CN104357484A - Black fungus liquid fermentation quick-producing high-yield melanin culture medium - Google Patents

Abstract

The invention relates to a black fungus liquid fermentation quick-producing high-yield melanin culture medium. The formula of the culture medium comprises the following raw materials: 17.27 g/L of a yeast extract, 1.92 g/L of tyrosine, 3.84 g/L of lactose, 1 g/L of NaCl, 2 g/L of MgSO4, 0.5 mg/L of biotin, and 1 g/L of KH2PO4, wherein the pH value is 6. According to the invention, the melanin is produced by black fungus liquid fermentation; with optimization of the fermentation conditions, the melanin production by fermentation can be ahead of time, and the yield of melanin can be increased. Under the optimized conditions, the yield of melanin can be up to 2.97 g/L, which is 2.14 times more than that of the existing culture medium; melanin can be produced on the third day during the culture process. The formula of the culture medium provided by the invention aims to shorten the melanin production time; the mycelial growth amount is less, and the melanin yield is high; the culture medium is more favorable to separate out and extract melanin, so as to reduce the follow-up working cost.

Description

A kind of black fungus liquid fermenting produces high yield nigrosine medium soon

Technical field

The present invention is specifically related to a kind of black fungus liquid fermenting and produces high yield nigrosine medium soon, and this culture medium prescription allows melanochrome produce in advance, the feature that melanin production is high.

Background technology

Melanochrome is the family macromolecule compound be polymerized by phenols or indoles substance oxidation, all can produce in animal, plant and microorganism.Melanochrome is not the prerequisite of biological growth and growth, but plays defencive function for biological Antagonistic Environment is coerced, and as uv-radiation, extreme temperature, therefore can improve the ability such as biological existence, competition.In addition, melanochrome also can be used for makeup manufacture, free radical scavenging and the protection of biotic pesticide light etc., and its application prospect is boundless, therefore studies natural black pigment significant.

Natural black pigment, except black, also shows other colors, as red, orange, yellow etc.Nicolaus etc. advocate melanochrome to be divided into 3 classes: the first kind is eumelanin (eumelanins), it is under the effect of tyrosine oxidase, tyrosine is catalyzed there is oxidation and polyreaction formation, and nitrogen atom and non-sulfur atom-containing, mainly present black or brown; Equations of The Second Kind is phaeomelanin (phaeomelanins), by halfcystine or gsh etc. participate in formed, route of synthesis and eumelanin similar, nitrogen atom and sulphur atom, have brown, even some presents yellow to redness; 3rd class is different melanocyte (allomelanins), is to be polymerized at polyphenol oxidase catalyzed lower polyphenol oxidase, be distributed in plant, nonnitrogenous, in black or brown.

Though the research of melanin structure early, because melanochrome is insoluble in majority of organic solvent and water, be unfavorable for that the analysis of structure measures, so do not obtain the complete structure of natural black pigment yet at present, the knowledge about melanic direct organization is also less.Current infer eumelanin and phaeomelanin structure is as follows.Different melanocyte structure type is determined not yet.

eumelanins phaeomelanins。

Black fungus ( a. auricula), the wood fungus that is otherwise known as, brightness program are a kind of edible funguses, belong to Basidiomycota sub-, Auriculariale, Auriculariaceae, Auricularia.Sporophore wide about 3-10 cm, thick about 2 mm, in ear, lobate or cup-shaped, edge wave wave-like; Color and luster is dark brown, and quality is soft.Extensively be distributed in the provinces such as China Zhejiang, Heilungkiang, Jilin, Fujian, Taiwan, Hubei, have wild also can by artificial growth.Black fungus has very high nutritive value, is famous mountain delicacy, has the good reputation of " meat or fish in element ", and Chinese common people eat for a long time and do not mind over the past thousands of years.Black fungus is not only nutritious, and also has that enhancing body resists disease ability, hypoglycemic blood fat, anticancer, radioprotective, health care wait for a long time biological function.These functions of black fungus and the black of its generation have substantial connection.As a kind of secondary metabolite, melanochrome is that black fungus grows into certain period generation, more needs the supply of complicated nutritive substance compared with primary metabolite.

