CN103937682B - Method for preparing monascus preparation by use of xylose mother liquor - Google Patents
Method for preparing monascus preparation by use of xylose mother liquor Download PDFInfo
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- CN103937682B CN103937682B CN201410152887.6A CN201410152887A CN103937682B CN 103937682 B CN103937682 B CN 103937682B CN 201410152887 A CN201410152887 A CN 201410152887A CN 103937682 B CN103937682 B CN 103937682B
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Abstract
The invention relates to a method for preparing a monascus preparation by use of xylose mother liquor. The method comprises the following steps: (1) inoculating a monascus strain on a potato dextrose agar (PDA) slant medium, and activating and cultivating to obtain an activated strain; (2) inoculating the activated strain in a PDA seed medium, and cultivating to obtain fermented seeds; (3) inoculating the fermented seeds in a monascus culture medium, and cultivating to obtain the monascus preparation. By taking the xylose mother liquor as a carbon source of a fermentation medium, the method disclosed by the invention can be used for recycling the waste xylose mother liquor; moreover, the method can be used for effectively reducing generation of citrinin, lowering a ratio of citrinin concentration to fermentation broth color value and improving safety of a monascus product.
Description
Technical field
The present invention relates to a kind of method utilizing xylose mother liquid to prepare monascus preparation, belong to technical field of microbial fermentation.
Background technology
Monascus purpureus (Monascus purpureus) is the important production bacterial strain of monascus red pigment and hypolipidemic, its fermentation
Product is also widely used for diet culinary art.Nineteen ninety-five, Blanc is found that nephrotoxin citrinin in red koji fermentation product
(citrinin), citrinin can cause kidney enlargement, tubular ectasia, and epithelial cell is downright bad, the most also has teratogenesis carcinogenic
Effect, therefore the safety of monascus product receives query.
Traditional monascus training method as raw material, has tune to there are about the Monas cuspurpureus Went of half in examining display Chinese market with rice (starch)
Product citrinin content exceeds standard (Yan li., 2012).
In order to reduce the yield of citrinin in monascus product, Monascus anka Nakazawa et sato is carried out researcher substantial amounts of strain improvement and fermentation technology changes
Good research.Have among these and monascus sp bacteria strain is carried out genetic engineering modified (Zhou Lihong, Southern Yangtze University), use 60Co irradiation to lure
Becoming (Jiangxi is flourish perhaps, Southern Yangtze University), having research and utilization Soy hydrolysate is that nitrogen source optimization fermentation technology reduction citrinin yield is (old
Accumulate, Southern Yangtze University).
Such as Chinese patent literature CN102987180A(application number 201210536354.9) disclose a kind of low citrinin Monas cuspurpureus Went
Production method, its use Monascus purpureus fermentation rice form, the processing step of the method is: (1) is big by clean up
Rice is put in the water added with citrinin inhibitor and is soaked；(2) rice after soaking is through draining, steam rice, lowering the temperature, inoculate, then heap
Aerlbic culture is proceeded to after long-pending cultivation；Make by adding water during described aerlbic culture the water content of material be maintained at 50wt%～
70wt%, described in the water added there was added metal-chelator；(3), before described aerlbic culture terminates, oxidant is added to material
To remove the citrinin on Monas cuspurpureus Went；After described aerlbic culture terminates, discharging is dried, and i.e. can get low citrinin Monas cuspurpureus Went.
Chinese patent literature CN1807576A(application number 200610033131.5) disclose a kind of functional without citrinin
The mycelial cultural method of Monascus anka Nakazawa et sato.The method is by regulation culturing room or plastics hothouse air output exhaust air rate or open type ventage
System, with low-concentration acetic acid spray in mycelial growth logarithmic (log) phase and lights Caulis et Folium Oryzae and produces sootiness and stimulate, and strengthen logical
Tolerance, alternating temperature, damping between making the Mycelium culture stage round the clock, regulation culture base material water content, keep starchiness culture base-material to loosen,
Accelerate mycelial growth speed and the synthesis of functional metabolic product Monacolin K and accumulation, the most effectively suppression
The formation of Monascidin A (Citrinin).But the studies above still can not meet the demand in market.
