, puffing method, acid-base hydrolysis and the use of oxidants or reducing agents are used to process waste feathers, which have large fixed investment, long capital recovery period, severe reaction conditions, high requirements for production equipment and high energy consumption
  • the product is prone to configuration changes or racemization, which results in extremely complex product components, serious amino acid damage, high product salt, and serious environmental pollution.
  • Amino acids can regulate and stimulate crop growth and development (promote seed germination and growth, promote root growth, regulate root morphology, promote leaf growth and increase leaf green content, etc.), improve crop quality, increase crop yield, improve crop disease resistance and stress resistance It also has the ability to promote crops' absorption of other nutrients such as nitrogen, phosphorus, and potassium. Amino acids can be used alone as biostimulants, amino acid nitrogen fertilizers, etc., or as biologically active synergists and chemical fertilizers (such as amino acid liquid fertilizer) to exert multiple synergies. Therefore, in agriculture, the application of amino acids is becoming more and more extensive.
  • the current common production method is to obtain the amino acid stock solution by enzymatic or acid hydrolysis of protein raw materials, or to concentrate the amino acid fermentation tail liquid.
  • These methods all have certain shortcomings, such as the use of acid hydrolysis, which causes greater environmental pollution and the higher content of chloride salts in the amino acid stock solution, which has a toxic effect on certain chlorine-sensitive plants; and the use of enzymatic hydrolysis, because The reason of enzymolysis raw material and process is that the content of amino acid in the enzymolysis solution is low, and it needs to be concentrated to increase the concentration of the amino acid stock solution, and the cost is higher; the amino acid tail liquid concentration method also has a higher cost, and the concentration of sodium ion in the concentrated product is too high , Sometimes causing problems such as salt damage.
  • the object of the present invention is to provide a biological method for producing amino acid liquid fertilizer using waste feathers in view of the above problems.
  • the invention intends to use a compound enzyme to enzymatically hydrolyze waste feathers so that the concentration of amino acids in the enzymolysis solution reaches more than 10%, and the national amino acid liquid fertilizer standard can be reached without concentration to prepare an environmentally friendly amino acid liquid fertilizer.
  • the feather meal is enzymatically hydrolyzed with a compound enzyme to obtain an enzymolysis solution, and then an acidic protease is added to the enzymolysis solution. After the enzymolysis is completed, the solution is filtered, and the filtrate is subjected to enzyme elimination to obtain an amino acid liquid fertilizer;
  • the complex enzymes include keratinase and amino acid peptidase.
  • amino acid peptidase refers to peptidase.
  • the complex enzymes include keratinase, neutral protease, alkaline protease and amino acid peptidase.
  • the composite enzyme includes keratinase 34-66%, alkaline protease 5-56%, neutral protease 5-56% and amino acid peptidase 5-56% in terms of enzyme activity.
  • the compound enzymes include keratinase 45.6%, alkaline protease 11.8%, neutral protease 11.6% and amino acid peptidase 31.0% in terms of enzyme activity ratio.
  • the feather powder is obtained by removing waste feathers and removing impurities, after high temperature cooking or sodium sulfite treatment, air drying or drying, and then crushing and grinding to obtain feather powder.
  • High temperature cooking or sodium sulfite treatment can help the proteolysis. It is further preferred that the high temperature cooking is feather cooking at 135°C and 0.4 MPa for 30 min.
  • the crushing and milling is crushing and milling through a 1 mm aperture sieve to obtain feather powder.
  • the enzymatic hydrolysis with the compound enzyme is adding feather powder to water, so that the mass fraction of the feather powder is above 30%, adjusting the pH value to 8.5-9.5, and then adding the compound enzyme after sterilization, the compound enzyme is in accordance with 9000U/ g Feather meal is added above, the enzymolysis temperature is 53 ⁇ 60°C, and the enzymolysis is over 36h.
  • the enzymatic hydrolysis with the compound enzyme is to add feather powder to water at a mass ratio of 3:10, adjust the pH to 9, and then add the compound enzyme after sterilization.
  • the compound enzyme is added according to 9000U/g feather powder, and the enzyme is hydrolyzed
  • the temperature is 53°C and the enzyme is hydrolyzed for 36h.
  • the acidic protease is hydrolyzed, the added amount is 2000 U/g feather powder, the enzymolysis conditions are 40° C., and the enzymolysis is 12 hours.
  • the enzyme inactivation is 90°C for 10 minutes.
  • the feathers are various poultry, such as chicken, duck and goose feathers.
  • the present invention has the following beneficial effects:
  • the method of the present invention can make the enzymatic hydrolysis rate of feathers reach more than 80%, the prepared amino acid liquid fertilizer contains 17 kinds of amino acids, and the variety is complete, and the amino acid content can reach more than 10.12% (mass fraction), and the small peptide content can reach 9.39% (mass Score), without concentration to reach the national amino acid liquid fertilizer standard, so that environmentally friendly amino acid liquid fertilizer can be prepared.
  • the preparation method of the invention can be industrialized, the process is simple and stable, can save the concentration process in the traditional amino acid liquid fertilizer preparation process, greatly reduce energy consumption and pollution; and has the advantages of wide source of raw materials, low price, and a full range of amino acid products.
  • the invention is an environment-friendly amino acid liquid fertilizer production technology.
  • Figure 1 shows the effect of different protease compositions on the rate of feather digestion (broken line) and amino acid content (histogram).
  • Figure 2 is a contour map of the interaction of various proteases affecting amino acid content.
  • Fig. 3 is the effect of different addition amounts of compound enzymes on the enzymatic hydrolysis rate (broken line) and amino acid content (bar graph) of feathers.
  • Figure 4 is the effect of system pH on the rate of feather digestion (broken line) and amino acid content (histogram).
  • Figure 5 is the effect of enzymatic hydrolysis temperature on the enzymatic hydrolysis rate (broken line) and amino acid content (histogram) of the feather.