The melanic output of microorganism is not only subject to the impact of this bacterial strain genetic material, also by the impact of culture condition.The temperature, pH, cell age etc. of the nitrogenous source of substratum, carbon source and carbon-nitrogen ratio thereof, culture condition decide the height of melanin production jointly.Substratum not only provides nutritive substance for the accumulation of the metabolism and growth product of biology, and its component also may affect the difference in pigment synthesis direction.Although the Microbial resources of producing natural black pigment are very abundant, there are many bacterial strains to be all found and report and can produce melanochrome, but up to the present also fail to realize the industrialization that microorganism melanochrome is produced, tracing it to its cause, it is lower to be that most bacterial strains produces melanochrome ability.Optimizing the culture condition producing melanochrome microorganism is the effective means improving biosynthesizing efficiency.

Summary of the invention

The object of the present invention is to provide a kind of black fungus liquid fermenting to produce high yield nigrosine medium soon, produce melanochrome fermentation condition by optimizing black fungus, make fermentation produce melanic time advance, melanin production improves.

A kind of black fungus liquid fermenting of the present invention produces high yield nigrosine medium soon, and composition of raw materials is as follows:

Yeast extract paste 17.27 g/L, tyrosine 1.92 g/L, lactose 3.84 g/L, NaCl 1 g/L, MgSO ₄2 g/L, vitamin H 0.5 mg/L, KH ₂pO ₄1 g/L, pH are 6.

Fermentation condition: inoculum size is 10 bacterium blocks (diameter d=8 mm), and liquid amount is 75 mL, 28 DEG C, 150 r/min shaking table constant temperature culture 8 d.

The present invention adopts black fungus liquid fermenting to produce melanochrome, by fermentation condition optimization, makes fermentation produce melanic time advance, improves melanin production.The present invention with optimal conditions melanin production can reach 2.97 g/L, and the substratum that comparatively sets out improves 2.14 times.General black fungus mycelium culture is 7-9 days, but culture medium prescription provided by the invention makes product melanochrome time advance, just starts to produce melanochrome when cultivating the 3rd day.The focusing on of culture medium prescription provided by the invention shortens produces the melanic time, and mycelial growth is few, and melanin production is high, is more conducive to melanochrome ground separation and extraction, reduces follow-up work cost.

Embodiment

The present invention's the following example further illustrates the present invention, but protection scope of the present invention is not limited to the following example.

embodiment 1:different Nutrition condition is on the impact of mycelial growth feature and melanochrome Formation rule

1 materials and methods

1.1 experiment material

1.1.1 bacterial strain

Black fungus ( a. auricula) (grandson is refined to wait quietly. produce screening and the physico-chemical property research thereof of natural pigment black fungus strain. and Agricultural University Of Jiangxi's journal, 2010,32 (1): 160).

1.1.2 substratum

(1) rich PDA solid medium is added: glucose 20 g/L, yeast powder 5 g/L, peptone 3 g/L, KH ₂pO ₄3 g/L, MgSO ₄1.5 g/L, VB ₁0.1 g/L, agar 20 g/L, pH nature.

(2) liquid perfect medium: lactose 10 g/L, yeast extract paste 10 g/L, tyrosine 1 g/L, CaCl ₂0.1 g/L, NaCl 5 g/L.

(3) carbon-free substratum: yeast extract paste 10 g/L, tyrosine 1 g/L, CaCl ₂0.1 g/L, NaCl 5 g/L.

(4) nitrogen-free agar: lactose 10 g/L, tyrosine 1 g/L, CaCl ₂0.1 g/L, NaCl 5 g/L.

