阅读说明：本技术 一种利用微生物或酶发酵或转化葵粕和葵壳增产黑色素的方法 (Method for increasing melanin production by fermenting or converting sunflower meal and sunflower shells by using microorganisms or enzymes ) 是由 陈育如 王屹东 王鸿飞 江慧玲 曾茜 钱者东 罗海波 于 2020-06-23 设计创作，主要内容包括：本发明公开了一种利用微生物或酶发酵或转化葵粕和葵壳增产黑色素的方法,葵粕和葵壳混合或分别加入微生物或酶,经发酵或转化产黑色素。本发明利用微生物对葵粕和葵壳发酵或酶转化深加工可大大增加黑色素的产量和提取效率。(The invention discloses a method for increasing melanin production by fermenting or converting sunflower meal and sunflower shells by using microorganisms or enzymes. The invention can greatly increase the yield and extraction efficiency of melanin by utilizing the fermentation or enzyme conversion deep processing of the microorganism sunflower meal and sunflower shell.)

1. A method for increasing melanin production by fermenting or converting sunflower meal and sunflower shells by using microorganisms or enzymes for producing polyphenol oxidase or cellulase, hemicellulase and the like, is characterized by comprising the following steps: mixing sunflower meal and sunflower shell, or adding microorganism or enzyme respectively, fermenting or converting to produce melanin.

2. The method for increasing melanin production by fermenting or converting sunflower meal and sunflower hulls with microorganisms or enzymes according to claim 1, wherein: the microorganism is Aspergillus niger (Aspergillus niger) and variants thereof.

3. The method for increasing melanin production by fermenting or converting sunflower meal and sunflower hulls with microorganisms or enzymes according to claim 1, wherein: the microorganism is Asterina coronaria (Eurotium cristatum) and its variant.

4. The method for increasing melanin production by fermenting or converting sunflower meal and sunflower hulls with microorganisms or enzymes according to claim 1, wherein: the microorganism is Trichoderma (Trichoderma spp.) and its variants.

5. The method for increasing melanin production by fermenting or converting sunflower meal and sunflower hulls with microorganisms or enzymes according to claim 1, wherein: the microorganism is Aspergillus oryzae (Aspergillus oryzae) and its variants.

6. The method for increasing melanin production by fermenting or converting sunflower meal and sunflower hulls with microorganisms or enzymes according to claim 1, wherein: the microorganism is yeast (Saccharomyces) and its variants.

7. The method for increasing melanin production by fermenting or converting sunflower meal and sunflower hulls with microorganisms or enzymes according to claim 1, wherein: the microorganism is Neurospora (Neurospora) and its variant.

8. The method for increasing melanin production by fermenting or converting sunflower meal and sunflower hulls with microorganisms or enzymes according to claim 1, wherein: the enzyme is Catechol oxidase (Catechol oxidase) or enzyme yeast of Catechol oxidase.

9. The method for increasing melanin production by fermenting or converting sunflower meal and sunflower hulls with microorganisms or enzymes according to claim 1, wherein: the enzyme is tyrosine lyase (Tyrosinase) or a yeast of the tyrosine lyase.

10. The method for increasing melanin production by fermenting or converting sunflower meal and sunflower hulls with microorganisms or enzymes according to claim 1, wherein: the enzyme is Laccase (Lactase) or fermentation enzyme yeast such as Coriolus versicolor and Schizophyllum commune containing Laccase.

Technical Field

The invention relates to a production method of melanin, in particular to a method for increasing the yield of the melanin by fermenting or converting sunflower meal and sunflower shells by using microorganisms or enzymes.

Background

Natural melanin is the origin of black color expression of plants, animals, fungi, etc., is widely distributed in plants, animals, and microorganisms, and is the most widely present pigment of all known biological pigments. Melanin was first named by Berzelius, a scientific scientist in switzerland, is a macromolecular substance with a complex and diverse structure formed by polymerizing polyhydroxy indole or polyhydroxy phenolic substances, is usually combined with biological macromolecules in cells, is amorphous, is insoluble in water, acidic solution and most organic solvents, is easily soluble in alkaline solution, and can be tightly combined with macromolecules such as proteins, tannins and carbohydrates. Under aerobic conditions, organisms oxidize polyphenols into quinones by polyphenol oxidase, which polymerize to produce melanin, and the polyphenol oxidase can be divided into three main groups: monophenol monooxygenase (Tyrosinase, ec.1.14.18.1), diphenol oxidase (Catechol oxidase cathechoxide, ec.1.10.3.2) and Laccase (lacgase, ec.1.10.3.1). Of the three main classes of polyphenol oxidases, catecholases are distributed mainly in plants, and polyphenol oxidases in microorganisms mainly include laccases and tyrosinases. Most of the literature now discloses polyphenol oxidases which are generally known as catechol oxidases and laccases.