  • Figure 6 is the effect of substrate concentration on the enzymatic hydrolysis rate (broken line) and amino acid content (histogram) of the feather
  • Figure 7 is the effect of enzymolysis time on the enzymatic hydrolysis rate (broken line) and amino acid content (histogram)
  • Figure 8 is a curved surface diagram of the interaction of different enzymatic hydrolysis conditions on amino acid content
  • Feathers are provided by Guangdong Bowater Biotechnology Co., Ltd. and belong to chicken feathers, which are thoroughly cleaned and dried before use.
  • Keratinase (100,000 U/g), independently produced; neutral protease (100,000 U/g), alkaline protease (100,000 U/g), lipase (200,000 U/g), papain (100,000 U/g), Yangshao Biochemical Technology Co., Ltd.; Amino Acid Peptidase (100,000 U/g, exopeptidase) Angel Yeast Co., Ltd.; Acid Protease (50,000 U/g), Yuanye Biotechnology Co., Ltd.; NaOH , HCl, sodium citrate, sulfosalicylic acid, trichloroacetic acid, potassium sulfate, EDTA-Na and other reagents are analytically pure; mixed amino acid standard solution, chromatographically pure.
  • L-8900 automatic amino acid analyzer was used for the determination.
  • the peptide content was determined according to the method in the national standard of soybean peptide powder (GB/T 22492-2008).
  • the enzymolysis solution was filtered through a 300-mesh aperture screen, dried at 105°C to constant weight, and the enzymolysis rate was calculated.
  • Feather enzymolysis rate (%) (feather addition amount-feather residue dry weight)/feather addition amount ⁇ 100%.
  • the specific processing method is:
  • the fixed total enzyme addition is 4500U/g feather powder, and the enzyme activity ratio of each enzyme is added.
  • a total of 13 protease compositions are selected to enzymatically hydrolyze the feather powder.
  • the specific composition is shown in Table 1.
  • the hydrolysis conditions are: the substrate concentration is 10g/100mL (that is, 10g feather powder is added to 100ml water), the enzyme amount is 4500U/g substrate, and the enzyme is hydrolyzed for 3d at 45°C, pH8.0, 180rpm, and free amino acids
  • the content is the main evaluation index, and the enzymatic hydrolysis rate of the feather is the auxiliary evaluation index.
  • the optimal protease composition of the amino acid-related products prepared by the enzymatic hydrolysis of the feather powder is selected, with 3 replicates per group.
  • the substrate mass concentration was 10g/100mL
  • the enzymolysis temperature was 45°C
  • the enzymolysis reaction system was pH8.0, 180rpm.
  • the optimal compound protease addition amount of amino acid-related products prepared from feather meal is 3 replicates per group.
  • the substrate concentration was 10g/100mL
  • the enzymolysis temperature was 45°C
  • the compound enzyme addition was 4500U/g feather powder
  • the enzymolysis was carried out at 180rpm for 3d. (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0)
  • the optimal compound protease dosage for related products is 3 replicates per group.
  • the substrate concentration was 10g/100mL
  • the enzymolysis reaction system was pH8.0
  • the addition amount of the complex enzyme was 4500U/g feather powder
  • the enzyme was hydrolyzed for 3d under the condition of 180rpm.
  • the effect of hydrolysis temperature (35, 40, 45, 50, 55, 60 °C) on the enzymatic hydrolysis of feather meal is based on the free amino acid content as the main evaluation index, and the enzymatic hydrolysis rate of feather on the water is used as an auxiliary evaluation index to screen out the enzymatic hydrolysis feather
  • the optimal amount of compound protease added for the preparation of amino acid related products by powder is 3 repeats per group.
  • the enzymolysis temperature was 45°C
  • the enzymolysis reaction system was pH8.0
  • the amount of the compound enzyme added was 4500U/g feather powder
  • the enzymolysis was carried out for 3 days at 180rpm. (5%, 10%, 15%, 20%, 25%, 30%, 35%, the substrate concentration refers to the mass of the substrate per 100g of water, for example, 5% refers to 5g of feather powder in 100g of water)
  • the content of free amino acids was used as the main evaluation index
  • the enzymatic hydrolysis rate of feather was used as the auxiliary evaluation index.
  • the substrate concentration is 10g/100mL
  • the enzymolysis temperature is 45°C
  • the enzymolysis reaction system is pH8.0
  • the amount of complex enzyme is 4500U/g feather powder
  • 180rpm conditions Under the enzymolysis, investigate the effect of enzymolysis time (12, 24, 36, 48, 60, 72, 84) on the enzymatic hydrolysis of feather meal, with free amino acid content as the main evaluation index, and the enzymatic hydrolysis rate of feather as the auxiliary evaluation index,
  • the optimal compound protease addition amount for enzymatic hydrolysis of feather meal to prepare amino acid related products was screened out, with 3 replicates per group.
  • the compound enzyme was used to enzymatically digest the feathers to obtain the preliminary enzymatic hydrolysis solution.
  • the amount of acid protease added is 2000U/g feather
  • the pH is natural (the initial enzymolysis solution pH is acidic)
  • the enzymolysis temperature is 40°C
  • the enzymolysis is 180 rpm for 12h.
  • the pretreatment method will affect the enzymatic hydrolysis effect of feathers.
  • Sulphuric acid is used to pretreat the feathers and then enzymatic hydrolysis, the highest enzymatic hydrolysis rate, and the highest content of amino acids in the enzymatic hydrolysate obtained at the same time, there is no significant difference from high temperature and high pressure cooking treatment, but it is different from ultrasonic treatment, microwave treatment, heating There are significant differences between treatment and ultra-low temperature treatment.
  • the reason for analysis is that the protein in feathers is mainly keratin, and the large amount of disulfide bonds in it is the reason why it is difficult to be degraded by ordinary proteases.
  • the sodium sulfite is a reducing agent, which helps to break the disulfide bonds in keratin protein and promote enzymatic hydrolysis.
  • the composition of complex enzymes is based on keratinase.
  • the composite protease composed of keratinase, alkaline protease, neutral protease and amino acid peptidase has the best effect, while the composite enzyme based on keratinase is superior to Single keratinase, especially the combination of keratinase + amino acid peptidase, and within a certain range, the more kinds of proteases, the better the effect.
