CN104357484B - A kind of black fungus liquid fermentation produces nigrosine medium - Google Patents
A kind of black fungus liquid fermentation produces nigrosine medium Download PDFInfo
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Abstract
High yield nigrosine medium is produced soon the present invention relates to a kind of black fungus liquid fermentation, and the composition of raw materials of the culture medium is：The g/L of yeast extract 17.27, the g/L of tyrosine 1.92, lactose 3.84 g/L, NaCl 1 g/L, MgSO42 g/L, biotin 0.5 mg/L, KH2PO41 g/L, pH are 6.The present invention produces melanin using black fungus liquid fermentation, by fermentation condition optimization, fermentation is produced the time advance of melanin, improves melanin production.Melanin production can reach 2.97 g/L to the present invention with optimal conditions, and the culture medium that relatively sets out improves 2.14 times, and begin to produce melanin when cultivating the 3rd day.The time for focusing on shortening product melanin of the culture medium prescription that the present invention is provided, and mycelial growth is few, melanin production is high, separates and extracts with being more beneficial for melanin, reduces follow-up work cost.
Description
Technical field
High yield nigrosine medium is produced soon present invention relates particularly to a kind of black fungus liquid fermentation, and the culture medium prescription allows
Melanin is produced and shifted to an earlier date, the characteristics of melanin production is high.
Background technology
Melanin is the family macromolecule compound by phenols or indoles material oxidation polymerization, in animal, plant
Can all be produced with microorganism.Melanin is not the necessary condition of biological growth and development, but is biological Antagonistic Environment stress
Defencive function, such as ultraviolet radioactive, extreme temperature are played, therefore the abilities such as biological existence, competition can be improved.Additionally, melanin is also
Can be used for cosmetics manufacture, radicals scavenging and the protection of biological insecticides light etc., its application prospect is boundless, therefore research
Natural black pigment is significant.
Natural black pigment also shows other colors, such as red, orange, yellow in addition to black.Nicolaus etc. is main
Open and melanin is divided into 3 classes：The first kind is eumelanin（eumelanins）, it is the tyrosine quilt in the presence of tyrosinase
Catalysis occurs what oxidation and polymerisation were formed, and nitrogen atom is main that black or brown is presented without sulfur atom-containing；Equations of The Second Kind is
Phaeomelanin（phaeomelanins）, being participated in being formed by cysteine or glutathione etc., route of synthesis is similar with eumelanin, contains
Nitrogen-atoms and sulphur atom, there is brown, red even some presentation yellow；3rd class is allomelanin（allomelanins）, be
Polyphenol oxidase catalyzed lower polyphenol oxidase is polymerized, and to be distributed in plant, not nitrogenous, in black or brown.
Though the research morning of melanin structure, because melanin is insoluble in most of organic solvent and water, it is unfavorable for structure
Analysis determine, so not obtaining the complete structure of natural black pigment, the knowledge of the direct organization on melanin yet at present
It is also less.Currently deduce eumelanin and phaeomelanin structure is as follows.Allomelanin structure type does not determine yet.
eumelanins phaeomelanins。
Black fungus（A. auricula）, the wood fungus that is otherwise known as, brightness program are a kind of edible funguses, and category Basidiomycota is sub-,
Auriculariale, Auriculariaceae, Auricularia.Fructification about 3-10 cm wide, about 2 mm of thickness, in ear, lobate or cup-shaped, edge is wavy；
Color and luster is dark brown, soft texture.The province such as China Zhejiang, Heilungkiang, Jilin, Fujian, Taiwan, Hubei is widely distributed in, there is wild
Also can be by artificial growth.Black fungus has nutritive value very high, is famous mountain delicacy, the good reputation for having " meat or fish in element ", 1,100
Chinese common people eat do not mind long over year.Black fungus is not only nutritious, and also has enhancing body to resist disease ability, hypoglycemic
Blood fat, anticancer, radioresistance, health care are waited for a long time biological function.The black that these functions and its of black fungus are produced have pass closely
System.Used as a kind of secondary metabolite, melanin is that black fungus grows into regular period generation, compared with metabolite more
Need the supply of complicated nutriment.
The yield of microorganism melanin is not only influenceed by the bacterial strain inhereditary material, is also influenceed by condition of culture.Training
Support the height that nitrogen source, carbon source and its carbon-nitrogen ratio, the temperature of condition of culture, pH, cell age of base etc. together decide on melanin production.
