A method for improving the yield of melanin in black fungus fermentation liquid and a black fungus strain

Abstract

The invention belongs to the technical field of biological fermentation, and specifically relates to a method for improving the yield of melanin in black fungus fermentation liquid and a black fungus strain. The invention provides a method for improving the yield of melanin in black fungus fermentation liquid, using tyrosine, salicylic acid and methyl jasmonate as inducers to induce fermentation culture of the black fungus strain, and combining solid induction culture and liquid induction culture to significantly improve the yield of melanin in the fermentation culture liquid.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and particularly relates to a method for improving the yield of melanin in black fungus fermentation liquid and a black fungus strain. Background Art

[0002] Natural melanin is a high molecular pigment with good biological activity formed by the oxidation polymerization of phenolic or indole compounds. It is widely found in plants, animals and microorganisms, especially in natural foods such as black sesame, black beans and black rice. Natural melanin has good biological activity and is mostly used in the production of foods such as beverages, cakes, chocolates or candies, and has great development value.

[0003] Black fungus is an important melanin source fungus with a high melanin content and high biological activity. There is no shortage of related research on the extraction of natural melanin from black fungus in the prior art. For example, Hou Ruolin et al. optimized the melanin extraction process from black fungus fruiting bodies, and the extraction rate of black fungus melanin can reach 10.48%. However, there are more colloids, polysaccharides and miscellaneous proteins in black fungus fruiting bodies, and it is difficult to extract melanin and the purity is not high. Black fungus mycelium fermentation has the advantages of fast fermentation speed and low cost. Zhang Min et al. prepared black fungus melanin by deep fermentation, and the yield of black fungus melanin can reach 170.37 mg·L -1 However, the melanin yield in the fermentation broth is still low and difficult to be applied in industrial production. In order to further improve the extraction yield of melanin in black fungus, it is very necessary for those skilled in the art to provide an induction method for improving the melanin yield in black fungus. Summary of the invention

[0004] The purpose of the present invention is to provide a method for improving the yield of melanin in black fungus fermentation broth. The method provided by the present invention can significantly improve the yield of melanin in black fungus fermentation broth.

[0005] The present invention provides a method for improving the yield of melanin in black fungus fermentation liquid, comprising the following steps: sequentially performing solid induction culture and liquid induction culture on the black fungus;

[0006] The solid induction culture medium comprises the following components: 90-110 g / L of potato, 3-8 g / L of glucose, 2.0-3.0 g / L of tyrosine, 0.05-0.2 g / L of potassium dihydrogen phosphate, 0.05-0.2 g / L of magnesium sulfate and 0.1-0.3 g / L of copper chloride;

[0007] The liquid induction culture medium comprises the following components: 17-19 mg / L tyrosine, 10-15 mg / L salicylic acid, 20-25 mg / L methyl jasmonate, 8-12 g / L peptone, 3.0-7.0 g / L wheat bran juice, 1.0-3.0 g / L soybean powder juice, 0.3-0.8 g / L copper chloride, 1.0-2.0 g / L magnesium sulfate and 1.0-2.0 g / L potassium dihydrogen phosphate.

[0008] Preferably, the solid induction culture comprises: subjecting the activated black fungus strain to solid induction in a solid culture medium; the solid induction comprises sequentially performing constant temperature and light-proof culture, high temperature induction culture at 40 to 50° C., and recovery culture.

[0009] Preferably, the high temperature induction culture is carried out for 0.5 to 4 hours.

[0010] Preferably, the temperature of the constant temperature and light-proof culture and the recovery culture are both 23-30° C.; the duration of the constant temperature and light-proof culture is 8-12 days, and the duration of the recovery culture is 2-4 days.

[0011] Preferably, the liquid induction culture comprises:

[0012] The black fungus strain cultured on solid medium is subjected to liquid induction in liquid medium;

[0013] The liquid induction comprises sequentially performing fed-batch culture and 40-50° C. high-temperature induction culture.

[0014] Preferably, the number of high temperature induction culture is ≥1; after each high temperature induction culture, tyrosine is supplemented, and the concentration of tyrosine is 8-12 mmol / L; the volume ratio of tyrosine to the initial amount of the liquid culture medium is (0.8-1.2): (18-22).

[0015] Preferably, the fed-batch culture is carried out on the 5th to 9th day of the liquid induction culture, and the fed-batch culture includes supplementing the culture medium, and the culture medium includes tyrosine, salicylic acid and methyl jasmonate.