  • each protease-specific cleavage site has specificity. After the class sites have been completely broken, even if the protease is not completely inactivated, or the protease is supplemented, the degree of hydrolysis is difficult to increase.
  • the purpose of this experiment is to develop amino acid foliar fertilizer, which belongs to deep enzymatic hydrolysis. Therefore, in order to further improve the degree of hydrolysis of feather keratin, increase the content of amino acids, the use of different protease specific cleavage sites is different, the use of multi-enzyme synergy digestion, increase the specific protease cleavage site, can effectively increase the amino acid content.
  • keratin proteolysis rate the composite protease composed of keratinase, alkaline protease, neutral protease and amino acid peptidase is also the best.
  • keratinase, alkaline protease, neutral protease and amino acid peptidase were selected to perform complex enzymatic hydrolysis of feathers.
  • the experimental design of the mixture is optimized for the compound enzyme ratio test, which is designed to be 4 factors and a total of 18 tests are carried out.
  • the experimental arrangement and results are shown in Table 5.
  • the compound enzyme ratio selected in this paper (according to the proportion of enzyme activity) is: keratinase, 45.6%; alkaline protease, 11.8%; neutral protease, 11.6%%; amino acid peptidase, 31.0%. Under this condition, the predicted amino acid content reached 16.34g/kg. as shown in picture 2.
  • the substrate mass concentration was 10g/100mL (that is, 10g feather powder was added to 100ml water)
  • the compound enzyme plus enzyme amount was added It is 4500U/g substrate
  • the compound enzyme ratio (according to the enzyme activity ratio) is: keratinase, 45.6%; alkaline protease, 11.8%; neutral protease, 11.6%%; amino acid peptidase, 31.0%, at 45 Enzymolysis at °C, pH8.0, 180rpm 3d), the measured amino acid content is 16.54g/kg, which is basically close to the predicted value, the deviation is 1.25%.
  • the protease first forms an enzyme-substrate complex with the substrate.
  • the enzyme concentration is proportional to the enzyme reaction rate. If the enzyme concentration is increased at this time, the reaction rate can be increased. Therefore, within a certain reaction time, the higher the protein digestion efficiency, the higher the amino acid conversion rate and protein digestion rate.
  • the greater the amount of enzyme added the protease itself may be hydrolyzed with each other, so that the enzyme activity is reduced, and the production cost will be higher. Therefore, considering the enzymatic hydrolysis rate, amino acid content and production cost of the feather, the optimum addition amount of the compound enzyme is 9000U/g feather.
  • the reason for the analysis is that when the pH of the reaction system is too high or low, the charging of the protease activity part changes, the binding ability of the protease and the substrate decreases, and the enzyme activity decreases.
  • Each enzyme has a specific optimal pH.
  • the complex enzyme contains a neutral protease. At pH 7.0, the neutral protease in the complex enzyme exhibits stronger enzyme activity. This may be a small peak of the complex enzyme at pH 7.0 One of the reasons. Therefore, considering the enzymatic hydrolysis rate and amino acid content of the feather, the optimal pH of the enzymatic hydrolysis system of the composite enzyme was determined to be 9.0.
  • the amino acid content in the enzymolysis solution decreased instead.
  • the feather digestion rate As far as the feather digestion rate is concerned, as the substrate concentration increases, the feather digestion rate continues to decrease. When the substrate concentration exceeds 25 g/100 mL, the rate of decrease in the feather digestion rate increases significantly. When the substrate concentration is 5g/100mL, the highest rate of enzymatic hydrolysis of the feather is 90.45%. Considering that the experimental purpose is to develop amino acid foliar fertilizer, and the concentration of amino acid is too low is an important factor to limit the enzymatic hydrolysis process. Therefore, the optimal substrate concentration of the compound enzymatic hydrolysis is determined to be 30g/100mL.
  • the reason for the analysis is that, as the enzymatic hydrolysis time is prolonged, on the one hand, the effective peptide bond concentration decreases, resulting in weakened keratin hydrolysis and slowed down the degree of hydrolysis; on the other hand, peptide molecules in the enzymatic hydrolysis products compete as proteases Substrate, which slows down the rate of enzymatic hydrolysis; in addition, as enzymatic hydrolysis progresses, the pH of the system decreases, while the activity of the protease decreases, thereby slowing the rate of enzymatic hydrolysis. Therefore, considering the production cost, feather hydrolysis rate and amino acid content required for enzymatic hydrolysis time, the optimal time for complex enzymatic hydrolysis was determined to be 36h.
  • Amino acid content 64.01+2.41A-2.81B-0.13C+3.14D+1.07AB-2.28AC-1.57AD-1.53BC+ 1.09BD-2.21A 2 -2.79B 2 -5.89C 2 -6.07D 2 .
  • the surface graph obtained using Design Expert 10.0 software is shown in Figure 8.
  • the enzymolysis solution of 1.4.5 (amino acid content is 66.35g/kg) was further enzymatically digested with acid protease.
  • the results are shown in Table 9 and Table 10.
  • the acid protease After further enzymolysis, the amino acid type of the enzymolysis solution did not change, and 17 kinds of amino acids were detected.
  • the content of free amino acids released after further enzymolysis increased to 101.23g/kg, reaching the relevant technical indicators of the national liquid products containing amino acid water-soluble fertilizers.
  • the content of small peptide is 93.94g/kg, and the enzymatic hydrolysis rate of feather is 80.19%.
  • the step-by-step compound enzymolysis method can be widely used in the processing of waste feathers to produce amino acid liquid fertilizer.