Culture medium is not only that biological growth and the accumulation of metabolite provide nutriment, and its component can also affect on pigment synthesis side
To difference.Although the microbial resources for producing natural black pigment are enriched very much, there are many bacterial strains all to have been found to and report energy
Melanin is produced, but up to the present has not been achievable the industrialization that microorganism melanin is produced, it is absolutely mostly to trace it to its cause
It is relatively low that several bacterial strains produces melanin ability.The condition of culture that melanin microorganism is produced in optimization is the effective of raising biosynthesis efficiency
Means.
The content of the invention
High yield nigrosine medium is produced soon it is an object of the invention to provide a kind of black fungus liquid fermentation, it is black by optimizing
Agaric produces melanin fermentation condition, fermentation is produced the time advance of melanin, and melanin production is improved.
A kind of black fungus liquid fermentation of the invention produces high yield nigrosine medium soon, and composition of raw materials is as follows：
The g/L of yeast extract 17.27, the g/L of tyrosine 1.92, lactose 3.84 g/L, NaCl 1 g/L, MgSO42 g/L, life
0.5 mg/L, KH of thing element2PO41 g/L, pH are 6.
Fermentation condition：Inoculum concentration is 10 bacterium blocks (diameter d=8 mm), and liquid amount is 75 mL, 28 DEG C, 150 r/min
Incubated 8 d of shaking table.
The present invention produces melanin using black fungus liquid fermentation, by fermentation condition optimization, make fermentation produce melanin when
Between in advance, improve melanin production.Melanin production can reach 2.97 g/L to the present invention with optimal conditions, culture of relatively setting out
Base improves 2.14 times.General black fungus cultural hypha is 7-9 days, but the culture medium prescription that the present invention is provided makes the product melanin time
In advance, begin to produce melanin when cultivating the 3rd day.The present invention provide culture medium prescription focus on shorten product melanin
Time, and mycelial growth is few, and melanin production is high, separates and extracts with being more beneficial for melanin, reduces follow-up work cost.
Specific embodiment
The present invention further illustrates the present invention with the following example, but protection scope of the present invention is not limited to following reality
Apply example.
Embodiment 1：Different Nutrition condition forms the influence of rule to mycelial growth feature and melanin
1 materials and methods
1.1 experiment materials
1.1.1 bacterial strain
Black fungus（A. auricula）（The refined screening for waiting product natural pigment black fungus strains quietly of grandson and its physicochemical property are ground
Study carefully Agricultural University Of Jiangxi journal, 2010,32 (1)：160）.
1.1.2 culture medium
(1) richness PDA solid mediums are added：The g/L of glucose 20, the g/L of dusty yeast 5, peptone 3 g/L, KH2PO4 3 g/
L, MgSO41.5 g/L, VB10.1 g/L, agar 20 g/L, pH nature.
(2) liquid complete medium：The g/L of lactose 10, the g/L of yeast extract 10, tyrosine 1 g/L, CaCl2 0.1 g/L,
NaCl 5 g/L。
(3) carbon-free culture medium：The g/L of yeast extract 10, tyrosine 1 g/L, CaCl2The g/L of 0.1 g/L, NaCl 5.
(4) nitrogen-free agar：The g/L of lactose 10, tyrosine 1 g/L, CaCl2The g/L of 0.1 g/L, NaCl 5.
(5) without tyrosine culture medium：The g/L of lactose 10, yeast extract 10 g/L, CaCl2The g/L of 0.1 g/L, NaCl 5.
(6) low-carbon high-nitrogen culture medium：The g/L of lactose 1, the g/L of yeast extract 10, tyrosine 1 g/L, CaCl20.1 g/L,
NaCl 5 g/L。
(7) low nitrogen high-carbon culture medium：The g/L of lactose 10, the g/L of yeast extract 1, tyrosine 1 g/L, CaCl20.1 g/L,
NaCl 5 g/L。
(8) the free medium：The g/L of lactose 10, the g/L of yeast extract 10, the g/L of tyrosine 1.
1.2 experimental techniques
1.2.1 strains tested activation
Preservation strain is accessed plus richness PDA inclined-planes, 28 DEG C of incubated 5 d.The new inclined-plane grown is forwarded to plus richness PDA
Flat board, 28 DEG C of incubated 7 d.
1.2.2 melanin is extracted
Black fungus nutrient solution is filtered off into mycelia, filtrate is acidified to pH value for 1.5 or so, 4 DEG C stood with 6 mol/L HCl
Night.10000 rpm are centrifuged 15 min, take precipitation.It is neutral pH to be precipitated to deionized water rinsing.10000 rpm are centrifuged 15 min,
Take precipitation normal-temperature vacuum to dry, obtain black fungus melanin crude product.
