CN110499338A - A kind of additive that can improve monascorubin yield while reducing citrinin yield - Google Patents

A kind of additive that can improve monascorubin yield while reducing citrinin yield Download PDF

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CN110499338A
CN110499338A CN201810485171.6A CN201810485171A CN110499338A CN 110499338 A CN110499338 A CN 110499338A CN 201810485171 A CN201810485171 A CN 201810485171A CN 110499338 A CN110499338 A CN 110499338A
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dhc
yield
cit
fermentation
mps
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邵彦春
张梦佳
陈福生
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Huazhong Agricultural University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/66—Aspergillus

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Abstract

The invention discloses the additives that a kind of raising Monascus Pigments yield reduces citrinin yield simultaneously --- dihydrocoumarin (DihydrocOumarin, DHC), add appropriate DHC in the medium, can be improved Monascus fermentation pigment (Monascus pigments, MPs) yield, while inhibit citrinin (CitRinin, CIT) generation.It is demonstrated experimentally that rising within the scope of a certain concentration with DHC concentration, MPs color value shows as downward trend after first rising, and CIT is increased with DHC concentration and reduced.Under the conditions of the solid-state of different scales and liquid state fermentation, the yield of MPs can be effectively improved by adding DHC in the medium, while reduce the yield of CIT.Wherein, in industrial 60t liquid fermentation fermentor, the yield about 10% for improving MPs can be stablized, reduce CIT yield about 90%.