(5) without tyrosine substratum: lactose 10 g/L, yeast extract paste 10 g/L, CaCl ₂0.1 g/L, NaCl 5 g/L.

(6) low-carbon high-nitrogen substratum: lactose 1 g/L, yeast extract paste 10 g/L, tyrosine 1 g/L, CaCl ₂0.1 g/L, NaCl 5 g/L.

(7) low nitrogen high-carbon substratum: lactose 10 g/L, yeast extract paste 1 g/L, tyrosine 1 g/L, CaCl ₂0.1 g/L, NaCl 5 g/L.

(8) the free medium: lactose 10 g/L, yeast extract paste 10 g/L, tyrosine 1 g/L.

1.2 experimental technique

1.2.1 strains tested activation

Preservation strain access is added rich PDA inclined-plane, 28 DEG C of constant temperature culture 5 d.The inclined-plane newly grown is forwarded to and adds rich PDA flat board, 28 DEG C of constant temperature culture 7 d.

1.2.2 melanochrome extracts

By black fungus nutrient solution elimination mycelia, it is about 1.5,4 DEG C of hold over night that filtrate is acidified to pH value with 6 mol/L HCl.Centrifugal 15 min of 10000 rpm, get precipitation.PH neutrality is precipitated to deionized water rinsing.Centrifugal 15 min of 10000 rpm, get precipitation normal-temperature vacuum dry, obtain black fungus melanochrome crude product.

1.2.3 Different Nutrition condition is on the impact of melanin production

Respectively liquid perfect medium, carbon-free substratum, nitrogen-free agar, without tyrosine substratum, the free medium, low-carbon high-nitrogen substratum, low nitrogen high-carbon substratum in access 10 bacterium sheets (d=8 mm), 28 DEG C, 150 r/min shaking table constant temperature culture 8 d, each process three is parallel.Melanochrome is extracted by 1.2.2 method.

1.2.4 Different Nutrition condition is on the impact of hypha form

1.2.4.1 hypha form is observed

There are being solid perfect medium, carbon-free substratum, nitrogen-free agar, inoculating a bacterium sheet (d=8 mm) without on tyrosine substratum, the free medium, low-carbon high-nitrogen substratum, low nitrogen high-carbon culture medium flat plate respectively.Inoculate and be completely placed in 28 DEG C of constant temperature culture 7 d.Measure the speed of growth, observe colonial morphology.

2 results and analysis

2.1 Different Nutrition conditions are on the impact of melanin production

The speed of growth of mycelia on seven kinds of different culture medias differs greatly, carbon-free the fastest with the speed of growth on low-carbon high-nitrogen substratum; Next is the free medium; The mycelial growth rate of nitrogen-free agar is the slowest.The melanin production of perfect medium is maximum, reaches 2.22 g/L; The free medium takes second place, and is 2.08 g/L.The melanic output of nitrogen-free agar is minimum, only 0.01 g/L, and which illustrating nitrogen has vital effect to melanic generation.And tyrosine is the substrate that melanochrome produces, so do not adding in the substratum of tyrosine, melanic output is only having 0.03 g/L.And in carbon-free substratum, melanic output is 0.85 g/L; In low-carbon (LC) substratum, output can reach 1.78 g/L, and this illustrates that the impact that carbon produces for melanochrome is less.

2.2 Different Nutrition conditions are on the impact of hypha form

2.2.1 hypha form is observed

On seven kinds of different substratum, the morphological differences of black fungus mycelia is comparatively large, and the colony diameter on carbon-free and low-carbon high-nitrogen substratum is maximum, edge is all comparatively neat, and bacterium colony is less on perfect medium, surface, in donut, illustrates that the carbon source of excessive concentrations is unfavorable for the growth of mycelia; The free medium mycelia is pure the whitest, but edge radially; Although low nitrogen substratum mycelia is thin and delicate, colony diameter is greater than perfect medium and without tyrosine substratum; Not only bacterium colony is minimum but also mycelia is very thin and delicate for nitrogen-free agar.