Among various natural pigments, melanin has excellent photo-thermal stability. With the continuous and intensive research on melanin, the bioactivity of melanin is more and more emphasized, including various bioactivities such as oxidation resistance, free radical scavenging, radiation resistance, aging resistance, immunity improvement and the like, and is widely applied in the fields of cosmetics, medicine, novel materials and the like (Zhouyu, Yi Dong Mei, Hu Wen fai and the like, research on physicochemical properties and functional characteristics of natural melanin advances [ J ] food industry science and technology, 2012, 33 (24): 445) 447). Synthetic melanin has a large toxic side effect, and biological melanin has a high safety factor, and is widely used for preparing safe and nontoxic melanin from microorganisms (Zou Y, Xie C, Fan G, et al. optimization of adsorbed-associated extraction of melanin from Auricularia auricula fruits [ J ]. Innovative Food Science and embedding Technologies, 2010, 11 (4): 0-615). The biological melanin can be used for human body antioxidation and immunoregulation, can also be used as a food or daily necessities additive, can be used for food coloring such as wines, beverages, candies and the like and in the cosmetic industry, and has great development potential and application prospect.

Biological melanin widely exists in plants, and is insoluble in water and organic solvents, and is a macromolecular substance with a complex structure. Melanin can be divided into two classes according to structure: one class is the end products of the metabolism of polyphenols and tyrosine and related compounds; another class is anthocyanins whose mother nucleus is a flavonoid. Plant melanin is mainly present in epidermal tissues of some black fruits or seeds, and currently, main research objects are black rice, black sesame, banana peel, prickly ash seed, apricot seed coat and the like.

Sunflower meal is a high-quality protein resource, and can be eaten and used as feed; sunflower shells are rich in cellulose, and black shell varieties are rich in melanin. The protein content of the hulled sunflower meal is higher than that of the soybean meal, the content of essential amino acids except lysine is higher than that of the soybean meal (Wang Jiu, Longqiang, Liehong, Zhang Bao sunflower meal and the nutritional value of the sunflower meal and the application in feed production [ J ]. Guangdong feed, 2010, 19 (01): 36-38), and compared with the soybean meal, the sunflower meal does not contain anti-nutritional factors which obviously influence the growth of animals, such as pepsin inhibition factor, oligosaccharide, lipoxygenase, phytohemagglutinin, saponin and the like (Xia Bright, xylonite, the nutritional value of the sunflower seed meal and the reasonable application of the sunflower seed meal in pig production [ J ]. feed research, 2019, 42 (04): 87-90). The sunflower meal and the sunflower shell are byproducts of sunflower seed oil production by using sunflower seeds, contain a large amount of protein, crude fiber and the like, are partially used as animal feed after rough processing, and have low additional value. In the prior art, deep processing of sunflower shells and sunflower meals is little, and reports of producing melanin by fermenting or enzymic methods of sunflower meals are not available.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a method for improving melanin yield and extraction efficiency by fermenting or converting sunflower meal and sunflower shells by using microorganisms or enzymes.

The technical scheme is as follows: the invention provides a method for increasing melanin production by fermenting or converting sunflower meal and sunflower shells by using microorganisms or enzymes. Enzyme is added or microorganism enzyme production is utilized to promote chlorogenic acid and other components in the sunflower meal to produce melanin, the sunflower shell improves the airtight defect of the sunflower meal, and the fermentation efficiency is improved. The sunflower meal presents black due to the oxidation blackening of chlorogenic acid and the like contained in the sunflower meal, and the sunflower meal is used as a culture medium for microbial fermentation, so that the content of melanin is increased, and the original melanin and the increased melanin are easier to extract. The sunflower meal has high protein content, is poor in ventilation when used as a fungus culture medium, can increase the ventilation of the culture medium after being mixed with the sunflower shells, and is beneficial to the growth of cultured microorganisms and the yield increase of melanin.

The microorganisms producing cellulase and the like can utilize sunflower shells as a culture medium, the sunflower shells are combined with sunflower meal and then fermented to increase the yield of melanin, and the melanin is obtained by extraction under alkaline conditions and the like and acid precipitation. The filtrate and residue left after extraction of melanin can be used as feed or fertilizer.

Further, the microorganism is Aspergillus niger (Aspergillus niger) and variants thereof.

Further, the microorganism is Asterina acutus (Eurotium cristatum) and its variant.

Further, the microorganism is Trichoderma (Trichoderma spp.) and its variants.

Further, the microorganism is aspergillus oryzae (asp. oryzae) and variants thereof.

Further, the microorganism is yeast (Saccharomyces) and its variant. Preferred are the melanin-producing yeasts, also known as Aureobasidium pullulans and variants thereof.

Further, the microorganism is Neurospora (Neurospora) and its variant. Preferably, the Neurospora for producing melanin is selected, the Neurospora hydrolyzes cellulose, etc., and the sunflower meal is fermented to promote melanin of phenolic acids, etc. and melanin in sunflower hull materials.