The plant materials such as corn stalk, corn cob, cotton seed hulls, bagasse, Caulis et Folium Oryzae, wheat stalk are carried out pressurized, heated pretreatment
And after acid hydrolysis, then hydrolyzed solution is carried out xylose condensing crystallizing, the waste liquid xylose mother liquid obtained after Crystallization Separation；Wood
Sugar mother solution uses film method to measure the production that its content of reducing sugar is 3%～80% and discards sugar liquid；Generally use high performance liquid chromatography
(HPLC) measuring its soluble sugar is xylose, glucose, the concentration of the various sugar of arabinose, and the Xylose Content of this waste liquid is
25～70%, glucose content is 5～25%, and arabinose content is 5～25%.For above-mentioned xylose mother liquid, there is presently no
Well Application way.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that a kind of method utilizing xylose mother liquid to prepare monascus preparation.
Technical scheme is as follows:
A kind of method utilizing xylose mother liquid to prepare monascus preparation, comprises the steps:
(1) monascus sp bacteria strain is inoculated on PDA slant medium, 25～35 DEG C of activation culture 4～5 days, prepares and live
Change bacterial strain；
(2) activated strains that step (1) prepares is inoculated in PDA seed culture medium, at 25～35 DEG C, 200rpm/min
Under the conditions of, cultivate 2～4 days, prepare fermentation seed；
(3) fermentation seed that step (2) prepares is inoculated in Fermentation Condition of Monascus spp culture medium according to volume ratio 1:10～1:5,
25～35 DEG C, under the conditions of 200rpm/min, cultivate 7～10 days, to obtain final product；
Fermentation Condition of Monascus spp culture medium in described step (3), every liter of component is as follows:
Xylose mother liquid 30～50mL, NaNO32～4g, yeast extract 1g, K2HPO41g, KCl0.5g, MgSO4 7H2O
0.5g, FeSO4 7H2O0.01g, water is settled to 1000mL, pH5～7.
According to currently preferred, every liter of component is as follows:
Xylose mother liquid 30mL, NaNO33g, yeast extract 1g, K2HPO41g, KCl0.5g, MgSO4 7H2O0.5g,
FeSO4 7H2O0.01g, water is settled to 1000mL, pH6.
According to currently preferred, the monascus sp bacteria strain in described step (1) manages purchased from Chinese industrial Microbiological Culture Collection
Center (CICC), culture presevation numbering 40943.Those skilled in the art can select different monascuses according to practical situation
Bacterial strain is prepared monascus preparation.
According to currently preferred, the PDA slant medium in described step (1), every liter of component is as follows:
Peeled potatoes 200g, stripping and slicing, the 600mL that adds water boils 30 minutes, eight layers of filtered through gauze, adds 20g glucose,
20g agar powder, is settled to 1000mL.
According to currently preferred, the PDA seed culture medium in described step (2), every liter of component is as follows:
Peeled potatoes 200g, stripping and slicing, the 600mL that adds water boils 30 minutes, eight layers of filtered through gauze, adds 20g glucose,
It is settled to 1000mL.
According to currently preferred, the component of described xylose mother liquid is as follows, is all weight percentage:
Xylose 40～50%, glucose 10%, arabinose 10%, surplus is water.DNS method record total reducing sugars concentration be 600～
700g/L。
Beneficial effect
1, the present invention utilizes xylose mother liquid as the carbon source of fermentation medium, is turned waste into wealth by xylose mother liquid, and effectively reduces
The generation of citrinin, reduces citrinin concentration and fermentation liquid color valency ratio, improves the safety of monascus product；
2, the present invention uses liquid fermentation method to produce monascus, compares solid fermentation handling by force, and material use efficiency is high,
Save the energy.