2.2.2 scanning electron microscopic observation

By scanning electronic microscope, seven kinds of mycelia that substratum produces are observed.Perfect medium mycelia is full, smooth surface and size is even; Carbon-free substratum mycelia partial collapse, size is more inconsistent; Nitrogen-free agar mycelia is broken comparatively serious, and mycelia is thin and delicate, not full, hyphal surface is rough; Without tyrosine substratum mycelia and perfect medium Hyphal form seemingly; The free medium mycelia is sturdy, surperficial many folds; Low-carbon high-nitrogen substratum mycelia is comparatively thin and delicate, thickness heterogeneity, and mycelia part is agglomerating; Low nitrogen high-carbon substratum mycelia thickness is more inconsistent, and hyphal surface is comparatively smooth.

3 brief summaries and discussion

Only have in the medium each nutritive ingredient concentration, proportioning all suitably time, microorganism could vigorous growth; When concentration is too low, microorganism normal growth is grown and be can not get enough nutritional supports; Then certain suppression even killing effect may be produced to microorganism growth during excessive concentration; Nutritive ingredient proportioning is incorrect, and microorganism just can not make full use of often kind of nutrition in process of growth, causes the waste even possibility underproduction.

Seven kinds of different substratum, what melanin production was the highest is perfect medium, but its mycelia is not dense; Although carbon-free substratum mycelia is dense, melanin production is very low; Low-carbon high-nitrogen substratum mycelia is the densest, and melanin production is a little less than perfect medium.This phenomenon indicates the growth of black fungus mycelia and carbon source concentration has much relations, and the carbon source of lower concentration is conducive to the growth of agaric mycelium; But formed for secondary metabolite, carbon source not only provides the energy needed for it, and provides the carbon skeleton of synthetic product, so carbon source concentration is too low be unfavorable for melanic synthesis.Although the free medium mycelia is pure white dense, edge is irregular, and the speed of growth is faster than perfect medium, and this may be have certain restraining effect due to the growth of inorganic salt to black fungus mycelia and can change its hypha form.But it is more to observe the sturdy fold of salt-free mycelia under Electronic Speculum, may be because salt ion deficiency causes osmotic pressure uneven, thus makes hypha form there occurs certain change.In nitrogen-free agar, bacterium colony is minimum, mycelia is very thin and delicate, produce melanochrome hardly, this is because nitrogenous source not only can be used as the important composition composition of microorganism cells structure, also be the source of the various enzyme of microorganism, nitrogenous source deficiency causes mycelial growth to be obstructed, and the synthesis of tyrosine oxidase can not be carried out, and then have impact on melanic generation.Can produce a small amount of melanochrome without tyrosine substratum is that tyrosine oxidase utilizes them to synthesize a small amount of melanochrome owing to having a small amount of tyrosine and similar compound generation in black fungus metabolic process; Tyrosine can be used as a kind of nutritional factor and is absorbed by black fungus mycelia, so less without tyrosine substratum bacterium colony, but does not affect hypha form without tyrosine.

embodiment 2:black fungus produces the optimization of melanochrome fermentation condition

1 materials and methods

1.1 experiment material

1.1.1 bacterial strain

Black fungus ( a. auricula) for preserving bacterial classification in this laboratory.

1.1.2 substratum

(1) PDA liquid nutrient medium: potato 200 g/L, glucose 20 g/L, pH nature.

(2) PDA solid medium: potato 200 g/L, glucose 20 g/L, agar 20 g/L, pH nature.

(3) rich PDA solid medium is added: glucose 20 g/L, yeast powder 5 g/L, peptone 3 g/L, KH ₂pO ₄3 g/L, MgSO ₄1.5 g/L, VB ₁0.1 g/L, agar 20 g/L, pH nature.