1.2.3 influence of the Different Nutrition condition to melanin production
Respectively liquid complete medium, carbon-free culture medium, nitrogen-free agar, without tyrosine culture medium, the free medium,
10 bacterium pieces (d=8 mm), 28 DEG C, 150 r/min shaking table constant temperature are accessed in low-carbon high-nitrogen culture medium, low nitrogen high-carbon culture medium
8 d are cultivated, each treatment three is parallel.Melanin is extracted by 1.2.2 methods.
1.2.4 influence of the Different Nutrition condition to hypha form
1.2.4.1 hypha form observation
Respectively have solid complete medium, carbon-free culture medium, nitrogen-free agar, without tyrosine culture medium, salt-free training
Support and a bacterium piece (d=8 mm) is inoculated with base, low-carbon high-nitrogen culture medium, low nitrogen high-carbon culture medium flat plate.Inoculation is finished and is placed in 28
DEG C incubated 7 d.The speed of growth is determined, colonial morphology is observed.
2 results and analysis
Influence of the 2.1 Different Nutrition conditions to melanin production
The speed of growth of the mycelia on seven kinds of different culture medias differs greatly, the life on carbon-free and low-carbon high-nitrogen culture medium
Length is fastest；Next to that the free medium；The mycelial growth rate of nitrogen-free agar is most slow.The melanin of complete medium is produced
Amount is maximum, reaches 2.22 g/L；The free medium takes second place, and is 2.08 g/L.The yield of nitrogen-free agar melanin is minimum, only
0.01 g/L, this illustrates that nitrogen has vital effect to melanin.And tyrosine is the bottom that melanin is produced
Thing, so in the culture medium without addition tyrosine, the yield of melanin only has 0.03 g/L.And in carbon-free culture medium,
The yield of melanin is 0.85 g/L；Up to 1.78 g/L, this explanation carbon is produced yield for melanin in low-carbon (LC) culture medium
Raw influence is smaller.
Influence of the 2.2 Different Nutrition conditions to hypha form
2.2.1 hypha form observation
On seven kinds of different culture mediums, the morphological differences of black fungus mycelia is larger, on carbon-free and low-carbon high-nitrogen culture medium
Colony diameter it is maximum, edge is all more neat, and bacterium colony is smaller on complete medium, and surface is in donut, illustrates too high
The carbon source of concentration is unfavorable for the growth of mycelia；The free medium mycelia is pure the whitest, but edge is radial；Low nitrogen culture medium
Although mycelia is thin and delicate, colony diameter is more than complete medium and without tyrosine culture medium；Not only bacterium colony is most for nitrogen-free agar
It is small and mycelia is very thin and delicate.
2.2.2 scanning electron microscopic observation
Mycelia is produced with SEM to seven kinds of culture mediums to observe.Complete medium mycelia is full, surface
It is smooth and uniform in size；Carbon-free culture medium mycelia partial collapse, size is not relatively consistent；The rupture of nitrogen-free agar mycelia is more tight
Weight, and mycelia is thin and delicate, not full, hyphal surface is rough；It is similar to complete medium mycelia without tyrosine culture medium mycelia；Nothing
Salt culture medium mycelia is sturdy, many folds in surface；Low-carbon high-nitrogen culture medium mycelia is more thin and delicate, thickness heterogeneity, mycelia part into
Group；Low nitrogen high-carbon culture medium mycelia thickness is not relatively consistent, and hyphal surface is relatively smooth.
3 brief summaries and discussion
Only in the medium each nutritional ingredient concentration, proportioning all where appropriate, microorganism could vigorous growth；Concentration is too low
When, the development of microorganism normal growth cannot get enough nutritional supports；During excessive concentration may then one be produced to growth of microorganism
Fixed suppression even killing effect；Nutritional ingredient proportioning is incorrect, and microorganism cannot make full use of every kind of in growth course
Nutrition, causes to waste the possibly even underproduction.
