CN104195179A - Producing method of food-safety type monascus color - Google Patents
Producing method of food-safety type monascus color Download PDFInfo
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- CN104195179A CN104195179A CN201410348938.2A CN201410348938A CN104195179A CN 104195179 A CN104195179 A CN 104195179A CN 201410348938 A CN201410348938 A CN 201410348938A CN 104195179 A CN104195179 A CN 104195179A
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Abstract
A producing method of food-safety type monascus color is disclosed. The method includes: subjecting monascus to slope seed culturing, liquid seed culturing and fermentation culturing, wherein a transforming agent is added during the fermentation culturing. The transforming agent is one or more selected from carotene, vitamin C, fulvic acid and EDTA-2Na. The content of citrinin in the monascus color produced by the method is largely reduced. The method has advantages of high safety, simple and convenient operation, and capability of being simple, feasible and efficient, and the like.
Description
The application is dividing an application of the application number submitted on October 29th, 2012 is 201210419781.9, denomination of invention is " a kind of production method of food safety type monascorubin " patent application.
Technical field
The invention belongs to biotechnology and technical field, be specifically related to a kind of production method of food safety type monascorubin.
Background technology
Monascorubin in state-owned long history, that pigment has is safe, to thermally-stabilised, to material colouring power is good, tone is scarlet, steady quality and cheap advantage, be synthetic food color and other natural pigment can not be than.But, in the process that monascorubin is produced, have Citrinin and supervene, Citrinin is a kind of mycotoxins, has renal toxicity, toxicity is obvious, can cause the symptoms such as kidney enlargement, hydrouria, tubular ectasia and epithelial cell degeneration necrosis of laboratory animal.Therefore, produce the security that copper production technique has obviously reduced natural monascorubin.
At present, the method that reduces Citrinin mainly concentrates on fermentation technology optimization, induction mutation of bacterium screening, three aspects of genetically engineered.First Blanc etc. have studied different nitrogenous sources affects Citrinin, but when reducing Citrinin, has also affected the generation of pigment.Hassan etc. carry out Monascus ruber liquid state fermentation under different ventilations and agitation condition, discovery is along with the increase of air flow or the raising of stirring velocity, the biomass of thalline and the output of secondary metabolite all increase to some extent, and the increase ratio of Citrinin is greater than the increase ratio of monascorubin.In recent years, scientific research personnel progressively focuses in the research of the synthetic genes involved of red colouring agent for food, also used as a Chinese medicine Citrinin and enzyme, be conducive to so the deep route of synthesis of understanding red colouring agent for food, also used as a Chinese medicine Citrinin, for fundamentally controlling in red colouring agent for food, also used as a Chinese medicine the citrinin content theory and technology means that provide the foundation.But, from the means of gene, to control Citrinin and inevitably will transform the gene of red colouring agent for food, also used as a Chinese medicine, this has just become now than more sensitive transgenosis pigment the natural pigment of former safety.In addition, also have part Study personnel to utilize chemical process to slough Citrinin, for example, adopt H
2o
2carry out detoxification, although by processing detoxification completely, also can affect in various degree the quality of monascorubin.If found, can stimulate monascus cell to discharge active oxygen, and in intracellular reactive oxygen species generation scavenge system, produce the transforming agent of prozyme, and don't affect pigment red colouring agent for food, also used as a Chinese medicine quality and security, thereby can fundamentally solve the bottleneck problem that pigment red colouring agent for food, also used as a Chinese medicine Citrinin pollutes.
Summary of the invention
In view of the deficiencies in the prior art, the present invention changes by the SOD enzymic activity in Active oxygen release and intracellular reactive oxygen species generation scavenge system in comparison monascus ruber cell, the generation of research active oxygen and pigment, the synthetic relation of Citrinin, obtain transforming agent and suppress the mechanism that Citrinin generated or promoted the nontoxic conversion of Citrinin, thereby the production method of the food safety type monascorubin that a kind of citrinin content is low is provided.