(4) tyrosine fermention medium: glucose 1 g/L, peptone 5 g/L, CaCl ₂0.1 g/L, NaCl 5 g/L, tyrosine 1 g/L, pH 7.0.

1.2 experimental technique

1.2.1 strains tested activation

Preservation strain access is added rich PDA inclined-plane, 28 DEG C of constant temperature culture 5 d.The inclined-plane newly grown is forwarded to and adds rich PDA flat board, 28 DEG C of constant temperature culture 7 d.

1.2.2 melanochrome extracts

By black fungus nutrient solution elimination mycelia, it is about 1.5,4 DEG C of hold over night that filtrate is acidified to pH value with 6 mol/L HCl.Centrifugal 15 min of 10000 rpm, get precipitation.PH neutrality is precipitated to deionized water rinsing.Centrifugal 15 min of 10000 rpm, get precipitation normal-temperature vacuum dry, obtain black fungus melanochrome crude product.

1.2.3 melanochrome production time curve

Get bacterium liquid supernatant at 1 d, 3 d that ferment, 5 d, 7 d, 9 d respectively, under 400 nm, measure its OD value.

1.2.4 inoculum size and liquid amount are on the impact of melanin production

Every bottle of substratum accesses 4,6,8,10,12 bacterium blocks respectively, surveys output after cultivating 8 d.Each process three is parallel.Melanochrome is extracted by 1.2.2 step.

Add substratum 50 mL, 75 mL, 100 mL, 125 mL in 250 mL triangular flasks respectively, after cultivating 8 d, survey output.Each process three is parallel.Melanochrome is extracted by 1.2.2 step.

1.2.5 additive is on the impact of melanin production

Substratum based on PDA liquid nutrient medium, adds tyrosine, adenylic acid, glycine, vitamins B respectively ₁, vitamins C, sulfanilamide (SN), acrylamide, addition is 1 g.Each process three is parallel.Melanochrome is extracted by 1.2.2 step.

1.2.6 carbon source is on the impact of melanin production

Substratum based on tyrosine fermention medium, adds sucrose respectively, glycerine, lactose, starch, N.F,USP MANNITOL, Semen Maydis powder, and oxysuccinic acid is as carbon source, and addition is 1 g.Each process three is parallel.Melanochrome is extracted by 1.2.2 step.

1.2.7 nitrogenous source is on the impact of melanin production

Substratum based on tyrosine fermention medium, adds with peptone, extractum carnis, yeast extract paste, urea, SODIUMNITRATE, methyllanthionine respectively, and ammonium chloride is as nitrogenous source, and addition is 1 g.Each process three is parallel.Melanochrome is extracted by 1.2.2 step.

2 results and analysis

2.1 melanochrome production time curves

The result of melanochrome production time shows, starts slow accumulation when melanochrome cultivates 3 days; The fastest accumulation in the 4 to 6 day; Be cultured to the 7th day, reach maximum value; Continue afterwards to cultivate, melanochrome absorbancy no longer improves.

2.2 inoculum sizes and liquid amount are on the impact of melanin production

When inoculum size is 4 bacterium blocks, melanin production is 0.39 g/L; When inoculum size is 6 bacterium blocks, melanin production is 0.48 g/L; When inoculum size is 8 bacterium blocks, melanin production is 0.83 g/L; When inoculum size is 10 bacterium blocks, melanin production is 0.95 g/L; When inoculum size is 12 bacterium blocks, melanin production is 0.73 g/L.As can be seen here, when cultivating same time within the specific limits, inoculum size is larger, and melanin production is higher; Exceeded this scope, melanin production declines on the contrary.When inoculum size is 10 bacterium blocks, melanin production is nearly 2.5 times of inoculation 4 bacterium blocks, and inoculum size has significant impact to melanin production.