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Abstract

一种利用废弃羽毛生产氨基酸液肥的方法。它是将羽毛粉用复合酶酶解，得到酶解液，然后向酶解液中加入酸性蛋白酶酶解，酶解完成后过滤，滤液经灭酶后得到氨基酸液肥；所述的复合酶包括角蛋白酶和氨基酸肽酶。该方法可以使羽毛酶解率达到80％以上，制备得到的氨基酸液肥含有17种氨基酸，种类齐全，且氨基酸含量能达到10.12％（质量分数）以上，小肽含量达到9.39％（质量分数），无需浓缩即达到国家氨基酸液肥标准，从而可以制备环境友好型氨基酸液肥。

Description

一种利用废弃羽毛生产氨基酸液肥的生物制方法 技术领域
本发明属于肥料加工技术领域，具体公开了一种利用角蛋白酶、中性蛋白酶、碱性蛋白酶、氨基酸肽酶及酸性蛋白酶分步复合酶解废弃羽毛制备氨基酸液体肥料的方法。
背景技术
羽毛是家禽处理后剩余的废弃物，在全世界的范围内，每年有数百万吨废弃羽毛产生，而资源化利用程度不到20％。这些羽毛除少量作为保暖衣填充料和动物饲料外，大部分被当作废弃物进行处理，不仅造成资源的浪费，且传统的焚烧、填埋等处理方法还会对环境造成严重的污染。因此，羽毛废弃物的管理问题成了家禽屠宰企业亟待解决的问题。然而羽毛废弃物是一种丰富的天然蛋白质资源，其粗蛋白含量高达85％～90％，羽毛蛋白中除赖氨酸、蛋氨酸的含量明显较低外，其它动物必需氨基酸的组成略高于鱼粉，这意味着利用蛋白质水解生产氨基酸类相关产品具有重大潜在的经济效益。禽类羽毛中的蛋白质主要由角质蛋白构成，角蛋白中含有较多的胱氨酸，它们之间形成大量的二硫键，再加上氢键和分子间作用力很难被普通蛋白酶(如碱性蛋白酶、胰蛋白酶、胃蛋白酶、木瓜蛋白酶等)水解。利用高温高压蒸煮水解、膨化法、酸碱水解和利用氧化剂或者还原剂等传统方法加工处理废弃羽毛，存在固定投资大，资金回收期长、反应条件剧烈，对生产设备要求较高且能耗大、产物极易发生构型变化或外消旋化，从而造成产物成分极为复杂和氨基酸破坏严重、产物盐分高、对环境污染严重等问题，而利用现代生物技术能很好的解决这些问题，如利用角蛋白酶对羽毛角蛋白进行降解，能降低能耗以及对环境的污染问题，并且提高水解产物的营养价值，形成以小肽和氨基酸为主要成分的酶解产物，具有巨大的小分子肽功能性饲料和氨基酸液肥潜力。
氨基酸具有调节和刺激作物生长发育(促进种子萌发及生长、促进根系生长、调节根系形态、促进叶片生长及提高叶绿色含量等)、改善作物品质、提高作物产量，提高作物的抗病及抗逆能力，还具有促进作物对其他营养成分如氮、磷、钾养分的吸收。氨基酸可作为生物刺激素、氨基酸态氮肥等单独使用，也可以作为生物活性增效剂与化学肥料配合施用(如氨基酸液肥)，发挥多重增效作用。因此，在农业上，氨基酸的应用越来越广泛。现在普遍的生产方法为对蛋白原料用酶或酸水解的方法获得氨基酸原液，或对氨基酸发酵尾液进行浓缩。而这几种方法均存在一定的缺点，如利用酸水解法，对环境污染较大，且氨基酸原液中氯盐含量较高，对某些氯敏感植物产生毒害作用；而利用酶法水解，由于酶 解原料及工艺的原因，酶解液中氨基酸含量较低，需进行浓缩来提高氨基酸原液的浓度，成本较高；氨基酸尾液浓缩法同样存在成本较高，浓缩产物中钠离子浓度过高，有时会造成盐害等问题。
发明内容
本发明的目的是针对上述问题提供一种利用废弃羽毛生产氨基酸液肥的生物制方法。本发明拟利用复合酶分步酶解废弃羽毛，使其酶解液中氨基酸浓度达到10％以上，无需浓缩即达到国家氨基酸液肥标准，制备环境友好型氨基酸液肥。
本发明的利用废弃羽毛生产氨基酸液肥的方法，其特征在于，包括以下步骤：
将羽毛粉用复合酶酶解，得到酶解液，然后向酶解液中加入酸性蛋白酶酶解，酶解完成后过滤，滤液经灭酶后得到氨基酸液肥；
所述的复合酶包括角蛋白酶和氨基酸肽酶。
所述的氨基酸肽酶指的是肽酶。
优选，所述的复合酶包括角蛋白酶、中性蛋白酶、碱性蛋白酶和氨基酸肽酶。
进一步优选，所述的复合酶按酶活占比计，包括角蛋白酶34～66％、碱性蛋白酶5～56％、中性蛋白酶5～56％和氨基酸肽酶5～56％。
进一步优选，所述的复合酶按酶活占比计，包括角蛋白酶45.6％、碱性蛋白酶11.8％、中性蛋白酶11.6％和氨基酸肽酶31.0％。
优选，所述的羽毛粉是将废弃羽毛除杂洗净，经高温蒸煮或亚硫酸钠处理后，经风干或烘干，然后粉碎碾磨，得到羽毛粉。高温蒸煮或亚硫酸钠处理能够有助于蛋白酶的酶解。进一步优选所述的高温蒸煮是羽毛在135℃，0.4MPa条件下蒸煮30min。
所述的粉碎碾磨是粉碎碾磨过1mm孔径筛，得到羽毛粉。
优选，所述的用复合酶酶解是羽毛粉加入到水中，使得羽毛粉的质量分数为30％以上，调pH值至8.5～9.5，灭菌后然后加入复合酶，复合酶是按照9000U/g羽毛粉以上加入，酶解温度是53～60℃，酶解36h以上。
优选，所述的用复合酶酶解是羽毛粉按质量比3:10加入到水中，调pH值至9，灭菌后然后加入复合酶，复合酶是按照9000U/g羽毛粉加入，酶解温度是53℃，酶解36h。
优选，所述的酸性蛋白酶酶解，其添加量为2000U/g羽毛粉，酶解条件为40℃，酶解12h。
优选，所述的灭酶为90℃，10min。
优选，所述的羽毛为各种家禽，例如鸡、鸭、鹅的羽毛。