The object of the present invention is achieved like this:
A kind of production method of food safety type monascorubin, comprise monascus ruber is carried out to inclined-plane seed culture, liquid seeds is cultivated, fermentation culture, wherein in fermentation culture process, add transforming agent, described transforming agent is selected from following one or more: carotene, vitamins C, xanthohumic acid, EDTA-2Na.
Preferably, the production method of described food safety type monascorubin, wherein, after fermentation culture 24h-96h, adds described transforming agent in the gradation in fermented liquid of the ratio of 0.2-100mg/L.
Further preferably, the step of described inclined-plane seed culture is: monascus ruber is inoculated in to solid slant culture base, in 32 ℃ of constant temperature culture 5 days, obtains inclined-plane first order seed; The formula of described solid slant culture base is: glucose 6%, peptone 2%, agar 3%, pH5.5 ~ 6.
Further preferably, the step that described liquid seeds is cultivated is: get the inclined-plane seed Guan Yizhi after cultivation, with physiological saline, thalline and spore are eluted in 100mL physiological saline, inoculum size by 10% is inoculated into secondary liquid seed culture medium, and at 250ml triangular flask, liquid amount is 100ml, add 20 granulated glass spherees, in 200rpm, cultivate 24h-48h, obtain liquid two stage seed for 28 ℃-32 ℃; The formula of described secondary liquid seed culture medium is: rice meal 3%, SODIUMNITRATE 0.5%, potassium primary phosphate 0.25%, magnesium sulfate heptahydrate 0.1%.
Further preferably, the step of described fermentation culture is: liquid two stage seed is inoculated in fermention medium by the inoculum size of 6-10%, and at 250ml triangular flask, liquid amount is 100ml, and in 32 ℃, 200rpm cultivates 6d-8d; After fermentation culture 24h-96h, in the gradation in fermented liquid of the ratio of 0.2-100mg/L, add described transforming agent; The formula of described fermention medium is: rice meal 9%, SODIUMNITRATE 0.5%, potassium primary phosphate 0.25%, magnesium sulfate heptahydrate 0.1%.
The present invention has carried out that Citrinin in Monascus Strains fermenting process is directed to be transformed and the research of transformation mechanism first, transforming agent is added in gradation respectively during the fermentation, detected the production of different fermentations time cell generation secondary metabolite pigment, Citrinin and the interior superoxide-dismutase of cell and free radical, illustrated these transforming factors and caused the variation of desmo enzyme work and the reason that cell secondary metabolite Citrinin reduces thereof.By contrast, add the fermented liquid look valency of different transforming factors and the detected value of Citrinin, relatively find that three kinds of transforming factor valencys of checking colors have raising in various degree, wherein add transforming factor B and C, look valency has improved respectively 50% left and right.On the impact of citrinin content clearly, Citrinin has declined respectively 94.77%, 94.26%, 96.23% to several transforming factors, has reached desirable effect, and the orientation that has realized food safety type monascorubin transforms.Through the mensuration to the free radical of SOD enzymic activity and generation in monascus cell, relatively find to have added transforming agent A, the fermented liquid SOD enzymic activity of B and C is relatively high, free radical significantly increases, this significantly reduces phenomenon with Citrinin and conforms to, the interpolation of transforming agent is described, stimulate monascus cell to discharge active oxygen, promoted cell interior to produce more free radical, played the effect of similar exciton, changed the redox enzyme system in born of the same parents, thereby show as and in culturing process, produce more SOD enzyme, impelled the resultant quantity of Citrinin to reduce, because transforming agent is foodstuff additive, safety non-toxic, and improved in various degree the look valency of pigment red colouring agent for food, also used as a Chinese medicine, quality and the security of monascorubin have been guaranteed, thereby fundamentally solve the bottleneck problem that the bent Citrinin of color red pollutes.