[0016] Preferably, the concentrations of tyrosine, salicylic acid and methyl jasmonate are all 8-12 mmol / L; the volume ratios of tyrosine, salicylic acid and methyl jasmonate to the initial amount of liquid culture medium are 1:50, (2-4): (80-120) and (0.8-1.2:18-22), respectively.

[0017] Preferably, the high temperature induction culture is carried out on the 3rd day after the fed-batch culture; the high temperature induction culture includes three times, each with an interval of 3 to 5 days, and each induction time is 1 to 3 hours.

[0018] The invention also provides a black fungus strain HW100, and the deposit number of the black fungus strain HW100 is CGMCC No.40192.

[0019] Beneficial effects:

[0020] The invention provides a method for improving the yield of melanin in black fungus fermentation liquid. Tyrosine, salicylic acid and methyl jasmonate are used as inducers to induce fermentation culture of black fungus strains, and solid induction culture and liquid induction culture are combined to significantly improve the yield of melanin in the fermentation culture liquid.

[0021] At the same time, the melanin obtained by the method provided by the present invention has higher antioxidant activity than conventional black fungus melanin.

[0022] The results of the embodiment show that the black fungus fermentation liquid obtained by the method of the present invention has a melanin yield of 430.43% higher in the supernatant after filtering the fermentation liquid than that in comparative example 1, and a melanin yield of 16.44% higher in the mycelium than that in comparative example 1.

[0023] Biodeposit Information:

[0024] Black fungus Auricularia auricularHW100 was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on May 19, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No.40192. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0026] Figure 1 This is a picture of the mycelium morphology before high temperature induction in the solid induction culture of Example 1;

[0027] Figure 2 This is a picture of the mycelium morphology after high temperature induction in the solid induction culture of Example 1;

[0028] Figure 3 This is the color picture of mycelium before high temperature induction in solid induction culture in Example 1;

[0029] Figure 4 This is the color picture of mycelium after high temperature induction in solid induction culture in Example 1;

[0030] Figure 5 The color change of mycelium in the liquid culture medium before and after the liquid induction culture in Example 1;

[0031] Figure 6 is the DPPH clearance rate of melanin in Example 1 and Comparative Example 1;

[0032] Figure 7 is the hydroxyl radical scavenging rate of melanin in Example 1 and Comparative Example 1;

[0033] Figure 8 is the ABTS clearance rate of melanin in Example 1 and Comparative Example 1;

[0034] Fig. 9 The present invention provides a technical roadmap for the method for increasing the yield of melanin in black fungus fermentation broth. DETAILED DESCRIPTION

[0035] The present invention provides a method for improving the yield of melanin in black fungus fermentation liquid, comprising the following steps: sequentially performing solid induction culture and liquid induction culture on the black fungus;

[0036] The solid induction culture medium comprises the following components: 90-110 g / L of potato, 3-8 g / L of glucose, 2.0-3.0 g / L of tyrosine, 0.05-0.2 g / L of potassium dihydrogen phosphate, 0.05-0.2 g / L of magnesium sulfate and 0.1-0.3 g / L of copper chloride;

[0037] The liquid induction culture medium comprises the following components: 17-19 mg / L tyrosine, 10-15 mg / L salicylic acid, 20-25 mg / L methyl jasmonate, 8-12 g / L peptone, 3.0-7.0 g / L wheat bran juice, 1.0-3.0 g / L soybean powder juice, 0.3-0.8 g / L copper chloride, 1.0-2.0 g / L magnesium sulfate and 1.0-2.0 g / L potassium dihydrogen phosphate.

[0038] Before the solid induction culture of the black fungus strain, the present invention preferably further comprises activating the black fungus strain to obtain an activated black fungus strain. The activated culture medium of the present invention is preferably a PDA culture medium, and the activation condition is a constant temperature of 28° C. and protected from light.

[0039] After obtaining the activated black fungus strain, the present invention performs solid induction culture on the activated black fungus strain to obtain a solid induced cultured black fungus strain. The solid induction culture of the present invention preferably includes: solid induction of the activated black fungus strain in a solid culture medium; constant temperature and light-proof culture, 40-50°C high temperature induction culture and recovery culture are performed on the solid culture medium in sequence. The temperature of the constant temperature and light-proof culture of the present invention is preferably 23-30°C, more preferably 28°C; the duration of the constant temperature and light-proof culture is preferably 8-12d, more preferably 10d. The present invention directly performs 40-50°C high temperature induction culture after the constant temperature and light-proof culture, and the temperature of the high temperature induction culture is preferably 42-49°C, more preferably 48°C; the time of the high temperature induction is preferably 0.5-4h, more preferably 0.8-3h, and most preferably 1-2h. The constant temperature and light-proof culture of the present invention has the effect of expanding the mycelium biomass. The present invention can perform recovery culture after the high temperature induction culture at 40-50°C. The temperature of the recovery culture is preferably 23-30°C, more preferably 28°C; the time of the recovery culture is preferably 2-4 days, more preferably 3 days. The high temperature induction culture in the solid induction culture of the present invention allows the strain to adapt to the environmental conditions of high temperature growth and induces the strain to metabolize and produce more melanin.