Accompanying drawing explanation
Fig. 1 be Monascus Strains fermented after fermentation liquid 500nm fermentation liquid color valency, citrinin concentration and citrinin concentration with
The block diagram of the ratio of fermentation liquid color valency；
Fig. 2 be Monascus Strains fermented after fermentation liquid mould fermentation liquid color valency, citrinin concentration and the Fructus Citri tangerinae of 400nm and 500nm
The block diagram of the ratio of element concentration and fermentation liquid color valency；
Detailed description of the invention
Below in conjunction with embodiment, technical scheme is described further, but institute of the present invention protection domain is not limited to this.
Culture medium
PDA slant medium in embodiment is prepared as follows:
Peeled potatoes 200g, stripping and slicing, the 600mL that adds water boils 30 minutes, eight layers of filtered through gauze, adds 20g glucose,
20g agar powder, is settled to 1000mL, after fully dissolving, and 121 DEG C of high pressure steam sterilization 30min；
PDA seed culture medium in embodiment is prepared as follows:
Peeled potatoes 200g, stripping and slicing, the 600mL that adds water boils 30 minutes, eight layers of filtered through gauze, adds 20g glucose,
It is settled to 1000mL, after fully dissolving, 121 DEG C of high pressure steam sterilization 30min.
Tunning index (fermentation liquid color valency, citrinin concentration) detection method is as follows:
Fermentation liquid color valency detection method: the dehydrated alcohol long-pending with fermentation liquid tetraploid extracts 1h in 60 DEG C of water-baths,
12000rpm/min is centrifuged 5min, takes supernatant, dilutes with appropriate dehydrated alcohol, does blank with dehydrated alcohol, measures
Light absorption value is measured at 400nm and 500nm.
Light absorption value × extension rate under color valency=500nm wavelength；
Or
Color valency=(light absorption value under light absorption value+500nm wavelength under 400nm wavelength) × extension rate
Citrinin detection method: the dehydrated alcohol long-pending with fermentation liquid tetraploid extracts 1h, 12000rpm/min in 60 DEG C of water-baths
Centrifugal 5min, takes supernatant, after the filter membrane microfiltration of 0.22 μm, carries out HPLC analysis.
Bacterium source: purchased from Chinese industrial Microbiological Culture Collection administrative center (CICC), culture presevation numbering 40943.
Xylose mother liquid is purchased from Shandong Longli Biology Science and Technology Co., Ltd, and after testing, the component of xylose mother liquid is as follows, all attaches most importance to
Amount percentage ratio:
Xylose 40～50%, glucose 10%, arabinose 10%, surplus is water.DNS method record content of reducing sugar be 600～
700g/L。
Embodiment 1
A kind of method utilizing xylose mother liquid to prepare monascus preparation, comprises the steps:
(1) monascus sp bacteria strain is inoculated on PDA slant medium, 30 DEG C of activation culture 4 days, prepares activated strains；
(2) activated strains that step (1) prepares is inoculated in PDA seed culture medium, at 30 DEG C, 200rpm/min bar
Under part, cultivate 3 days, prepare fermentation seed；
(3) fermentation seed that step (2) prepares is inoculated in Fermentation Condition of Monascus spp culture medium according to volume ratio 1:10,30
DEG C, under the conditions of 200rpm/min, cultivate 8 days, to obtain final product；
Fermentation Condition of Monascus spp culture medium in described step (3), every liter of component is as follows:
Xylose mother liquid 30mL, NaNO33g, yeast extract 1g, K2HPO41g, KCl0.5g, MgSO4 7H2O0.5g,
FeSO4 7H2O0.01g, water is settled to 1000ml, pH6.0.