Description

A kind of additive that can improve monascorubin yield while reducing citrinin yield
Technical field
The invention belongs to technical field of food biotechnology, be related to a kind of raising monascorubin (Monascus pigments, MPs) yield, at the same reduce citrinin (CitRinin, CIT) yield additive.
Background technique
Monascus, also known as monascus (Monascus spp.) are the important dual-purpose of drug and food microbial resources in China.Because of its energy Generation food color MPs and drop blood ester ingredient Mo Nakelin K (Monacolin K, MK) etc. beneficial to secondary metabolite, therefore It is widely used in producing food color, Chinese patent drug and health care product etc..But nineteen ninety-five France scholar Blanc finds, Mou Xiehong Aspergillus can generate a kind of mycotoxin --- CIT and pollute its fermented product (red yeast rice), to make the safety of product by matter It doubts.Therefore, how to promote the generation of MPs and MK etc., reduce CIT and generate the important research direction for having become Monascus.
Studies have shown that can be by changing medium component, adjusting the yeastings such as pH, the side such as mutation breeding and genetic engineering Method improves the beneficial metabolic product of Monascus, while reducing the generation of CIT.But the method effect for changing yeasting is not shown It writes, process is complicated and time-consuming；Mutation breeding technologies are more mature but without specific directionality, and heavy workload still has after mutation It may reply, it cannot be guaranteed that effect；Although genetic engineering has certain specificity and directionality, but people are to genetic engineering Obtained product is not to trust very much, and the safety of genetically modified organism is still to be assessed, needs more effective by other and pacifies thus Full method regulates and controls the metabolism of Monascus, improves the generation of MPs and MK etc., while inhibiting the generation of CIT.
Present invention discover that a kind of food additives dihydrocoumarin (DihydrocOumarin, DHC), MPs can be significantly improved Yield, while CIT yield is reduced, compared to the above method, there are easy to operate, the advantages such as at low cost.
Summary of the invention
The present invention provides a kind of additive for improving MPs yield while CIT being inhibited to synthesize, which is a kind of food additive Add agent dihydrocoumarin (DHC).
The preparation method of DHC are as follows: the DHC of liquid is dissolved in suitable dehydrated alcohol, is completely dissolved to DHC, 0.22 μ is crossed It after m miillpore filter degerming, is added in solid-state or liquid red yeast rice bacterium culture medium according to a certain concentration, inoculation Monascus is cultivated.
In the above method, in addition to adding certain density DHC, the condition of culture and culture formula of Monascus are without being appointed What is adjusted.
The experiment proves that addition DHC can be effectively improved under the conditions of different scales solid-state and liquid state fermentation MPs yield reduces CIT yield simultaneously.Wherein, it can stablize under the conditions of industrial 60t extensive liquid state fermentation and improve about 10% MPs yield, while reducing about 90% CIT yield.
Detailed description of the invention
Influence of Fig. 1 various concentration DHC to different Monascus Strains MPs color value in rice medium
Wherein, the label in accompanying drawing is: A. feathering Monascus (M.pilosus) MS-1 color value intracellular in rice medium；B. Feathering Monascus (M.pilosus) YDJ color value intracellular in rice medium.
" 0mM, 2mM, 4mM " etc. indicate that DHC concentration is 0mM, 2mM, 4mM in the box of the upper right corner in figure.
Influence of Fig. 2 various concentration DHC to different Monascus Strains MPs color value in different liquids culture medium
Wherein, the label in accompanying drawing is: the color value of A. Monascus ruber (M.ruber) M7 fermentation liquid in PDB；B.M7 is in PDB In color value intracellular；C.M7 fermentation liquid color value in IM-1；D.M7 color value intracellular in IM-1；E. purple Monascus (M.purpureus) JM fermentation liquid color value in PDB；F.JM color value intracellular in PDB；G.JM fermentation liquid color value in IM-1； H.JM color value intracellular in IM-1；I. purple Monascus (M.purpureus) R6 fermentation liquid color value in PDB；J.R6 is in PDB Color value intracellular；K.R6 fermentation liquid color value in IM-2；L.R6 color value intracellular in IM-2.
" 0mM, 2mM, 4mM " etc. indicate that DHC concentration is 0mM, 2mM, 4mM in the box of the upper right corner in figure.
Influence of Fig. 3 various concentration DHC to different Monascus Strains liquid state fermentation CIT
Wherein, the label in accompanying drawing is: A.JM fermentation liquid CIT in PDB；B.R6 fermentation liquid CIT in PDB.
Fig. 4 DHC produces the influence of CIT to R6 in 15L fermentation cylinder for fermentation
Fig. 5 DHC to R6 60t fermentation cylinder for fermentation MPs yield influence
Wherein, the label in accompanying drawing is: " CK " indicates blank control group in the box of the upper right corner in figure, the fermentation for being not added with DHC is taken The average value of tank color value；" 0.1mM DHC " indicates the experimental group of addition 0.1mM DHC.
Influence of Fig. 6 DHC to R6 industry 60t fermentation MPs and CIT yield
Wherein, the label in accompanying drawing is: influence of the A.DHC to MPs yield；Influence of the B.DHC to CIT yield.
" CK " indicates blank control group in the box of the upper right corner in figure, that is, is not added with the fermentor of DHC；" 0.1mM DHC " expression adds Add the experimental group of 0.1mM DHC.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is all from unless otherwise specified Conventional biochemical reagent quotient.Present invention be described in more detail with reference to the accompanying drawing.
Influence of the 1 various concentration DHC of embodiment to different Monascus Strains MPs color value in rice medium: by specification Setting debita spissitudo makes an addition in the rice medium after sterilizing after DHC is crossed film sterilizing by method, and MS-1 and YDJ are made 106Cfu/mL spore suspension is inoculated in rice medium respectively by 1% inoculum concentration and carries out solid state fermentation, and preceding 3d is at 30 DEG C Fermentation, 3d are placed on 28 DEG C of fermentations and dry red yeast rice in 50 DEG C of baking ovens after fermentation to 10d, measure its color value intracellular. Experiment discovery, when DHC concentration is 2mM, MS-1 and YDJ solid state fermentation color value intracellular reaches maximum, set dense in experiment It spends in range, it is regular: to be increased with DHC concentration, solid State Fermentation of Monascus color value intracellular first rises declines (Fig. 1) afterwards.