The method of existing reduction Citrinin has gene knockout, and acid-alkali treatment etc. are compared with these methods, the present invention and the monascorubin produced of monascorubin production method in citrinin content significantly reduce, have have safe, easy and simple to handle, simple, the advantage such as efficient.
Accompanying drawing explanation
Fig. 1. the influence curve figure of SOD, radical pair Citrinin in monascus ruber cell.
Fig. 2. the influence curve figure of transforming agent A to SOD, free radical and Citrinin in red colouring agent for food, also used as a Chinese medicine cell in embodiment 1.
Fig. 3. the influence curve figure of transforming agent B to SOD, free radical and Citrinin in red colouring agent for food, also used as a Chinese medicine cell in embodiment 2.
Fig. 4. the influence curve figure of transforming agent C to SOD, free radical and Citrinin in red colouring agent for food, also used as a Chinese medicine cell in embodiment 3.
Fig. 5. the influence curve figure of transforming agent D to SOD, free radical and Citrinin in red colouring agent for food, also used as a Chinese medicine cell in embodiment 4.
concrete embodiment
The raw materials used rice of the present invention is long-grained nonglutinous rice morning, and bacterial classification is the bacterial classification that laboratory filters out from Red kojic rice, and transforming agent A is carotene, and transforming agent B is vitamins C, and transforming agent C is xanthohumic acid, and transforming agent D is EDTA-2Na.Form is described in further detail foregoing of the present invention again by the following examples, but this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following embodiment, all technology realizing based on foregoing of the present invention all belong to scope of the present invention.
embodiment 1
(1) monascus ruber is inoculated in to solid slant culture base, in 32 ℃ of constant temperature culture 5 days, obtains inclined-plane first order seed, solid slant culture base: glucose 6%, peptone 2%, agar 3%, pH5.5 ~ 6;
(2) get one, inclined-plane seed after cultivation, with physiological saline, thalline and spore are eluted in 100ml physiological saline, the inoculum size by 10% is inoculated into secondary liquid seed culture medium, at 250ml triangular flask, liquid amount is 100ml, adds 20 granulated glass spherees, in 200rpm, cultivate 24h-48 hour for 32 ℃, secondary seed medium is: rice meal 3%, SODIUMNITRATE 0.5%, potassium primary phosphate 0.25%, magnesium sulfate heptahydrate 0.1%, lactic acid is adjusted pH5.5 ~ 6;
(3) the secondary liquid seeds of cultivating after 48 h is inoculated in fermention medium by the inoculum size of 6-10%, at 300ml triangular flask, liquid amount is 100ml, in 32 ℃, 200rpm cultivates 6d, and fermention medium is: rice meal 9%, SODIUMNITRATE 0.5%, potassium primary phosphate 0.25%, magnesium sulfate heptahydrate 0.1%, lactic acid is adjusted pH5.5 ~ 6;
(4) gradation in the fermented liquid of cultivating after 24h is added to the transforming agent A of 100 μ L 100mg/ml;
(5) during the fermentation, at 12h, 36h, 60h, 84h, 108h, 132h, 144h, 156h, 168h, 192h, take out sample determination look valency, Citrinin, superoxide-dismutase (SOD) and free radical respectively.Result is referring to Fig. 2.
embodiment 2
In the method for step (1)-(3) and embodiment 1, (1)-(3) are identical.
(4) gradation in the fermented liquid of cultivating after 24h is added to the transforming agent B of 100 μ l 2mg/ml;
(5) during the fermentation, at 12h, 36h, 60h, 84h, 108h, 132h, 144h, 156h, 168h, 192h, take out sample determination look valency, Citrinin, superoxide-dismutase (SOD) and free radical respectively.Result is referring to Fig. 3.
embodiment 3
In the method for step (1)-(3) and embodiment 1, (1)-(3) are identical.Result is referring to Fig. 4.
(4) gradation in the fermented liquid of cultivating after 24h is added to 100ul 5mg/ml transforming agent C.