Xylose mother liquid Xylose Content is used to be 468g/L through analyzing this；Glucose content is 114g/L；DNS method records also
Raw sugar amount is 618g/L
According to currently preferred, the monascus sp bacteria strain in described step (1) manages purchased from Chinese industrial Microbiological Culture Collection
Center (CICC), culture presevation numbering 40943.Those skilled in the art can select different monascuses according to practical situation
Bacterial strain is prepared monascus preparation.
The fermentation liquid color valency that monascus sp bacteria strain involved in the present invention produces under above-mentioned fermentation process is 17.2U/mL, citrinin
Content is 0.598mg/L, and citrinin concentration is 0.03 μ g/U with the ratio of fermentation liquid color valency.
Embodiment 2
A kind of method utilizing xylose mother liquid to prepare monascus preparation, comprises the steps:
(1) monascus sp bacteria strain is inoculated on PDA slant medium, 30 DEG C of activation culture 4 days, prepares activated strains；
(2) activated strains that step (1) prepares is inoculated in PDA seed culture medium, at 30 DEG C, 200rpm/min bar
Under part, cultivate 4 days, prepare fermentation seed；
(3) fermentation seed that step (2) prepares is inoculated in Fermentation Condition of Monascus spp culture medium according to volume ratio 1:10,30
DEG C, under the conditions of 200rpm/min, cultivate 8 days, to obtain final product；
Fermentation Condition of Monascus spp culture medium in described step (3), every liter of component is as follows:
Xylose mother liquid 20mL, NaNO33g, yeast extract 1g, K2HPO41g, KCl0.5g, MgSO4 7H2O0.5g,
FeSO4 7H2O0.01g, water is settled to 1000ml, pH6.0.
The fermentation liquid color valency that monascus sp bacteria strain involved in the present invention produces under above-mentioned fermentation process is 6.54U/mL, citrinin
Content is 0.548mg/L, and citrinin concentration is 0.083 μ g/U with the ratio of fermentation liquid color valency.
Embodiment 3
A kind of method utilizing xylose mother liquid to prepare monascus preparation, comprises the steps:
(1) monascus sp bacteria strain is inoculated on PDA slant medium, 30 DEG C of activation culture 4 days, prepares activated strains；
(2) activated strains that step (1) prepares is inoculated in PDA seed culture medium, at 30 DEG C, 200rpm/min bar
Under part, cultivate 4 days, prepare fermentation seed；
(3) fermentation seed that step (2) prepares is inoculated in Fermentation Condition of Monascus spp culture medium according to volume ratio 1:10,30
DEG C, under the conditions of 200rpm/min, cultivate 8 days, to obtain final product；
Fermentation Condition of Monascus spp culture medium in described step (3), every liter of component is as follows:
Xylose mother liquid 30mL, NaNO33g, yeast extract 1g, K2HPO41g, KCl0.5g, MgSO4 7H2O0.5g,
FeSO4 7H2O0.01g, water is settled to 1000ml, pH6.0.
The application of the Fermentation Condition of Monascus spp culture medium that above-mentioned low citrinin produces, step is as follows:
The fermentation liquid color valency that monascus sp bacteria strain involved in the present invention produces under above-mentioned fermentation process is 6.3U/mL, and citrinin contains
Amount is 0.47mg/L, and citrinin concentration is 0.075 μ g/U with the ratio of fermentation liquid color valency.
Embodiment 4
A kind of method utilizing xylose mother liquid to prepare monascus preparation, comprises the steps:
(1) monascus sp bacteria strain is inoculated on PDA slant medium, 30 DEG C of activation culture 4 days, prepares activated strains；
(2) activated strains that step (1) prepares is inoculated in PDA seed culture medium, at 30 DEG C, 200rpm/min bar
Under part, cultivate 4 days, prepare fermentation seed；
(3) fermentation seed that step (2) prepares is inoculated in Fermentation Condition of Monascus spp culture medium according to volume ratio 1:10,30
DEG C, under the conditions of 200rpm/min, cultivate 8 days, to obtain final product；
Fermentation Condition of Monascus spp culture medium in described step (3), every liter of component is as follows:
Xylose mother liquid 50mL, NaNO33g, yeast extract 1g, K2HPO41g, KCl0.5g, MgSO4 7H2O0.5g,
FeSO4 7H2O0.01g, water is settled to 1000ml, pH6.0.