When liquid amount is 50 mL, melanin production is 0.89 g/L; When liquid amount is 75 mL, melanin production is 1.14 g/L; When liquid amount is 100 mL, melanin production is 0.99 g/L; When liquid amount is 125 mL, melanin production is 0.43 g/L.Within the specific limits, along with the increase of liquid amount, melanin production presents the trend first increasing and reduce afterwards; When liquid amount is 75 mL, output reaches the highest.

2.3 additives are on the impact of melanin production

Different additive is different to different bacterial classification roles.For black fungus, add tyrosine and can improve melanic output, therefore tyrosine is the additive of its best.

2.4 carbon sources are on the impact of melanin production

Take carbon source as the substratum of lactose, melanin production is high compared with the substratum of other carbon sources.Therefore lactose is chosen as the melanic carbon source of product black fungus.

2.5 nitrogenous sources are on the impact of melanin production

Organic nitrogen source is more conducive to melanic generation than inorganic nitrogen-sourced.With the substratum of yeast extract paste, melanin production is the highest.Therefore choosing yeast extract paste is that black fungus produces melanic optimum nitrogen source.

3 brief summaries and discussion

Microbial secondary metabolism did not produce in the growing microorganism phase, was generally just to produce in the stable growth phase.In experiment, melanochrome starts slow accumulation when cultivation 3 days; Be cultured to the 7th day, absorbancy reaches maximum value; Melanin concentration no longer improves afterwards, so the optimal period that black fungus is produced is 7 days.

Inoculum size and liquid amount all can affect melanic output.Within the specific limits, inoculum size is larger, and cultivate same time, pigment production is higher, this is because inoculum size is too small, growth is slow, and fermentation period extends, and the accumulation of pigment is slower; If but inoculum size is excessive, the thalline brought into is more, and old thalline accounting example may be caused large, and the thalline ratio of therefore early ageing is large, arrives fermentation termination too early, causes pigment production to decline; In addition, inoculum size is excessive also may cause under-nutrition, and thalli growth cannot be met thus output is reduced.Along with the increase of liquid amount, pigment production presents the trend first increasing and slow down afterwards.This is black fungus is a kind of aerobic fungi, and mycelial growing needs certain oxygen to provide; Exceed maximum dissolved oxygen demand, continued to increase air flow and not only can not improve output, even may change pathways metabolism and cause production declining.

In various additive, tyrosine can significantly improve melanic output.Screen the carbon nitrogen source kind in fermention medium, discovery organic carbon source, nitrogenous source are more conducive to the melanic generation of black fungus.

Determine with yeast extract paste, tyrosine and the lactose factor for major effect black fungus melanin production by Plackett-Burman experiment; Carry out steepest hill climbing test on this basis, determine region, optimal response face; The addition adopting Box-Behnken design to obtain 3 kinds of factors is again: yeast extract paste 17.27 g/L, tyrosine 1.92 g/L, lactose 3.84 g/L.All the other conditions are respectively NaCl 1 g/L; PH is 6; MgSO ₄2 g/L; Vitamin H 0.5 mg/L; KH ₂pO ₄1 g/L.Under this condition, melanin production can reach 2.97 g/L, and the substratum that comparatively sets out improves 2.14 times, illustrates that model is reliable, proves that response surface optimization method has advantage fast and efficiently in improvement melanochrome liquid fermentation medium.

Claims (2)

1. black fungus liquid fermenting produces a nigrosine medium, it is characterized in that: the composition of raw materials of described substratum is as follows:

Yeast extract paste 17.27 g/L, tyrosine 1.92 g/L, lactose 3.84 g/L, NaCl 1 g/L, MgSO ₄2 g/L, vitamin H 0.5 mg/L, KH ₂pO ₄1 g/L, pH are 6.

2. black fungus liquid fermenting according to claim 1 produces nigrosine medium, and it is characterized in that: fermentation condition: liquid amount is 75 mL, inoculum size is the bacterium block of 10 diameter=8 mm, 28 DEG C, 150 r/min shaking table constant temperature culture 8 d.