[0040] The culture medium for the solid induction culture of the present invention comprises the following components: 90-110 g / L of potato, 3-8 g / L of glucose, 2.0-3.0 g / L of tyrosine, 0.05-0.2 g / L of potassium dihydrogen phosphate, 0.05-0.2 g / L of magnesium sulfate and 0.1-0.3 g / L of copper chloride, preferably 100 g / L of potato, 5 g / L of glucose, 2.5 g / L of tyrosine, 0.1 g / L of potassium dihydrogen phosphate, 0.1 g / L of magnesium sulfate and 0.2 g / L of copper chloride. The tyrosine in the culture medium of the present invention is a substrate for melanin synthesis, and combined with the induction conditions of the present invention, black fungus can be induced to produce more melanin.

[0041] After obtaining the black fungus strain cultured in solid induction culture, the present invention inoculates the black fungus strain cultured in solid induction culture into a liquid culture medium for liquid induction culture to obtain a liquid induction culture solution, wherein the liquid induction culture solution includes melanin.

[0042] The culture medium for the liquid induction culture of the present invention comprises the following components: 17-19 mg / L tyrosine, 10-15 mg / L salicylic acid, 20-25 mg / L methyl jasmonate, 8-12 g / L peptone, 3.0-7.0 g / L wheat bran juice, 1.0-3.0 g / L soybean powder juice, 0.3-0.8 g / L copper chloride, 1.0-2.0 g / L magnesium sulfate and 1.0-2.0 g / L potassium dihydrogen phosphate, preferably comprising 18.1 mg / L tyrosine, 13.8 mg / L salicylic acid, 22.4 g / L methyl jasmonate, 10 g / L peptone, 5.0 g / L wheat bran juice, 2.0 g / L soybean powder juice, 0.5 g / L copper chloride, 1.5 g / L magnesium sulfate and 1.5 g / L potassium dihydrogen phosphate.

[0043] The present invention preferably performs feed-batch culture and 40-50°C high temperature induction culture on the liquid culture medium in sequence. The present invention preferably further comprises dividing the black fungus strain of the solid induction culture into blocks before the inoculation to obtain a block black fungus strain. The diameter of the block black fungus strain of the present invention is preferably 0.8-1.2 cm, more preferably 1 cm. The present invention preferably inoculates the block black fungus strain into a liquid induction culture medium for feed-batch culture to obtain a feed culture solution. The volume ratio of the number of the block black fungus strain of the present invention to the liquid induction culture medium is preferably 3-8 blocks: 80-120 mL, more preferably 5 blocks: 100 mL. The present invention preferably performs the feed-batch culture when the block black fungus strain is inoculated into the liquid induction culture medium for 5-9 days, more preferably 7 days. The feed-batch culture of the present invention preferably further includes a shake flask, and the temperature of the shake flask is preferably 23-30°C, more preferably 25°C; the rotation speed of the shake flask is preferably 140-170r / min, more preferably 160r / min; the shake flask has the function of expanding the culture and increasing the mycelium biomass. The feed-batch culture of the present invention preferably includes a supplementary culture medium, and the culture medium preferably includes tyrosine, salicylic acid and methyl jasmonate. The tyrosine of the present invention is preferably present in the form of a solution, and the concentration of the tyrosine solution is preferably 8-12mmol / L, more preferably 10mmol / L; the volume ratio of the tyrosine solution to the initial amount of the liquid culture medium is preferably (0.8-1.2): (45-54), more preferably 1:50, such as when the initial amount of the liquid culture medium is 100mL, the supplementary dosage of the tyrosine solution is 2mL. The salicylic acid of the present invention is also supplemented in the form of a solution, and the concentration of the salicylic acid solution is preferably 8-12 mmol / L, more preferably 10 mmol / L; the volume ratio of the salicylic acid solution to the initial amount of the liquid culture medium is preferably (2-4): (80-120), more preferably 3:100. The methyl jasmonate of the present invention is also supplemented in the form of a solution, and the concentration of the methyl jasmonate solution is preferably 8-12 mmol / L, more preferably 10 mmol / L; the volume ratio of the methyl jasmonate solution to the initial amount of the liquid culture medium is preferably (0.8-1.2): (18-22), more preferably 1:20. The supplementary culture material of the present invention is preferably completed in a clean bench. The present invention preferably performs a first recovery culture after completing the supplementary culture to obtain a feed culture solution; the first recovery culture is preferably carried out in the form of a shake flask culture, and the temperature and speed of the shake flask culture are the same as those of the above-mentioned shake flask, and no further description is given. The tyrosine of the present invention is a raw material for synthesizing melanin. Tyrosine, salicylic acid and methyl jasmonate all have the function of melanin inducers. By supplementing tyrosine, salicylic acid and methyl jasmonate in the liquid culture stage, black fungus can be further induced to produce melanin, thereby improving the melanin yield.