The fermentation liquid color valency that monascus sp bacteria strain involved in the present invention produces under above-mentioned fermentation process is 6.5U/mL, and citrinin contains
Amount is 0.58mg/L, and citrinin concentration is 0.09 μ g/U with the ratio of fermentation liquid color valency.
Comparative example 1
(1) monascus sp bacteria strain is inoculated on PDA slant medium, 30 DEG C of activation culture 4 days, prepares activated strains；
(2) activated strains that step (1) prepares is inoculated in PDA seed culture medium, at 30 DEG C, 200rpm/min bar
Under part, cultivate 3 days, prepare fermentation seed；
(3) fermentation seed that step (2) prepares is inoculated in Fermentation Condition of Monascus spp culture medium according to volume ratio 1:10,30
DEG C, under the conditions of 200rpm/min, cultivate 8 days, to obtain final product；
Fermentation Condition of Monascus spp culture medium in described step (3), every liter of component is as follows:
Xylose 30g, NaNO33g, yeast extract 1g, K2HPO41g, KCl0.5g, MgSO4 7H2O0.5g, FeSO4 7H2O
0.01g, water is settled to 1000ml, pH6.0.
The fermentation liquid color valency that monascus sp bacteria strain involved by comparative example 1 produces under above-mentioned fermentation process is 8.7U/mL, citrinin
Content is 0.348mg/L, and citrinin concentration is 0.03 μ g/U with the ratio of fermentation liquid color valency.
Comparative example 2
(1) monascus sp bacteria strain is inoculated on PDA slant medium, 30 DEG C of activation culture 4 days, prepares activated strains；
(2) activated strains that step (1) prepares is inoculated in PDA seed culture medium, at 30 DEG C, 200rpm/min bar
Under part, cultivate 3 days, prepare fermentation seed；
(3) fermentation seed that step (2) prepares is inoculated in Fermentation Condition of Monascus spp culture medium according to volume ratio 1:10,30
DEG C, under the conditions of 200rpm/min, cultivate 8 days, to obtain final product；
Fermentation Condition of Monascus spp culture medium in described step (3) uses PDA culture medium as a comparison, and every liter of component is as follows:
Peeled potatoes 200g, stripping and slicing, the 600mL that adds water boils 30 minutes, eight layers of filtered through gauze, adds 20g glucose,
It is settled to 1000mL.
The fermentation liquid color valency that monascus sp bacteria strain involved by comparative example 2 produces under above-mentioned fermentation process is 20.8U/mL, and Fructus Citri tangerinae is mould
Cellulose content is 8.62mg/L, and citrinin concentration is 0.41 μ g/U with the ratio of fermentation liquid color valency.
Comparative example 3
(1) monascus sp bacteria strain is inoculated on PDA slant medium, 30 DEG C of activation culture 4 days, prepares activated strains；
(2) activated strains that step (1) prepares is inoculated in PDA seed culture medium, at 30 DEG C, 200rpm/min bar
Under part, cultivate 4 days, prepare fermentation seed；
(3) fermentation seed that step (2) prepares is inoculated in Fermentation Condition of Monascus spp culture medium according to volume ratio 1:10,30
DEG C, under the conditions of 200rpm/min, cultivate 8 days, to obtain final product；
Fermentation Condition of Monascus spp culture medium in described step (3), reference literature report method prepare (Xing Shujie, Zhou Ying, Liu Kaihua.
The research of Corncob hydrolysate fermenting and producing monascorubin and condition optimizing [J]. China brewages, 2010(9): 130-132) go out
Corncob hydrolysate, adds Corncob hydrolysate configuration fermentation medium according to reducing sugar amount, and reducing sugar amount is same as in Example 2,
Remaining components unchanged of culture medium.