Color value method intracellular is detected in examples detailed above to include the following steps: to grind broken mycelium after taking 0.05g to dry It is fitted into centrifuge tube, 1.5mL 70% (v/v) ethyl alcohol is added, ultrasound 10min, is put into 45 DEG C of water-baths and extracts after shaking uniformly 30min is centrifuged 5min under 10000r/min revolving speed, collects supernatant.1mL 70% (v/v) ethyl alcohol is added into precipitating, Aforesaid operations are repeated, supernatant is collected.0.5mL 70% (v/v) ethyl alcohol is added into precipitating, repeatedly aforesaid operations, in collection Clear liquid.The supernatant extracted three times is summarized, total volume is in terms of 3mL (1.5+1+0.5), 70% (v/v) second of leaching liquor Alcohol dilutes suitable multiple, with ultraviolet-uisible spectrophotometer using 70% (v/v) ethyl alcohol as blank control, respectively in 390nm, Light absorption value (OD value) is measured under 460nm, 505nm wavelength.Calculation formula: color value (U/g)=OD × extension rate × 3 ÷ intracellular 0.05。
Influence of the 2 various concentration DHC of embodiment to different Monascus Strains MPs color value in different liquids culture medium: normally Setting debita spissitudo makes an addition in the culture medium after sterilizing after DHC is crossed film sterilizing by bright book method, is inoculated with Monascus M7, JM, R6 It is made 106Cfu/mL spore suspension is inoculated in PDB culture medium or industrial culture medium (IM-1, IM-2) by 1% inoculum concentration respectively In, its color value is measured after 32 DEG C of 200r/min fermentation 7d.Experiment discovery, M7 is 32 DEG C in the PDB and IM-1 of the DHC containing various concentration 200r/min fermentation 7d, making the highest DHC concentration of fermentation liquid color value is respectively 4mM and 1mM, keeps the highest DHC of color value intracellular dense Degree is respectively 2mM and 2mM；JM 32 DEG C of 200r/min fermentation 7d in the PDB and IM-1 of the DHC containing various concentration, make fermentation liquid color The highest DHC concentration of valence is respectively 4mM and 1mM, and making the highest DHC concentration of color value intracellular is respectively 2mM and 0.5mM；R6 is containing 32 DEG C of 200r/min fermentation 7d in the PDB and IM-2 of various concentration DHC, making the highest DHC concentration of fermentation liquid color value is respectively 4mM And 0.1mM, making the highest DHC concentration of color value intracellular is respectively 2mM and 0.1mM.There is universal law: testing set concentration model In enclosing, with the raising of DHC, Monascus fermentation MPs yield first increases to be declined afterwards, i.e. low concentration DHC can promote MPs and generate (Fig. 2).
Fermentation liquid color value method is detected in examples detailed above to include the following steps: after collecting fermentation liquid, is diluted with distilled water Certain multiple, with ultraviolet-uisible spectrophotometer, using distilled water as blank control, respectively in 390nm, 460nm, 505nm wavelength Lower measurement light absorption value (OD value).Calculation formula: fermentation liquid color value (U/mL)=OD value × extension rate.
Color value method intracellular is detected in examples detailed above with the detection method of color value intracellular in example 1.
Influence of the 3 various concentration DHC of embodiment to different Monascus Strains liquid state fermentation CIT: JM and R6 are made 106cfu/ ML spore suspension is inoculated in the PDB containing various concentration DHC respectively by 1% inoculum concentration, and 32 DEG C of 200r/min fermentation 7d are surveyed Determine CIT yield.Experiment discovery increases, Monascus (JM, R6) liquid state fermentation in experimental setup concentration range with DHC concentration CIT yield significantly reduces (Fig. 3).
In examples detailed above, CIT detection method includes the following steps: after fermentation, Monascus fermentation to be collected into dry Dry red yeast rice powder, accurately weighs 0.2g, with 80% first after the dry rear mycelium or solid state fermentation for grinding and being uniformly mixed Alcohol extracting, solid-liquid ratio 1: 10 (w/v).Ultrasonic extraction 40min (40kHz) after mixing, under 12000r/min revolving speed from Heart 5min, takes supernatant.80% methanol of 10 times of volumes, ultrasonic extraction 20min after mixing is added into precipitating again.Exist again It is centrifuged 5min under 12000r/min revolving speed, merge the supernatant extracted twice and is settled to 10mL, liquid crosses 0.22 μm of filter membrane, uses It is analyzed in HPLC.
Above-mentioned efficient liquid phase chromatographic analysis condition are as follows: Shimadzu LC-20AT high performance liquid chromatograph, C18Chromatographic column, PDA detection Device, Detection wavelength 331nm, mobile phase are acetonitrile: 0.1% phosphoric acid=70: 30,30 DEG C of column temperature, flow velocity 0.8mL/min, and sample volume 20μL。
Embodiment 4DHC produces the influence of CIT to R6 in 15L fermentation cylinder for fermentation: R6 is made 106Cfu/mL spore suspension, It is inoculated in seed liquid culture medium by 10% inoculum concentration, seed liquor is made in fermentation 48h, then is inoculated with seed liquor with 10% inoculum concentration In 15L fermentor, experimental group adds the DHC after 0.1mM sterilizing, measures CIT after fermentation.Experiment discovery, adds the reality of DHC Testing group CIT yield reduces by 42% or so (Fig. 4) than blank control.
In examples detailed above, CIT detection method is identical as detection method in embodiment 3.
Influence of the embodiment 5DHC to R6 industry 60t fermentation MPs and CIT yield: R6 is inoculated in industrial 60t fermentor In, experimental group adds 0.1mM to cross the DHC after film sterilizing, and respectively for 24 hours, 48h and 72h measure MPs color value and CIT.By experimental group with The color value data of daily fermentation monitoring compare, and calculate average value, as a result, it has been found that, 22,30,46,54,70,78,94,102 With 118 (h), addition DHC tank color value distinguish than blank tank it is high by 15.34%, 24.95%, 7.99%, 5.6%, 7.58%, 10.73%, 12.05%, 9.3%, 9.07% (Fig. 5).Experiment discovery, the experimental group MPs yield for adding DHC compare blank control (CK) high about 10%, CIT yield reduces about 90% (Fig. 6) than blank control.
In examples detailed above, in addition to adding DHC, other operations are with industrial production indifference, and color value measuring method is referring to work Factory's routine testing method, CIT detection method is referring to GB 5009.222-2016.
The embodiment of the present invention is described in detail above, but the present invention is not restricted to above embodiments.This Outside, made equivalent change and modification in the case where not departing from method and range of the invention, all should be contained within the scope of the invention.