Further, the enzyme is catechol oxidase or a catechol oxidase enzyme koji.

Further, the enzyme is a tyrosine lyase or a tyrosine lyase koji.

Further, the enzyme is Laccase (Lactase) or fermentation enzyme yeast such as Coriolus versicolor and Schizophyllum commune containing Laccase.

Tyrosinase (Tyrosinase) plays an important role in melanin biosynthesis, is the main rate-limiting enzyme in the process of converting tyrosine (Tyr) and dopa to melanin, and is widely distributed in microorganisms, animals, plants and human bodies. In microorganisms and human bodies, tyrosine is oxidized into dopaquinone under the catalytic action of tyrosinase; dopaquinone is automatically oxidized to generate DOPA and dopachrome, DOPA is also a substrate of tyrosinase, and is catalyzed to generate dopaquinone again, and dopaquinone enters DOPA to finally form melanin. In plants, a large number of phenolic compounds are available as substrates, and tyrosinase oxidizes tissues to brown when they are damaged. The DOPA synthesis pathway of microbial melanin is as follows:

catecholamine (cathecoxidase), also known as cresol enzyme, tyrosinase, phenol oxidase, catecholamine, and the like, is widely present in plant organs or tissues, is not strictly expressed in specific tissue parts of plants, and its content and activity are also changed accordingly as plants grow. When plant tissues are exposed to the air, catechol oxidase exists in the plant tissues to catalyze the oxidation of various phenols (catechol, tannic acid, tyrosine and the like) to form quinones, and the quinones are polymerized into melanin, so that the tissue color is gradually deepened from brown to black.

Laccase (lacgase) acts as a copper-containing polyphenol oxidase enzyme that is widely present in fungi, bacteria, insects and plants. Unlike tyrosinase and catechol oxidase, laccase does not catalyze tyrosine reactions and can catalyze phenolic compounds and their derivatives to produce the corresponding benzoquinones and water. Laccase catalyzes substrates in a wide range and low specificity, and can catalyze substances such as ascorbic acid, phenylenediamine and the like to be oxidized besides phenolic substances. Laccase catalyzes the oxidative polymerization of 3, 4-levodopa (L-DOPA) to form melanin.

Melanin produced by microorganisms is mainly divided into intracellular, extracellular and mural combined melanin. The microorganism passes through DOPA pathway, and tyrosine is catalyzed by tyrosinase in the form of DOPA, DOPA, quinone, indoloquinone, etc. Finally, melanin is produced. Many molds, bacteria and actinomycetes can produce melanin, and the microbial production and extraction of melanin has the advantages that the raw materials are not limited by regions and seasons, and the industrial production is easy. The fungus melanin is a heterogeneous polyphenol polymer widely existing on the mycelium cell walls of various fungi, has biological functions of photoprotection, radiation resistance, combination of metal ions, free radical removal, oxidation resistance and the like, and plays a vital role in improving the survival capability of the fungi in a stress environment.

Aspergillus niger (Aspergillus niger) is a filamentous fungus with a strong protein secretion ability, belonging to the phylum Ascomycota, Ascomycetes, Ascomycetales, Tricholomataceae, Aspergillus. Aspergillus niger has dark spore color, covered with a layer of melanin, plays a key role in stress resistance, and has the functions of resisting ultraviolet radiation, absorbing energy, changing hydrophobicity and static electricity.

Neurospora (Neurospora) is a member of the Ascomycota class, Ascomycetes order, Fagaceae family. It has loose netted long hypha with septum, branches and multiple cores, and is asexually propagated to form conidium, generally oval, with branch chain on the top of aerial hypha. The cellulase can degrade cellulose into cellobiose, glucose and other small molecular substances. Neurospora has melanin producing capacity.

The Eurotium cristatum, also known as Eurotium cristatum, is usually metabolized to produce a large amount of pigment substances during growth, the primary pigment is yellow pigment, and a large amount of melanin is produced along with the growth of culture time.

Black yeast is also known as Aureobasidium pullulans (Aureobasidium pullulans), a pleomorphic fungus with yeast-type and hyphal-type forms, and chlamydospores of the pleomorphic fungus can accumulate melanin.

Trichoderma spp is considered as one of the best cellulase-producing strains, and the cellulase system produced by Trichoderma spp is in a proper proportion and can effectively hydrolyze cellulosic materials.

Has the advantages that: the invention can greatly increase the yield of melanin by utilizing the fermentation or enzyme conversion deep processing of the sunflower meal and the sunflower shell by microorganisms, and compared with the method for simply extracting the sunflower meal, the yield of the melanin is improved by about 72 to 350 percent, and the extraction efficiency is improved by about 22 to 78 percent. The sunflower meal and the sunflower shells are fully utilized for fermentation, so that resources can be fully utilized, the production cost is reduced, and products such as melanin with high added value are obtained.

Detailed Description