与现有技术相比，本发明具有如下有益效果：
本发明的方法，可以使羽毛酶解率达到80％以上，制备得到的氨基酸液肥含有17种氨基酸，种类齐全，且氨基酸含量能达到10.12％(质量分数)以上，小肽含量达到9.39％(质量分数)，无需浓缩即达到国家氨基酸液肥标准，从而可以制备环境友好型氨基酸液肥。本发明的制备方法可进行工业化生产，工艺简单且稳定，能省去传统氨基酸液肥制备工艺中浓缩过程，大大减少能耗及污染；且具有原料来源广泛、价格低廉、产品氨基酸种类齐全等优点。本发明为一种环境友好型氨基酸液肥生产技术。
附图说明
下面结合附图对本发明作进一步的说明。
图1是不同蛋白酶组成对羽毛酶解率(折线)及氨基酸含量(柱状图)的影响。
图2是各蛋白酶交互作用影响氨基酸含量等高线图。
图3是复合酶的不同添加量对羽毛酶解率(折线)及氨基酸含量(柱状图)的影响。
图4是体系pH对羽毛酶解率(折线)及氨基酸含量(柱状图)的影响。
图5是酶解温度对羽毛酶解率(折线)及氨基酸含量(柱状图)的影响。
图6是底物浓度对羽毛酶解率(折线)及氨基酸含量(柱状图)的影响
图7是酶解时间对羽毛酶解率(折线)及氨基酸含量(柱状图)的影响
图8是不同酶解条件交互作用影响氨基酸含量的曲面图
具体实施方式
以下结合说明书附图和具体实施例进一步解释本发明，但不对本发明构成任何限定。以下实施例中除特殊说明外，均为本领域常规试剂和方法步骤。
实施例1：
1.1材料与试剂
羽毛：羽毛由广东博沃特生物科技有限公司提供，属于鸡羽毛，经彻底清洗烘干备用。
角蛋白酶(10万U/g)，自主生产；中性蛋白酶(10万U/g)、碱性蛋白酶(10万U/g)、脂肪酶(20万U/g)、木瓜蛋白酶(10万U/g)，仰韶生化科技有限公司；氨基酸肽酶(10万U/g，外肽酶)安琪酵母股份有限公司；酸性蛋白酶(5万U/g)，源叶生物科技有限公司；NaOH、HCl、柠檬酸钠、磺基水杨酸、三氯乙酸、硫酸钾、EDTA-Na等试剂为分析纯；混合氨基酸标准溶液，色谱纯。
1.2仪器与设备
统筹实验室仪器、氨基酸自动分析仪、凯氏定氮仪、离心机等
1.3方法
1.3.1工艺流程
收集羽毛——洗净——0.4MPa蒸煮30min(预处理)——烘干——粉碎——过1mm孔径筛——第一步酶解——第二步酶解——过滤(2层纱布)——灭酶(90℃，10min)——冷却——包装
1.3.2游离氨基酸测定
按含氨基酸水溶肥料农业行业标准(NY/T 1975-2010)中的方法测定，使用L-8900全自动氨基酸分析仪进行测定。
1.3.3肽含量测定：
按大豆肽粉国家标准(GB/T 22492-2008)中的方法测定肽含量。
1.3.4酶解率测定
酶解液经300目孔径筛过滤，105℃烘干至恒重，计算酶解率。
羽毛酶解率(％)＝(羽毛添加量-羽毛残渣干重)/羽毛添加量×100％。
1.3.5羽毛最佳预处理方式的确定
采用高温蒸煮处理、超声波处理、微波处理、亚硫酸钠处理5种方式对羽毛进行预处理，然后烘干粉碎，再用角蛋白酶对其进行酶解，确定羽毛预处理的最佳方式。
具体处理方式为：
收集废弃羽毛，将羽毛除杂洗净，经预处理，自然风干，然后粉碎碾磨过1mm孔径筛，得到羽毛粉。
其中以高温蒸煮(将羽毛在135℃，0.4MPa条件下蒸煮30min)作为预处理方式后得到的羽毛粉进行以下的各种实验。
1.3.6最佳蛋白酶组成的选择
固定总酶添加量为4500U/g羽毛粉，各酶等酶活比添加，选择共计13种蛋白酶组成分别对羽毛粉进行酶水解，具体组成见表1。水解条件为：底物质量浓度为10g/100mL(即将10g羽毛粉加入到100ml水中)，加酶量为4500U/g底物，在45℃、pH 8.0、180rpm条件下酶解3d，以游离氨基酸含量为主要评价指标，以羽毛酶解率为辅助评价指标，筛选出酶解羽毛粉制备氨基酸相关产品的最佳蛋白酶组成，每组3个重复。
表1不同复合酶组成成分表
Figure PCTCN2019096125-appb-000001
1.3.7复合酶配比的选择
根据筛选的最佳蛋白酶组成，采用混料设计(mixture)，根据单纯型重心实验设计原理(Simplex Centroid)，利用软件Design-Expert 10对角蛋白酶、碱性蛋白酶、中性蛋白酶、氨基酸肽酶4种蛋白酶配比进行优化。根据前期实验结果，确定复合酶的组分以及用量范围，组分及其范围见表2。以游离氨基酸含量为主要评价指标，以羽毛酶解率为辅助评价指标。最后对优化复合酶配方进行3次验证试验。
表2各组分考察范围设置表
Figure PCTCN2019096125-appb-000002
1.3.8酶解单因素试验
1.3.8.1复合蛋白酶用量对酶解的影响
利用筛选出的复合蛋白酶组成及其配比，在底物质量浓度为10g/100mL，酶解温度为45℃、酶解反应体系为pH8.0、180rpm条件下酶解3d，考察复合酶添加量(1500、3000、4500、6000、7500、9000、10500U/g羽毛粉)对羽毛粉酶解的影响，以游离氨基酸含量为主要评价指标，以羽毛酶解率为辅助评价指标，筛选出酶解羽毛粉制备氨基酸相关产品的最佳复合蛋白酶添加量，每组3个重复。
1.3.8.2体系PH对酶解的影响