Claims (5)

1. a kind of monascorubin yield that improves reduces the additive of citrinin yield simultaneously.
2. additive as described in claim 1, it is characterised in that: additive is food additives --- dihydrocoumarin (DihydrocOumarin, DHC).
3. DHC as claimed in claim 2, it is characterised in that: DHC is dissolved in suitable dehydrated alcohol, wait be completely dissolved, warp After 0.22 μm of miillpore filter degerming, it is added directly in the culture medium of Monascus.
4. DHC additive amount as claimed in claim 3, it is characterised in that: the optimum concentration of DHC in the film solid medias such as rice For 0.1-2mM；The optimum concentration of DHC is 2-4mM in the liquid culture mediums such as PDB.
5. the red yeast rice and its Related product that are obtained by claim 4 the method processing post-fermentation.
CN201810485171.6A 2018-05-17 2018-05-17 A kind of additive that can improve monascorubin yield while reducing citrinin yield Pending CN110499338A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101774990A (en) * 2010-02-09 2010-07-14 上海应用技术学院 Method for preparing dihydrocoumarin without phenol
CN104498528A (en) * 2014-12-25 2015-04-08 天津科技大学 Monascus fermentation method for low-yield citrinin and high-yield monascorubin
CN106755171A (en) * 2017-02-09 2017-05-31 江南大学 A kind of method for promoting red yeast rice strains liquid fermentation to produce red yeast rice citraurin
WO2017108799A1 (en) * 2015-12-23 2017-06-29 Dsm Ip Assets B.V. Production of sterols in modified yeast
CN106967665A (en) * 2017-05-24 2017-07-21 福州大学 A kind of method that external source adds antioxidant liquid state fermentation monascus low-yield citrinin

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101774990A (en) * 2010-02-09 2010-07-14 上海应用技术学院 Method for preparing dihydrocoumarin without phenol
CN104498528A (en) * 2014-12-25 2015-04-08 天津科技大学 Monascus fermentation method for low-yield citrinin and high-yield monascorubin
WO2017108799A1 (en) * 2015-12-23 2017-06-29 Dsm Ip Assets B.V. Production of sterols in modified yeast
CN106755171A (en) * 2017-02-09 2017-05-31 江南大学 A kind of method for promoting red yeast rice strains liquid fermentation to produce red yeast rice citraurin
CN106967665A (en) * 2017-05-24 2017-07-21 福州大学 A kind of method that external source adds antioxidant liquid state fermentation monascus low-yield citrinin

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YAN HU等: "NAD+-dependent HDAC inhibitor stimulates Monascus pigment production but inhibit citrinin", 《AMB EXPRESS》 *

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