(5) during the fermentation, at 12h, 36h, 60h, 84h, 108h, 132h, 144h, 156h, 168h, 192h, respectively take out one bottle respectively and measure look valency, Citrinin, superoxide-dismutase (SOD) and free radical.Result is referring to Fig. 4.
embodiment 4
In the method for step (1)-(3) and embodiment 1, (1)-(3) are identical.
(4) gradation in the fermented liquid of cultivating after 24h is added to 100ul 3.72mg/ml transforming agent D.
(5) during the fermentation, at 12h, 36h, 60h, 84h, 108h, 132h, 144h, 156h, 168h, 192h, respectively take out one bottle respectively and measure look valency, Citrinin, superoxide-dismutase (SOD) and free radical.Result is referring to Fig. 5.
comparative example
Except do not add any transforming agent in fermentation culture process, other steps are all same as embodiment 1.
The mensuration of Monas cuspurpureus Went fermentation liquid look valency adopts spectrophotometer method; Citrinin content is measured the bioassay standard (GB/T 5009.222-2008) according to citrinin in national standard red colouring agent for food, also used as a Chinese medicine series products, adopts RP-HPLC HPLC-fluorimetric detector (RP-HPLC-FLD) to detect.The mensuration of superoxide-dismutase adopts superoxide dismutase; The mensuration of free radical adopts the method for azanol oxidation.
Different transforming agents is different to red koji fermentation process influence, by contrast, add the fermented liquid look valency of different transforming agents and the detected value (in Table 1) of Citrinin, can clearly see that four kinds of transforming agents all increase to monascorubin, wherein add transforming agent A, B, the effect of C is all fine, and look valency has improved 50% left and right nearly, but adds the look valency of D to fall a bit.In addition, several transforming agents on the impact of citrinin content all clearly, have declined respectively 82.1%, 95.32%, 93.36%, 91.69%.Consider Citrinin and look valency, determine that transforming agent B is optimum transforming agent.
Table 1. look valency and Citrinin measurement result (fermentation 144h)
Through the mensuration (see figure 1) to SOD enzymic activity and free radical, we can see the fermented liquid that does not add transforming agent clearly, the content of free radical is between 0-84h, slowly increase, to 84h, reach a peak value, and SOD enzymic activity becomes downtrending at 0-60h, rising afterwards, the rising of SOD enzymic activity has been induced in the generation of this explanation free radical.See again the variation tendency of Citrinin, very mild between 0-108h, almost there is no the generation of what Citrinin, but after there is peak in free radical, Citrinin increases sharply after 108h, 144h reaches peak value, now free radical also reaches peak value again, SOD is at a low ebb, 168h drops to level while starting, if this explanation is not adding under the condition of transforming agent, as spent we after 168h Extending culture time, SOD enzymic activity has by a small margin and raises, Citrinin can decline under the effect of red colouring agent for food, also used as a Chinese medicine self, so when producing pigment, the content of Citrinin is unstable, mainly because fermentation period problem.In liquid state fermentation process at ordinary times, general fermentation time is in 144h left and right, so Citrinin is all higher on the occasion of the Citrinin of peak monascorubin in the past.And add after transforming agent us can at any time stop fermentation and do not affect the content of Citrinin.From the above mentioned, the generation of Citrinin has been described fully, free radical plays very important exciton effect.
Pass through Fig. 1, contrast the detection figure that all the other several width have added transforming agent, we find in Fig. 2 that also free radical has reached a peak value before Citrinin produces, but when producing Citrinin, SOD enzymic activity has reached peak value, free radical is but in low ebb, and Citrinin will be well below the content of control group Citrinin after testing.And by Fig. 3, Fig. 4, we have further verified that Citrinin produces this conclusion that excites that will lean on free radical, because we see, in this two width figure, at 0-108h free radical, all do not produce peak value, so lacked the exciton that produces Citrinin, after testing Citrinin certain output lacking very.And in Fig. 5, transforming agent D is a kind of SOD activity inhibitor, adding of D greatly suppressed SOD enzymic activity, thereby lost the ability of removing free radical, makes free radical basic maintenance between 84-156h constant, not fluctuation, maintain a higher level, this explanation ought be during the fermentation, if if SOD always in a very low activity, can reduce the generation of Citrinin equally, but also affect the accumulation of pigment simultaneously.