利用筛选出的复合蛋白酶组成及其配比，在底物质量浓度为10g/100mL，酶解温度为45℃、复合酶添加量为4500U/g羽毛粉、180rpm条件下酶解3d，考察体系pH(6.0、6.5、7.0、7.5、8.0、8.5、9.0)对羽毛粉酶解的影响，以游离氨基酸含量为主要评价指标，以羽毛酶解率为辅助评价指标，筛选出酶解羽毛粉制备氨基酸相关产品的最佳复合蛋白酶添加量，每组3个重复。
1.3.8.3酶解温度对酶解的影响
利用筛选出的复合蛋白酶组成及其配比，在底物质量浓度为10g/100mL，酶解反应体系为pH 8.0、复合酶添加量为4500U/g羽毛粉、180rpm条件下酶解3d，考察酶解温度(35、40、45、50、55、60℃)对羽毛粉酶解的影响，以游离氨基酸含量为主要评价指标，以水以羽毛酶解率为辅助评价指标，筛选出酶解羽毛粉制备氨基酸相关产品的最佳复合蛋白酶添加量，每组3个重复。
1.3.8.4底物浓度对酶解的影响
利用筛选出的复合蛋白酶组成及其配比，在酶解温度为45℃，酶解反应体系为pH 8.0、复合酶添加量为4500U/g羽毛粉、180rpm条件下酶解3d，考察底物浓度(5％、10％、15％、20％、25％、30％、35％，底物浓度指的是每100g水中含有底物的质量，例如5％指的是100g水中含有5g羽毛粉)对羽毛粉酶解的影响，以游离氨基酸含量为主要评价指标，以羽毛酶解率为辅助评价指标，筛选出酶解羽毛粉制备氨基酸相关产品的最佳复合蛋白酶添加量，每组3个重复。
1.3.8.5酶解时间对酶解的影响
利用筛选出的复合蛋白酶组成及其配比，在底物质量浓度为10g/100mL，酶解温度为45℃，酶解反应体系为pH 8.0、复合酶添加量为4500U/g羽毛粉、180rpm条件下酶解，考察酶解时间(12、24、36、48、60、72、84)对羽毛粉酶解的影响，以游离氨基酸含量为主要评价指 标，以羽毛酶解率为辅助评价指标，筛选出酶解羽毛粉制备氨基酸相关产品的最佳复合蛋白酶添加量，每组3个重复。
通过上述实验，确定最佳单因素水平。
1.3.9氨基酸含量响应面优化实验
根据单因素试验结果，采用响应面设计实验，运用根据Box-Benhnken的中心组合实验设计原理，选取对氨基酸含量影响显著的因素，酶解温度、体系pH、底物浓度和酶用量4因素做3水平的响应面分析实验。最后对优化的酶解条件进行3次验证试验。
表3相应面优化实验因素与水平
Figure PCTCN2019096125-appb-000003
1.3.10酸性蛋白酶第二步酶解实验
根据响应面优化的最佳酶解条件，利用复合酶在此条件下酶解羽毛得到初步酶解液。根据所选酸性蛋白酶的最适酶解条件，酸性蛋白酶添加量为2000U/g羽毛，pH自然(初步酶解液pH为酸性)，酶解温度为40℃，180rpm酶解12h。
1.4结果与分析
1.4.1羽毛预处理方式的选择
表4不同预处理方式对羽毛酶解率及氨基酸含量的影响
Figure PCTCN2019096125-appb-000004
由表4可知，预处理方式会影响羽毛的酶解效果。采用亚硫酸对羽毛进行预处理然后进 行酶解，酶解率最高，同时所得的羽毛酶解液中氨基酸含量最高，与高温高压蒸煮处理不存在显著性差异，但与超声波处理、微波处理、加热处理及超低温处理存在显著性差异。分析原因为，羽毛中蛋白主要为角蛋白，其中大量存在的二硫键是导致其很难被普通蛋白酶降解的原因。而亚硫酸钠为还原剂，有助于打断角蛋白质中二硫键，促进酶解。
1.4.2复合酶组成的选择
由于羽毛角蛋白的特殊结构，基本不能被普通蛋白酶(如单独用氨基酸肽酶无法降解)所降解，所以复合酶的组成均以角蛋白酶为基础。由图1可知，就酶解液中氨基酸含量而言，以角蛋白酶、碱性蛋白酶、中性蛋白酶和氨基酸肽酶组成的复合蛋白酶效果最好，同时以角蛋白酶为基础的复合酶均优于单一角蛋白酶，特别是角蛋白酶+氨基酸肽酶的组合，而且在一定范围内，蛋白酶的种类越多效果越好，这是因为每一种蛋白酶特异性酶切位点有专一性，当这类位点已经全部断裂以后，即使蛋白酶没有完全失活，或补充蛋白酶，水解度也难以提高。本实验的目的是为了开发氨基酸叶面肥，属于深度酶解。因此，为了进一步提高提高羽毛角蛋白的水解度，增加氨基酸含量，利用不同蛋白酶的特异性酶切位点不同，采用多酶协同酶解，增加蛋白酶的特异性酶切位点，可有效提高氨基酸含量。就羽毛角蛋白酶解率而言，同样是以角蛋白酶、碱性蛋白酶、中性蛋白酶和氨基酸肽酶组成的复合蛋白酶效果最好。综合考虑羽毛酶解率以及酶解液中氨基酸含量，选择角蛋白酶、碱性蛋白酶、中性蛋白酶和氨基酸肽酶对羽毛进行复合酶解。
1.4.3复合酶配比的选择
混料实验设计优化复合酶配比试验，设计为4因素，共进行18次试验。实验安排与结果见表5。采用Design-Expert 10.0对实验结果数据统计及回归分析，得到回归方程为：氨基酸含量(g/kg)＝1.158A+0.059B+0.059C-6.2249 D+49.096AB+49.096A C+69.098AD+37.447BD+37.447CD。
表5单纯型重心设计实验表及结果
Figure PCTCN2019096125-appb-000005
Figure PCTCN2019096125-appb-000006
表6氨基酸含量回归模型方差分析表
Figure PCTCN2019096125-appb-000007