Seven kinds of different culture mediums, melanin production highest is complete medium, but its mycelia is not dense；It is carbon-free
Culture medium mycelia is although dense, but melanin production is very low；Low-carbon high-nitrogen culture medium mycelia is the densest, and melanin production is omited
Less than complete medium.This phenomenon indicates the growth of black fungus mycelia and carbon source concentration has much relations, low concentration
Carbon source is conducive to the growth of agaric mycelium；But for secondary metabolite formation, carbon source is not only provided needed for it
Energy, and there is provided the carbon skeleton of synthetic product, so the too low synthesis for being unfavorable for melanin of carbon source concentration.The free medium
Mycelia is although pure white dense, but edge is irregular, and the speed of growth is faster than complete medium, this be probably due to
Growth of the inorganic salts to black fungus mycelia has certain inhibitory action and can change its hypha form.Observed under Electronic Speculum salt-free
But the sturdy fold of mycelia is more, it may be possible to which, because salt ion deficiency causes osmotic pressure uneven, hypha form occurs
Certain change.Bacterium colony is minimum in nitrogen-free agar, and mycelia is very thin and delicate, melanin is hardly produced, because nitrogen source is not only
Can be also the source of the various enzymes of microorganism as the important composition composition of microbial cell structure, nitrogen source deficiency causes mycelia
Body growth retardation, the synthesis of tyrosinase can not be carried out, and then have impact on the generation of melanin.Can be produced without tyrosine culture medium
A small amount of melanin is produced due to having a small amount of tyrosine and similar compound in black fungus metabolic process, and tyrosinase is utilized
They synthesize a small amount of melanin；Tyrosine can be hacked fungus mycelium and absorb as a kind of trophic factors, so without tyrosine culture
Base bacterium colony is smaller, but does not influence hypha form without tyrosine.
Embodiment 2：Black fungus produces the optimization of melanin fermentation condition
1 materials and methods
1.1 experiment materials
1.1.1 bacterial strain
Black fungus（A. auricula）For this laboratory preserves strain.
1.1.2 culture medium
(1) PDA liquid medium：The g/L of potato 200, glucose 20 g/L, pH nature.
(2) PDA solid mediums：The g/L of potato 200, the g/L of glucose 20, agar 20 g/L, pH nature.
(3) richness PDA solid mediums are added：The g/L of glucose 20, the g/L of dusty yeast 5, peptone 3 g/L, KH2PO4 3 g/
L, MgSO41.5 g/L, VB1 0.1 g/L, agar 20 g/L, pH nature.
(4) tyrosine fermentation medium：The g/L of glucose 1, peptone 5 g/L, CaCl2The g/ of 0.1 g/L, NaCl 5
L, tyrosine 1 g/L, pH 7.0.
1.2 experimental techniques
1.2.1 strains tested activation
Preservation strain is accessed plus richness PDA inclined-planes, 28 DEG C of incubated 5 d.The new inclined-plane grown is forwarded to plus richness PDA
Flat board, 28 DEG C of incubated 7 d.
1.2.2 melanin is extracted
Black fungus nutrient solution is filtered off into mycelia, filtrate is acidified to pH value for 1.5 or so, 4 DEG C stood with 6 mol/L HCl
Night.10000 rpm are centrifuged 15 min, take precipitation.It is neutral pH to be precipitated to deionized water rinsing.10000 rpm are centrifuged 15 min,
Take precipitation normal-temperature vacuum to dry, obtain black fungus melanin crude product.
1.2.3 melanin production time curve
1 d respectively in fermentation, 3 d, 5 d, 7 d, 9 d take bacterium solution supernatant, and its OD value is determined under 400 nm.
1.2.4 the influence of inoculum concentration and liquid amount to melanin production
Every bottle of culture medium is respectively connected to 4,6,8,10,12 bacterium blocks, and yield is surveyed after 8 d of culture.Each treatment three is parallel.
Melanin is extracted by 1.2.2 steps.
The mL of culture medium 50,75 mL, 100 mL, 125 mL are separately added into 250 mL triangular flasks, are surveyed after 8 d of culture and produced
Amount.Each treatment three is parallel.Melanin is extracted by 1.2.2 steps.
1.2.5 influence of the additive to melanin production
The culture medium based on PDA liquid medium, adds tyrosine, adenosine monophosphate, glycine, vitamin respectively
B1, vitamin C, sulfanilamide (SN), acrylamide, addition be 1 g.Each treatment three is parallel.Melanin is extracted by 1.2.2 steps.
1.2.6 influence of the carbon source to melanin production
The culture medium based on tyrosine fermentation medium, is separately added into sucrose, and glycerine, lactose, starch, mannitol is beautiful
Ground rice, used as carbon source, addition is 1 g to malic acid.Each treatment three is parallel.Melanin is extracted by 1.2.2 steps.
1.2.7 influence of the nitrogen source to melanin production
The culture medium based on tyrosine fermentation medium, is separately added into peptone, beef extract, yeast extract, urea, nitre
Sour sodium, methyllanthionine, used as nitrogen source, addition is 1 g to ammonium chloride.Each treatment three is parallel.Black is extracted by 1.2.2 steps
Element.
2 results and analysis
2.1 melanin production time curves
The result of melanin production time shows, slow accumulation is started during melanin culture 3 days；In accumulation in the 4th to 6 day most
Hurry up；Culture reached maximum to the 7th day；Continue to cultivate afterwards, melanin absorbance is no longer improved.