[0044] After obtaining the feed culture solution, the present invention preferably subjects the feed culture solution to high temperature induction culture at 40-50°C to obtain a high temperature induction culture solution. The high temperature induction culture of the present invention preferably includes three high temperature induction cultures, namely, a first high temperature induction culture, a second high temperature induction culture and a third high temperature induction culture.

[0045] The present invention preferably performs the first high temperature culture 2 to 4 days after the feed culture, more preferably 3 days. The temperature of the first high temperature induction culture of the present invention is preferably 42 to 48°C, more preferably 43 to 46°C, and most preferably 45°C; the duration of the first high temperature induction culture is preferably 0.5 to 4h, more preferably 0.8 to 3h, and most preferably 1h.

[0046] After completing the first high temperature induction culture, the present invention preferably performs the second high temperature induction culture 2 to 6 days after the first high temperature induction culture, more preferably 2.5 to 5.5 days, further preferably 3 to 5 days, and most preferably 3 days. The temperature of the second high temperature induction culture of the present invention is preferably 42 to 48°C, more preferably 43 to 46°C, and most preferably 45°C; the duration of the second high temperature induction culture is preferably 0.5 to 4 hours, more preferably 0.8 to 3.5 hours, and most preferably 3 hours.

[0047] After completing the second high temperature induction culture, the present invention preferably performs the third high temperature induction culture 2 to 6 days after the second high temperature induction culture, more preferably 2.5 to 5.5 days, further preferably 3 to 5 days, and most preferably 3 days. The temperature of the third high temperature induction culture of the present invention is preferably 41 to 49°C, more preferably 42 to 48°C, and most preferably 48°C; the duration of the second high temperature induction culture is preferably 0.5 to 4 hours, more preferably 0.8 to 3.5 hours, and most preferably 3 hours.

[0048] The present invention preferably further comprises supplementing tyrosine after each high temperature induction culture in the liquid induction culture stage. The tyrosine is preferably present in the form of a solution, and the concentration of the tyrosine solution is preferably 8-12 mmol / L, more preferably 10 mmol / L; the volume ratio of the tyrosine solution to the initial amount of the liquid culture medium is preferably (0.8-1.2):(17-23), more preferably 1:20. The present invention preferably supplements tyrosine in an ultra-clean workbench. The present invention preferably performs fed-batch recovery culture after each tyrosine supplementation, and the conditions of each fed-batch recovery culture are the same as those of the first recovery culture, and will not be repeated.

[0049] The present invention preferably further comprises, after the last tyrosine supplementation, post-processing the obtained induced liquid to obtain melanin. The post-processing of the present invention is preferably carried out 2 days after the last tyrosine supplementation. The post-processing of the present invention preferably comprises filtration and melanin extraction. The present invention preferably filters the obtained induced fermentation liquid to obtain fermentation liquid and mycelium. The particle size of the filtering device of the present invention is preferably 100 meshes. The present invention does not specifically limit the filtering method, and the conventional filtering method in the art can be used. The present invention preferably uses alkali dissolution and acid precipitation to extract the fermentation liquid and mycelium respectively. The alkaline dissolution pH value in the alkali dissolution and acid precipitation method of the present invention is preferably 10 to 14, and more preferably 12.0. The acid precipitation pH in the alkali dissolution and acid precipitation method of the present invention is preferably 1.0 to 3.5, and more preferably 2.0. The present invention does not specifically limit the method of the alkali dissolution and acid precipitation method, and the conventional method in the art can be used. The black fungus strains used in the method of the present invention preferably include black fungus strains HW100, Heiwei No. 15, and black fungus No. 1, more preferably black fungus strains HW100 and Heiwei No. 15, and most preferably HW100.