由表6可知，此模型的P<0.0001，表明此回归模型到达极其显著水平；相关系数R 2＝0.9842，说明该回归方程回归效果比较好，该模型能够解释98.42％的总变异，仅有1.58％变异无法用该模型解释。模型决定系数越大表示该模型预测值与实测值拟合越好，该系数必须大于0.80。模型Adj.R 2＝0.9702表明模型是显著的。通过比较方程一次项系数绝对值的大小，可以判断各因素对响应值影响的主次性。由方程可知，各组分酶对氨基酸含量的贡献程度如下：D(氨基酸肽酶)>A(角蛋白酶)>B(碱性蛋白酶)＝C(中性蛋白酶)。由表6可知，交互作用AB、AC、AD、BD、CD是极显著的，表明其相互配比可以显著影响氨基酸含量。
根据评价指标的目标范围和组分配比范围，优化出12个符合要求的配方以及相应指标的预测值。为方便实际操作，本文选取的复合酶配比(按酶活占比)为：角蛋白酶，45.6％；碱性蛋白酶，11.8％；中性蛋白酶，11.6％％；氨基酸肽酶，31.0％。在此条件下预测的氨基酸含量达到16.34g/kg。如图2所示。为验证模型预测的可靠性，在最佳复合酶配比下进行3次验证试验(具体是将底物质量浓度为10g/100mL(即将10g羽毛粉加入到100ml水中)，添加复合酶加酶量为4500U/g底物，复合酶配比(按酶活占比)为：角蛋白酶，45.6％；碱性蛋白酶，11.8％；中性蛋白酶，11.6％％；氨基酸肽酶，31.0％，在45℃、pH 8.0、180rpm条件下酶解3d)，测得的氨基酸含量为16.54g/kg，与预测值基本接近，偏差为1.25％。
1.4.4单因素实验
1.4.4.1复合蛋白酶用量对酶解的影响
由图3可知，就氨基酸含量而言，随着复合酶的添加量的增加，酶解液中氨基酸含量逐渐升高，当复合酶的添加量达到9000U/g羽毛时，氨基酸含量达到最大值29.30g/kg。随着复合酶含量继续增加，氨基酸含量略微降低。就羽毛酶解率而言，随着复合酶的添加量的增加，羽毛酶解率逐渐升高，当复合酶的添加量超过9000U/g羽毛时，酶解率增加的速率减慢。这是因为蛋白酶进行反应时首先与底物形成酶-底物复合物。当底物浓度远远大于酶浓度时，酶浓度与酶反应速率成正比，如果此时增加酶浓度，可增加反应速率。因此，在一定的反应时间内，蛋白质的酶解效率越高，同时其氨基酸转化率和蛋白质酶解率也越高。但是加酶量越大，可能导致蛋白酶自身相互水解，使之酶活力下降，同时生产成本也会越高。因此，综合考虑羽毛酶解率、氨基酸含量以及生产成本，确定复合酶的最适添加量为9000U/g羽毛。
1.4.4.2体系pH对酶解的影响
由图4可知，就氨基酸含量而言，随着pH 6.5～7.0区间内随着体系pH的增加，酶解液中氨基酸含量呈递增趋势，pH 7.0时氨基酸含量达到11.91g/kg，在pH 7.0～9.0区间内，氨基酸含量呈先降低后上升的趋势，在pH 9.0时氨基酸含量达到最大值12.92g/kg，当pH再增加 时氨基酸含量呈略微递减趋势。同时，羽毛酶解率呈现相似的变化趋势，在pH 9.0时，羽毛酶解率达到最大值85.65％。分析原因为，当反应体系pH偏高或偏低时，蛋白酶活力部分带电情况发生变化，蛋白酶与底物的结合能力降低，从而使酶活力下降。每种酶都有特定的最适pH，复合酶中含有中性蛋白酶，在pH 7.0时，复合酶中的中性蛋白酶表现更强的酶活，这可能是复合酶在pH 7.0时出现小峰值的原因之一。因此，综合考虑羽毛酶解率和氨基酸含量，确定复合酶的最适酶解体系pH为9.0。
1.4.4.3酶解温度对酶解的影响
由图5可知，随着酶解温度的升高，羽毛酶解率和酶解液中氨基酸含量呈先递增后降低的趋势，55℃时复合酶的羽毛酶解率(87.86％)和酶解液中氨基酸含量(31.66g/kg)达到最大值。这是因为蛋白酶属于一种蛋白质类生物催化剂，受温度影响较大，在一定的温度范围内，催化反应速率随温度的上升而增加，但温度过高会导致酶变性，使催化活性降低。因此，综合考虑羽毛酶解率和氨基酸含量，确定复合酶的最适酶解温度为55℃。
1.4.4.4底物浓度对酶解的影响
由图6可知，就酶解液中氨基酸含量而言，随着底物浓度的增大，氨基酸含量呈先上升后下降的趋势。在底物浓度为30g/100mL时，氨基酸含量最高，达到29.58g/kg；当底物浓度继续增大时，氨基酸含量反而略微下降，可能是因为由于过高的底物浓度从而降低了酶解体系中有效水浓度，从而降低了分子的扩散和运动，从而对水解反应产生抑制，另一方面酶解体系中过高浓度的蛋白质底物可能与蛋白酶形成无活性的中间产物，从而抑制水解反应，是的酶解液中氨基酸含量反而下降。就羽毛酶解率而言，随着底物浓度的增大，羽毛酶解率持续降低，当底物浓度超过25g/100mL时，羽毛酶解率降低速率显著增大。在底物浓度为5g/100mL时，羽毛酶解率最高为90.45％。考虑实验目的为开发氨基酸叶面肥，而氨基酸的浓度过低是限制酶解工艺的一个重要因素，因此，确定其复合酶解的最适底物浓度为30g/100mL。
1.4.4.5酶解时间对酶解的影响