The influence of 2.2 inoculum concentrations and liquid amount to melanin production
When inoculum concentration is 4 bacterium blocks, melanin production is 0.39 g/L；When inoculum concentration is 6 bacterium blocks, melanin production is
0.48 g/L；When inoculum concentration is 8 bacterium blocks, melanin production is 0.83 g/L；When inoculum concentration is 10 bacterium blocks, melanin production
It is 0.95 g/L；When inoculum concentration is 12 bacterium blocks, melanin production is 0.73 g/L.As can be seen here, cultivate within the specific limits
During same time, inoculum concentration is bigger, and melanin production is higher；This scope is exceeded, melanin production declines on the contrary.Connecing
When planting amount for 10 bacterium blocks, melanin production is 4 nearly 2.5 times of bacterium block of inoculation, and inoculum concentration has significantly to melanin production
Influence.
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, with the increase of liquid amount, melanin production is presented the trend for first increasing and reducing afterwards；It is in liquid amount
During 75 mL, yield reaches highest.
Influence of 2.3 additives to melanin production
Different additives are different to the role of different strain.For black fungus, tyrosine is added to improve
The yield of melanin, therefore tyrosine is its optimal additive.
Influence of 2.4 carbon sources to melanin production
Culture medium with carbon source as lactose, melanin production is high compared with the culture medium of other carbon sources.Therefore lactose conduct is chosen
Produce the carbon source of black fungus melanin.
Influence of 2.5 nitrogen sources to melanin production
Organic nitrogen source is than the inorganic nitrogen-sourced generation for being more beneficial for melanin.With the culture medium of yeast extract, melanin production is most
It is high.Therefore it is the optimum nitrogen source that black fungus produces melanin to choose yeast extract.
3 brief summaries and discussion
Microbial secondary metabolism is not produced in the growing microorganism phase, is usually just produced stablizing growth period.In experiment, black
Element starts slow accumulation when cultivating 3 days；To the 7th day, absorbance reached maximum for culture；Melanin concentration is no longer carried afterwards
Height, so the optimal period of black fungus production is 7 days.
Inoculum concentration and liquid amount can all influence the yield of melanin.Within the specific limits, inoculum concentration is bigger, when cultivating identical
Between, pigment production is higher, because inoculum concentration is too small, growth is slow, fermentation period extension, the accumulation of pigment compared with
Slowly；But if inoculum concentration is excessive, the thalline brought into is more, and old thalline accounting example may be caused big, therefore the thalline ratio of early ageing is big,
Fermentation termination is early arrived at, causes pigment production to decline；Additionally, inoculum concentration is excessive to also can result in subalimentation, thalli growth
Cannot be met so that yield is reduced.With the increase of liquid amount, pigment production is presented the trend for first increasing and slowing down afterwards.This
It is that black fungus is a kind of aerobic fungi, mycelial growing needs certain oxygen to provide；Maximum dissolved oxygen is exceeded
Demand, continuing increase throughput can not only improve yield, in some instances it may even be possible to change metabolic pathway and cause yield to decline.
In various additives, tyrosine can significantly improve the yield of melanin.To the carbon nitrogen source kind in fermentation medium
Class is screened, it is found that organic carbon source, nitrogen source are more beneficial for the generation of black fungus melanin.
Tested by Plackett-Burman and determine that with yeast extract, tyrosine and lactose be main influence black fungus black
The factor of plain yield；Steepest hill climbing test is carried out on this basis, determines optimal response face region；Box-Behnken is used again
Design obtains the addition of 3 kinds of factors：The g/L of yeast extract 17.27, the g/L of tyrosine 1.92, the g/L of lactose 3.84.Remaining
Part is respectively the g/L of NaCl 1；PH is 6；MgSO42 g/L；The mg/L of biotin 0.5；KH2PO41 g/L.Black under the conditions of this
Plain yield can reach 2.97 g/L, and the culture medium that relatively sets out improves 2.14 times, illustrates that model is reliable, it was demonstrated that response surface
Optimization method has fast and efficiently advantage in melanin liquid fermentation medium is improved.
Claims (1)
1. a kind of black fungus liquid fermentation produces nigrosine medium, it is characterised in that：The composition of raw materials of the culture medium is as follows：
The g/L of yeast extract 17.27, the g/L of tyrosine 1.92, lactose 3.84 g/L, NaCl 1 g/L, MgSO42 g/L, biotin 0.5
mg/L、KH2PO41 g/L, pH are 6.
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