[0050] The invention also provides a black fungus strain HW100, and the deposit number of the black fungus strain HW100 is CGMCC No.40192.

[0051] The black fungus strain HW100 of the present invention is preferably collected from the field in Shangzhi City, Heilongjiang Province in 2015 and separated by tissue to obtain a purified black fungus strain. The strain and the black fungus strain registered in Heilongjiang Province have antagonism, indicating that the strain is a new black fungus strain. The mycelium of the strain grows faster in PDA culture medium, which is 3.4±0.21mm / d. It is easy to produce melanin in solid culture medium, and the melanin production is significantly improved after high temperature induction. After fermentation and cultivation in liquid culture medium for 3 days, the fermentation liquid begins to turn black and begins to produce melanin. The black fungus strain HW100 of the present invention is used, and the yield of melanin in black fungus can be further improved in combination with the method described in the present invention.

[0052] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] Example 1

[0054] A method for improving the yield of melanin in black fungus fermentation liquid, comprising the following steps:

[0055] 1) The black fungus strain HW100 was activated in a PDA culture dish and transferred to a solid induction medium for culture. Cultured at 25°C in the dark, transferred to 48°C for high temperature induction for 1 hour after 13 days, and then transferred to 28°C for recovery culture for 3 days, which was used to inoculate the liquid culture medium;

[0056] The formula of solid induction medium: 100g potato, 5g glucose, 2.5g tyrosine, 0.1g potassium dihydrogen phosphate, 0.1g magnesium sulfate, 0.2g copper chloride, add distilled water to 1000mL.

[0057] 2) The strains that have undergone solid induction culture were punched with a 1 cm hole puncher and inoculated into a triangular flask containing 100 mL of liquid culture medium, with 5 inoculation blocks in each flask for liquid induction culture;

[0058] Liquid induction medium formula: tyrosine 18.1 mg, salicylic acid 13.8 mg, methyl jasmonate 22.4 mg, peptone 10 g, wheat bran juice 5.0 g, soybean powder juice 2.0 g, copper chloride 0.5 g, magnesium sulfate 1.5 g, potassium dihydrogen phosphate 1.5 g, add distilled water to 100 mL.

[0059] 3) The inoculated liquid induction culture was placed in a shaking flask at 25°C and 160 r / min;

[0060] Fed-batch culture: After 7 days of liquid induction culture, add sterilized reagents in the clean bench: 2 mL of 10 mmol / L tyrosine solution, 3 mL of 10 mmol / L salicylic acid solution, 5 mL of 10 mmol / L methyl jasmonate solution, and resume culture at 25°C and 160 r / min;

[0061] First high temperature induction culture: After 10 days of liquid induction culture, high temperature induction was performed at 45°C for 1 hour. After induction, 5 mL of 10 mmol / L tyrosine solution was added to each bottle of liquid culture medium in the clean bench, and the culture was resumed at 25°C and 160 r / min.

[0062] Second high temperature induction culture: After 15 days of liquid induction culture, high temperature induction was performed at 45°C for 3 hours. After induction, 5 mL of 10 mmol / L tyrosine solution was added to each bottle of liquid culture medium in the clean bench, and the culture was resumed at 25°C and 160 r / min.

[0063] Third, high temperature induction culture: When the liquid induction culture was 18 days old, high temperature induction was performed at 48°C for 3 hours. After the induction was completed, 5 mL of 10 mmol / L tyrosine solution was added to each bottle of liquid culture medium in the clean bench, and the culture was resumed at 25°C and 160 r / min to obtain the induced fermentation liquid. The specific process is as follows: Fig. 9 shown.

[0064] 4) Post-treatment: After 20 days of liquid induction culture, the induced fermentation liquid was filtered, and the fermentation liquid and mycelium were collected separately. The melanin in the fermentation liquid and mycelium was extracted respectively by alkali dissolution and acid precipitation method.

[0065] Example 2

[0066] The method in Example 1 was used to induce black fungus to produce melanin, except that the fungus strain used was Heiwei No. 15.

[0067] Example 3

[0068] The method in Example 1 was used to induce black fungus to produce melanin, except that the fungus strain used was black fungus No. 1.

[0069] Comparative Example 1

[0070] The method in Example 1 was used to induce black fungus to produce melanin, except that after the black fungus was cultured in step 1) and step 2), it was cultured in a shaking flask at 25° C. and 160 r / min for 20 days, and then post-treated in step 4).

[0071] Comparative Example 2

[0072] The method in Example 1 was used to induce black fungus to produce melanin, except that the first high temperature induction culture in step 3) was omitted and replaced by shaking flask culture at 25°C and 160 r / min.