由图7可知，随着酶解时间的延长，酶解率及氨基酸含量均呈增长趋势，在0-36h内，酶解率及氨基酸含量呈快速增长趋势，当酶解时间为36h时，酶解率及氨基酸含量分别为82.98％和12.06g/kg。当酶解时间超过36h，酶解率及氨基酸含量呈缓慢增长趋势。分析原因为，随着酶解时间延长，一方面，可作用的有效肽键浓度降低从而导致角蛋白水解作用减弱、水解程度放缓；另一方面，酶解产物中肽分子竞争作为蛋白酶的作用底物，从而使酶解速率放缓；此外，随着酶解的进行，体系pH发生降低，同时蛋白酶活力下降，从而使酶解速率放缓。 因此，综合考虑酶解时间所需的生产成本、羽毛酶解率和氨基酸含量，确定其复合酶解的最佳时间为36h。
1.4.5响应面优化
表7相应面分析方案及实验结果
Figure PCTCN2019096125-appb-000008
Figure PCTCN2019096125-appb-000009
表8氨基酸含量回归模型方差分析表
Figure PCTCN2019096125-appb-000010
利用Design Expert 10.0软件对所得数据进行回归分析，得到的回归方程为：
氨基酸含量(g/kg)＝64.01+2.41A-2.81B-0.13C+3.14D+1.07AB-2.28AC-1.57AD-1.53BC+ 1.09BD-2.21A 2-2.79B 2-5.89C 2-6.07D 2。利用Design Expert 10.0软件得到的曲面图如图8所示。
由表8可知，此模型的P＝0.0001，表明响应面回归模型达到极显著水平；相关系数R 2＝0.9058，说明该回归方程回归效果比较好，实验误差小。在酶解时间为36h的条件下，经响应面分析得出复合酶酶解羽毛的最优条件为：9623U/g羽毛粉、酶解温度53℃、体系pH8.96，羽毛底物浓度为30.85％，在此条件下预测的氨基酸含量达到65.319g/kg。为了检验该模型预测的可靠性，并根据实际操作的方便性，将最佳酶解条件略微修改为复合酶(按酶活占比角蛋白酶45.6％；碱性蛋白酶11.8％；中性蛋白酶11.6％％；氨基酸外肽酶31.0％)9600U/g羽毛粉、酶解温度53℃、体系pH9.0，羽毛底物浓度为30％(即100ml水中含有30g羽毛粉)，酶解时间为36h，在此条件下进行了3次验证试验，测得酶解液中氨基酸含量达到66.35g/kg。
1.4.6第二步酶解实验及酶解液组分分析
对1.4.5的酶解液(氨基酸含量为66.35g/kg)用酸性蛋白酶进行进一步酶解，其结果如表9和表10所示，从表9和表10可以看出，经过酸性蛋白酶的进一步酶解，酶解液的氨基酸种类未发生变化，都检测出17种氨基酸，进一步酶解后释放的游离氨基酸含量增加，达到101.23g/kg，达到国家含氨基酸水溶肥料液体产品相关技术指标，小肽含量93.94g/kg，羽毛酶解率为80.19％。
表9酸性蛋白酶酶解液成分分析表
Figure PCTCN2019096125-appb-000011
表10酸性蛋白酶酶解液氨基酸成分分析表
Figure PCTCN2019096125-appb-000012
Figure PCTCN2019096125-appb-000013
综上所述，该分步复合酶解方法可广泛用于废弃羽毛生产加工氨基酸液肥的加工过程。

Claims (10)

  1. 一种利用废弃羽毛生产氨基酸液肥的方法，其特征在于，包括以下步骤：
    将羽毛粉用复合酶酶解，得到酶解液，然后向酶解液中加入酸性蛋白酶酶解，酶解完成后过滤，滤液经灭酶后得到氨基酸液肥；
    所述的复合酶包括角蛋白酶和氨基酸肽酶。
  2. 根据权利要求1所述的方法，其特征在于，所述的复合酶包括角蛋白酶、中性蛋白酶、碱性蛋白酶和氨基酸肽酶。
  3. 根据权利要求2所述的方法，其特征在于，所述的复合酶按酶活占比计，包括角蛋白酶34～66％、碱性蛋白酶5～56％、中性蛋白酶5～56％和氨基酸肽酶5～56％。
  4. 根据权利要求3所述的方法，其特征在于，所述的复合酶按酶活占比计，包括角蛋白酶45.6％、碱性蛋白酶11.8％、中性蛋白酶11.6％和氨基酸肽酶31.0％。
  5. 根据权利要求1所述的方法，其特征在于，所述的羽毛粉是将废弃羽毛除杂洗净，经高温蒸煮或亚硫酸钠处理后，经风干或烘干，然后粉碎碾磨，得到羽毛粉。
  6. 根据权利要求5所述的方法，其特征在于，所述的高温蒸煮是羽毛在135℃，0.4MPa条件下蒸煮30min。
  7. 根据权利要求1、2、3或4所述的方法，其特征在于，所述的用复合酶酶解是羽毛粉加入到水中，使得羽毛粉的浓度为30g/100g水以上，调pH值至8.5～9.5，灭菌后然后加入复合酶，复合酶是按照9000U/g羽毛粉以上加入，酶解温度是53～60℃，酶解36h以上。
  8. 根据权利要求7所述的方法，其特征在于，所述的用复合酶酶解是羽毛粉按质量比3:10加入到水中，调pH值至9，灭菌后然后加入复合酶，复合酶是按照9000U/g羽毛粉加入，酶解温度是53℃，酶解36h。
  9. 根据权利要求1、2、3或4所述的方法，其特征在于，所述的酸性蛋白酶酶解，其添加量为2000U/g羽毛粉，酶解条件为40℃，酶解12h；所述的灭酶为90℃，10min。
  10. 根据权利要求1所述的方法，其特征在于，所述的羽毛为鸡、鸭和/或鹅的羽毛。
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