The fermentation liquid color valency that monascus sp bacteria strain involved by comparative example 3 produces under above-mentioned fermentation process is 5.0U/mL, citrinin
Content is 0.44mg/L, and citrinin concentration is 0.08 μ g/U with the ratio of fermentation liquid color valency.
Interpretation of result
By the above results it can be seen that use xylose mother liquid (embodiment 1) to cultivate (comparative example 1) relative to pure xylose, have
Relatively High color values, keeps relatively low citrinin concentration and color valency ratio simultaneously；Relative to PDA culture medium (comparative example 2), this
Bright can reduce citrinin yield greatly；And relative to method (comparative example 3) present invention of document report, there is higher sending out
Ferment liquid color valency, citrinin concentration and fermentation liquid color valency ratio relatively low (embodiment 2,3,4), meanwhile, when xylose mother liquid adds
When dosage is 30mL/L (embodiment 3), ferment effect is optimal.
Although the application is disclosed above with preferred embodiment, so it is not limited to the scope that the application implements, according to the application
Claims and the simple equivalence change made of description with modify, still fall within the scope of technical scheme.
Claims (5)
1. one kind utilizes the method that xylose mother liquid prepares monascus preparation, it is characterised in that comprise the steps:
(1) monascus sp bacteria strain is inoculated on PDA slant medium, 25～35 DEG C of activation culture 4～5 days, prepares activated strains；
(2) activated strains that step (1) prepares is inoculated in PDA seed culture medium, 25～35 DEG C, under the conditions of 200rpm, cultivate 2～4 days, prepared fermentation seed；
(3) fermentation seed that step (2) prepares is inoculated in Fermentation Condition of Monascus spp culture medium according to volume ratio 1:10～1:5,25～35 DEG C, under the conditions of 200rpm, cultivate 7～10 days, to obtain final product；
Fermentation Condition of Monascus spp culture medium in described step (3), every liter of component is as follows:
Xylose mother liquid 30～50mL, NaNO32～4g, yeast extract 1g, K2HPO41g, KCl 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, water is settled to 1000mL, pH5～7；
The component of described xylose mother liquid is as follows, is all weight percentage:
Xylose 40～50%, glucose 10%, arabinose 10%, surplus is water.
2. the method for claim 1, it is characterised in that the Fermentation Condition of Monascus spp culture medium in described step (3), every liter of component is as follows:
Xylose mother liquid 30mL, NaNO33g, yeast extract 1g, K2HPO41g, KCl 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, water is settled to 1000mL, pH6.
3. the method for claim 1, it is characterised in that the monascus sp bacteria strain in described step (1) is purchased from Chinese industrial Microbiological Culture Collection administrative center, culture presevation numbering 40943.
4. the method for claim 1, it is characterised in that the PDA slant medium in described step (1), every liter of component is as follows:
Peeled potatoes 200g, stripping and slicing, the 600mL that adds water boils 30 minutes, eight layers of filtered through gauze, adds 20g glucose, 20g agar powder, is settled to 1000mL.
5. the method for claim 1, it is characterised in that the PDA seed culture medium in described step (2), every liter of component is as follows:
Peeled potatoes 200g, stripping and slicing, the 600mL that adds water boils 30 minutes, eight layers of filtered through gauze, adds 20g glucose, is settled to 1000mL.
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Non-Patent Citations (3)
| Title |
|---|
| HPLC法测定木糖母液的组成;刘建伟等;《安徽农业科学》;20091231;第37卷(第5期);1881-1882 * |
| Morphology control of Monascus cells and scale-up of pigment fermentation;Hyun Jung Kim, et al.;《Process Biochemistry》;20021231;第38卷;649-655 * |
| 玉米芯水解液发酵生产红曲色素的研究及条件优化;邢淑婕等;《中国酿造》;20101231(第9期);130-133 * |
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