[0073] Comparative Example 3

[0074] The method in Example 1 was used to induce black fungus to produce melanin, except that the second high temperature induction culture in step 3) was omitted and replaced by shaking flask culture at 25°C and 160 r / min.

[0075] Comparative Example 4

[0076] The method in Example 1 was used to induce black fungus to produce melanin, except that the third high-temperature induction culture in step 3) was omitted and replaced by shaking flask culture at 25°C and 160 r / min.

[0077] Comparative Example 5

[0078] The method in Example 1 was used to induce black fungus to produce melanin, except that the fed-batch culture in step 3) was omitted and replaced by a shake flask culture at 25° C. and 160 r / min.

[0079] Comparative Example 6

[0080] The method in Example 1 was used to induce black fungus to produce melanin, except that the fungus strain used was black fungus No. 2.

[0081] Application Example 1

[0082] 1. The mycelium in the solid culture medium before and after high temperature induction in the solid induction culture of Example 1 was observed under a microscope. The results are as follows: Figures 1-2 As shown, Figure 1 This is the mycelium morphology before high temperature induction in solid induction culture. Figure 2 This is the morphology of mycelium after high temperature induction in solid induction culture. Figures 1-2 The scale in the figure is 40×.

[0083] Compared with the mycelium before high temperature induction in solid induction culture, the diameter of the black fungus mycelium after high temperature induction became thicker, and some mycelium became tangled and aggregated together. The color of the mycelium at the tangled part became darker, indicating that the black fungus has responded to high temperature stress and produced melanin through metabolism to cope with high temperature stress.

[0084] 2. Observe the color change of mycelium in the solid culture medium before and after the solid induction culture in Example 1. The results are as follows: Figures 3-4 As shown, Figure 3 This is the color picture of mycelium before high temperature induction in solid induction culture. Figure 4 This is the color picture of mycelium after high temperature induction in solid induction culture.

[0085] Depend on Figures 3-4 It can be seen that compared with the mycelium before high-temperature induction, the color of the black fungus mycelium after high-temperature induction is significantly darker, indicating that after being subjected to high-temperature stress, the black fungus mycelium produces secondary metabolite melanin to cope with external high-temperature stress.

[0086] 3. Observe the color change of mycelium in the liquid culture medium before and after the liquid induction culture in Example 1. The results are as follows: Figure 5 As shown, from left to right are the mycelium color picture before liquid induction culture, the mycelium color picture after the first high temperature induction, the mycelium color picture after the second high temperature induction and the mycelium color picture after the third high temperature induction.

[0087] Depend on Figure 5 It can be seen that with the increase of high-temperature induction times, the color of black fungus mycelium gradually deepens, indicating that the more high-temperature induction times, the higher the melanin yield.

[0088] Application Example 2

[0089] The chromaticity values ​​of the fermentation broths (filtrate after centrifugation to remove mycelium) in Examples 1 to 3 and Comparative Examples 1 to 6 were measured using a colorimeter (cm-2500d), where L indicates brightness, and the smaller the L value, the darker the color, and the darker the color, the higher the melanin content. The results are shown in Table 1:

[0090] Table 1 Chroma of fermentation liquid of Examples 1 to 3 and Comparative Examples 1 to 6

[0091]

[0092] The L value represents the brightness value. The smaller the L value, the darker the color. As shown in Table 1, the L value of the induced fermentation broth in Example 1 is the smallest, which is 35.49±1.2. The L value in the fermentation broth of Comparative Example 6 is the largest, which is 56.35±0.41. The L values ​​of Examples 2 to 3 are second. The L values ​​of the comparative examples are all higher than those of the embodiments. The L value of Comparative Example 1 is the largest, the L values ​​of Comparative Examples 2 to 4, which lack one high-temperature induction, are second, and the L value of Comparative Example 5, which does not supplement the culture medium, is the smallest. The one-way ANOVA found that the L value of Example 1 was significantly different from that of Examples 2 to 3 and Comparative Examples 1 to 6 (P<0.05). Therefore, the color of the fermentation broth is significantly increased after high-yield melanin induction, and the mycelium metabolism produces more melanin.