Therefore, it is close associated that we have confirmed that in the red colouring agent for food, also used as a Chinese medicine liquid state fermentation process generation of Citrinin and the content of free radical and SOD enzymic activity has, and wants to control Citrinin, must control the content of free radical, keeps SOD enzymic activity.Transforming agent add quality and the security that has guaranteed monascorubin, thereby fundamentally solve the bottleneck problem that the bent Citrinin of color red pollutes.
Claims (4)
1. the production method of a food safety type monascorubin, comprise monascus ruber is carried out to inclined-plane seed culture, liquid seeds is cultivated, fermentation culture, it is characterized in that: after fermentation culture 24h, in the gradation in fermented liquid of the ratio of 0.2-100mg/L, add transforming agent, described transforming agent is xanthohumic acid.
2. the production method of food safety type monascorubin according to claim 1, is characterized in that the step of described inclined-plane seed culture is: monascus ruber is inoculated in to solid slant culture base, in 32 ℃ of constant temperature culture 5 days, obtains inclined-plane first order seed; The formula of described solid slant culture base is: glucose 6%, peptone 2%, agar 3%, pH5.5 ~ 6.
3. the production method of food safety type monascorubin according to claim 1, it is characterized in that the step that described liquid seeds is cultivated is: get the inclined-plane seed Guan Yizhi after cultivation, with physiological saline, thalline and spore are eluted in 100mL physiological saline, inoculum size by 10% is inoculated into secondary liquid seed culture medium, and at 250ml triangular flask, liquid amount is 100ml, add 20 granulated glass spherees, in 200rpm, cultivate 24h-48h, obtain liquid two stage seed for 28 ℃-32 ℃; The formula of described secondary liquid seed culture medium is: rice meal 3%, SODIUMNITRATE 0.5%, potassium primary phosphate 0.25%, magnesium sulfate heptahydrate 0.1%.
4. the production method of food safety type monascorubin according to claim 1, the step that it is characterized in that described fermentation culture is: liquid two stage seed is inoculated in fermention medium by the inoculum size of 6-10%, at 250ml triangular flask, liquid amount is 100ml, in 32 ℃, 200rpm cultivates 6d-8d; After fermentation culture 24h-96h, in the gradation in fermented liquid of the ratio of 0.2-100mg/L, add described transforming agent; The formula of described fermention medium is: rice meal 9%, SODIUMNITRATE 0.5%, potassium primary phosphate 0.25%, magnesium sulfate heptahydrate 0.1%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748074A (en) * | 2008-12-22 | 2010-06-23 | 东莞市天益生物工程有限公司 | Recombined monascus purpureus with characteristics of low citrinin expression and high haematochrome expression |
| CN101880691A (en) * | 2010-01-14 | 2010-11-10 | 陈豪锋 | Preparation method for brewing functional red yeast rice with low-yield citrinin |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748074A (en) * | 2008-12-22 | 2010-06-23 | 东莞市天益生物工程有限公司 | Recombined monascus purpureus with characteristics of low citrinin expression and high haematochrome expression |
| CN101880691A (en) * | 2010-01-14 | 2010-11-10 | 陈豪锋 | Preparation method for brewing functional red yeast rice with low-yield citrinin |
Non-Patent Citations (2)
| Title |
|---|
| 曹岚等: "红曲生产工艺中桔霉素的控制", 《肉类研究》 * |
| 杨涛等: "高色价低桔霉素红曲色素的提取研究", 《食品科技》 * |
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