[0093] Application Example 3

[0094] The tyrosinase activity in the fermentation broth and mycelium in Examples 1 to 3 and Comparative Examples 1 to 6 was determined using a tyrosinase kit (BC4050, Solarbio). The results are shown in Table 2:

[0095] Table 2 Tyrosinase activity in fermentation broth and mycelium of Examples 1 to 3 and Comparative Examples 1 to 6

[0096]

[0097] Studies in this field have found that tyrosinase is a key enzyme in melanin synthesis, and tyrosinase activity is positively correlated with melanin yield. It can be concluded from Table 2 that the tyrosinase activity in the fermentation broth and mycelium of Example 1 is the highest, which are 12.41U / g and 35.45U / g, respectively. The tyrosinase activity in the fermentation broth and mycelium of Example 1 is higher than that in Examples 2 to 3 and Comparative Examples 1 to 6. One-way analysis of variance found that the tyrosinase activity in the fermentation broth and mycelium of Example 1 was significantly higher than that in Examples 2 to 3 and Comparative Examples 1 to 6 (P < 0.05). Therefore, the tyrosinase activity in the fermentation broth and mycelium is significantly increased after high-yield melanin induction, thereby promoting melanin synthesis.

[0098] Application Example 4

[0099] The melanin in the fermentation broth and mycelium induced in Examples 1 to 3 and Comparative Examples 1 to 6 was extracted by alkali dissolution and acid precipitation. The preparation method is as follows:

[0100] Melanin extraction steps in fermentation broth:

[0101] 1. Take the filtered fermentation liquid and adjust the pH value to 12.0 with 1mol / LNaOH to completely dissolve the melanin;

[0102] 2. Adjust the pH to 2.0 with 1 mol / L HCl to precipitate the melanin and let it stand for 10 minutes;

[0103] 3. Centrifuge at 8000rpm for 20min, collect the precipitate and measure the mass of the precipitate.

[0104] 4. Repeat steps 1 to 3 to purify melanin 3 to 5 times.

[0105] Dissolve the precipitate in step 4 in 0.1 mol / L NaOH, measure the absorbance at 210 nm, and calculate the amount of melanin according to the standard curve. The regression equation of the standard curve of the melanin standard is Y=0.0534X-0.01753, where Y is the melanin content and X is the absorbance.

[0106] Melanin extraction steps from mycelium:

[0107] 1. Grind the dried mycelium into powder and select particles with a size of 0.18-0.23 mm for later use.

[0108] 2. Mix mycelium powder and water at a ratio of 1:50 (mass ratio), adjust the pH value to 12.0 with 1 mol / L NaOH, and extract in a water bath at 70°C for 1 h.

[0109] 3. Centrifuge at 8000 rpm for 20 min, take the supernatant, adjust the pH to 2.0 with 1 mol / L HCl to precipitate the melanin, and let it stand for 10 min.

[0110] 4. Centrifuge at 8000rpm for 20min, collect the precipitate and measure the mass of the precipitate.

[0111] 5. Repeat steps 2 to 4 to purify melanin 3 to 5 times.

[0112] 6. Dissolve the precipitate in step 5 in 0.1 mol / L NaOH, measure the absorbance at 210 nm, and calculate the amount of melanin according to the standard curve. The regression equation of the standard curve of the melanin standard is Y=0.0534X-0.01753, where Y is the melanin content and X is the absorbance.

[0113] The results are shown in Table 3:

[0114] Melanin yield = melanin mass / fermentation liquid or mycelium mass × 100%.

[0115] Table 3 Melanin yield in fermentation broth and mycelium of Examples 1 to 3 and Comparative Examples 1 to 6

[0116]

[0117]

[0118] It can be concluded from Table 3 that: among the examples, Example 1 has the highest melanin yield from the fermentation broth, which is 1.22%, and Examples 2-3 have the second highest melanin yield. Among the comparative examples, Comparative Example 6 did not extract melanin from the fermentation broth, and the lowest melanin yield from the mycelium was 1.1%. The melanin yield in the fermentation broth of Comparative Example 1 was the second lowest. Compared with Comparative Example 1, the melanin yield in the fermentation broth of Example 1 increased by 430.43%, and the melanin yield in the mycelium increased by 16.44%. The melanin yield in the fermentation broth of Comparative Example 5 without supplementary culture medium was only higher than that of Comparative Example 1, indicating that supplementary culture medium can promote the production of melanin. The melanin yield in the fermentation broth of Comparative Examples 2-4, which lacked one high-temperature induction, was higher than that of Comparative Examples 1 and Comparative Example 5, indicating that high-temperature induction can increase the melanin yield. One-way ANOVA found that the melanin yield in the fermentation broth of Example 1 was significantly higher than that of other groups (P < 0.05). The melanin yield from mycelium in Example 1 was the highest, at 8.5%, and the melanin yield from mycelium in Comparative Examples 1 to 6 was lower than that in Example 1. Single factor ANOVA analysis found that the melanin yield from mycelium in Example 1 was significantly higher than that in other groups (P < 0.05). Therefore, the method of the present invention significantly improves the melanin yield in the fermentation broth and mycelium.

[0119] Application Example 5

[0120] The antioxidant capacity of melanin extracted from Example 1 and Comparative Example 1 was determined using the DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity and ABTS scavenging capacity detection kits produced by Nanjing Jiancheng Biotechnology Co., Ltd., with Vc as the control. The results are shown in Tables 4 to 6 and Figures 6 to 8 As shown, Figures 6 to 8 The high-yield induced melanin in the above example represents the melanin obtained in Example 1, and the melanin in the above example represents the melanin obtained in Comparative Example 1.

[0121] Table 4 DPPH clearance of melanin in Example 1 and Comparative Example 1

[0122]

[0123] Table 5 Hydroxyl radical scavenging rate of melanin in Example 1 and Comparative Example 1

[0124]

[0125] Table 6 ABTS clearance rate of melanin in Example 1 and Comparative Example 1

[0126]

[0127] From Tables 4 to 6 and Figures 6 to 8 The results showed that when the concentration of melanin was 1.5-3.0 mg / mL, the melanin in Example 1 had a significantly enhanced ability to scavenge DPPH free radicals compared with the melanin in Comparative Example 1 (P < 0.05); the melanin in Example 1 had a certain degree of improvement in the scavenging ability of hydroxyl free radicals and ABTS compared with the melanin in Comparative Example 1.

[0128] It can be concluded from the above examples that the method of the present invention can significantly increase the yield of melanin in the fermentation culture broth. At the same time, the obtained melanin has higher antioxidant activity than conventional black fungus melanin.

[0129] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for increasing the yield of melanin in black fungus fermentation broth, characterized in that: The method comprises the following steps: sequentially performing solid induction culture and liquid induction culture on black fungus; The solid induction culture medium comprises the following components: 90-110 g / L of potato, 3-8 g / L of glucose, 2.0-3.0 g / L of tyrosine, 0.05-0.2 g / L of potassium dihydrogen phosphate, 0.05-0.2 g / L of magnesium sulfate and 0.1-0.3 g / L of copper chloride; The liquid induction culture medium comprises the following components: 17-19 mg / L tyrosine, 10-15 mg / L salicylic acid, 20-25 mg / L methyl jasmonate, 8-12 g / L peptone, 3.0-7.0 g / L wheat bran juice, 1.0-3.0 g / L soybean powder juice, 0.3-0.8 g / L copper chloride, 1.0-2.0 g / L magnesium sulfate and 1.0-2.0 g / L potassium dihydrogen phosphate; The solid induction culture comprises: solid induction of the activated black fungus strain in a solid culture medium; the solid induction comprises sequentially performing constant temperature and light-proof culture, 40-50° C. high temperature induction culture and recovery culture; the duration of the high temperature induction culture is 0.5-4 hours; the temperature of the constant temperature and light-proof culture and the recovery culture are both 23-30° C.; the duration of the constant temperature and light-proof culture is 8-12 days, and the duration of the recovery culture is 2-4 days; The liquid induction culture comprises: liquid induction of the black fungus strain in solid induction culture in a liquid culture medium; The liquid induction comprises sequentially performing fed-batch culture and 40-50° C. high-temperature induction culture; The high temperature induction culture is carried out on the 3rd day after the fed-batch culture; the high temperature induction culture includes three times, each with an interval of 3 to 5 days, and each induction time is 1 to 3 hours; after each high temperature induction culture, tyrosine is supplemented, and the concentration of tyrosine is 8 to 12 mmol / L; the volume ratio of tyrosine to the initial amount of the liquid culture medium is (0.8 to 1.2): (18 to 22); The black fungus is the black fungus strain HW100, and the preservation number of the black fungus strain HW100 is CGMCC No.40192.

2. The method according to claim 1, characterized in that The feed-batch culture is carried out on the 5th to 9th day of the liquid induction culture. The feed-batch culture includes supplementing culture material, and the culture material includes tyrosine, salicylic acid and methyl jasmonate.

3. The method according to claim 2, characterized in that The concentrations of the tyrosine, salicylic acid and methyl jasmonate are all 8-12 mmol / L; the volume ratios of the tyrosine, salicylic acid and methyl jasmonate to the initial amount of the liquid culture medium are 1:50, (2-4):(80-120) and (0.8-1.2):(18-22) respectively.

4. A black fungus strain HW100, characterized in that The deposit number of the black fungus strain HW100 is CGMCC No.40192.

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