CN104263758A - Production method for food safety type monascorubin - Google Patents
Production method for food safety type monascorubin Download PDFInfo
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- CN104263758A CN104263758A CN201410348963.0A CN201410348963A CN104263758A CN 104263758 A CN104263758 A CN 104263758A CN 201410348963 A CN201410348963 A CN 201410348963A CN 104263758 A CN104263758 A CN 104263758A
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Abstract
The invention discloses a production method for food safety type monascorubin. The production method comprises the steps of subjecting monascorubin to slant seed culture, liquid seed culture and fermentation culture. A transforming agent is added in the fermentation culture process; and the transforming agent is one or more selected from carotin, vitamin C, fulvic acid and EDTA-2Na. The content of citrinin in the monascorubin prepared by the production method is greatly reduced; and the production method has high safety, is simple to operate, is simple and practicable, and is efficient.
Description
The application is the divisional application that the application number submitted on October 29th, 2012 is 201210419781.9, denomination of invention is the patent application of " a kind of production method of food safety monascorubin ".
Technical field
The invention belongs to biotechnology and technical field, be specifically related to a kind of production method of food safety monascorubin.
Background technology
Monascorubin in state-owned long history, pigment have security high, to thermally-stabilised, to substance stain ability is good, tone is scarlet, steady quality and cheap advantage, be synthetic food color and other natural pigment can not be than.But, have Citrinin in the process that monascorubin is produced to supervene, Citrinin is a kind of mycotoxins, has renal toxicity, toxicity is obvious, can cause the symptoms such as the kidney enlargement of laboratory animal, hydrouria, tubular ectasia and epithelial cell degeneration necrosis.Therefore, the security that copper production technique obviously reduces natural monascorubin is produced.
At present, the method reducing Citrinin mainly concentrates on fermentation technology optimization, and induction mutation of bacterium screens, genetically engineered three aspects.First Blanc etc. have studied different nitrogenous sources affects Citrinin, but also have impact on the generation of pigment while reducing Citrinin.Monascus ruber is carried out liquid state fermentation by Hassan etc. under different ventilations and agitation condition, find along with the increase of air flow or the raising of stirring velocity, the biomass of thalline and the output of secondary metabolite increase all 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 on the genes involved of red colouring agent for food, also used as a Chinese medicine Citrinin synthesis and the research of enzyme, being conducive to the deep route of synthesis understanding red colouring agent for food, also used as a Chinese medicine Citrinin like this, providing the foundation theory and technology means for fundamentally controlling citrinin content in red colouring agent for food, also used as a Chinese medicine.But control Citrinin from the means of gene and inevitably will transform the gene of red colouring agent for food, also used as a Chinese medicine, this becomes now than more sensitive transgenosis pigment with regard to making the natural pigment of safety originally.In addition, also have some investigators to utilize chemical process to slough Citrinin, such as, adopt H
2o
2carry out detoxification, although can detoxification completely by process, also can affect the quality of monascorubin in various degree.Monascus cell can be stimulated to discharge active oxygen if found, 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, thus fundamentally can solve the bottleneck problem of pigment red colouring agent for food, also used as a Chinese medicine Citrinin pollution.
Summary of the invention
In view of the deficiencies in the prior art, the present invention is by comparing the SOD enzyme activity change in monascus ruber cell in Active oxygen release and intracellular reactive oxygen species generation scavenge system, the relation that the generation of research active oxygen and pigment, Citrinin synthesize, obtain transforming agent suppress Citrinin to generate or promote the mechanism of the nontoxic conversion of Citrinin, thus the production method of the food safety 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 monascorubin, comprise and monascus ruber is carried out inclined-plane seed culture, liquid seeds is cultivated, fermentation culture, wherein add transforming agent in fermentation culture process, 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 monascorubin, wherein after fermentation culture 24h-96h, adds described transforming agent in ratio gradation in fermented liquid of 0.2-100mg/L.
Further preferably, the step of described inclined-plane seed culture is: monascus ruber is inoculated in solid slant culture base, in 32 DEG C 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 DEG C-32 DEG C; 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: be inoculated in fermention medium by liquid two stage seed by the inoculum size of 6-10%, and at 250ml triangular flask, liquid amount is 100ml, and in 32 DEG C, 200rpm cultivates 6d-8d; After fermentation culture 24h-96h, add described transforming agent in ratio gradation in fermented liquid of 0.2-100mg/L; 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 the research of the conversion of Citrinin orientation and transformation mechanism in Monascus Strains fermenting process first, transforming agent is added in gradation respectively during the fermentation, have detected the production that different fermentations time cell produces secondary metabolite pigment, Citrinin and intracellular superoxide dismutase and free radical, illustrate change that these transforming factors cause desmo enzyme to live and the reason that cell secondary metabolite Citrinin reduces thereof.Add the fermented liquid look valency of different transforming factor and the detected value of Citrinin by contrast, comparing discovery three kinds of transforming factor valencys of checking colors has raising in various degree, and wherein add transforming factor B and C, look valency improves about 50% respectively.On the impact of citrinin content clearly, Citrinin have dropped 94.77%, 94.26%, 96.23% to several transforming factor respectively, reaches desirable effect, and the orientation achieving food safety monascorubin transforms.Through the mensuration to the free radical of SOD enzyme activity and generation in monascus cell, relatively discovery with the addition of transforming agent A, the fermented liquid SOD enzyme activity of B and C is relatively high, free radical significantly increases, this and Citrinin significantly reduce phenomenon and conform to, the interpolation of transforming agent is described, stimulate monascus cell release active oxygen, facilitate cell interior and produce more free radical, serve the effect of similar exciton, change the redox enzyme system in born of the same parents, thus show as in culturing process and produce more SOD enzyme, the resultant quantity of Citrinin has been impelled to reduce, because transforming agent is foodstuff additive, safety non-toxic, and improve the look valency of pigment red colouring agent for food, also used as a Chinese medicine in various degree, ensure that quality and the security of monascorubin, thus fundamentally solve the bottleneck problem that the bent Citrinin of color red pollutes.
The method of existing reduction Citrinin has gene knockout, acid-alkali treatment etc., compared with these methods, the present invention and the monascorubin produced of monascorubin production method in citrinin content significantly reduce, have and have security high, 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. in embodiment 1, transforming agent A is to the influence curve figure of SOD, free radical and Citrinin in red colouring agent for food, also used as a Chinese medicine cell.
Fig. 3. in embodiment 2, transforming agent B is to the influence curve figure of SOD, free radical and Citrinin in red colouring agent for food, also used as a Chinese medicine cell.
Fig. 4. in embodiment 3, transforming agent C is to the influence curve figure of SOD, free radical and Citrinin in red colouring agent for food, also used as a Chinese medicine cell.
Fig. 5. in embodiment 4, transforming agent D is to the influence curve figure of SOD, free radical and Citrinin in red colouring agent for food, also used as a Chinese medicine cell.
concrete embodiment
The raw materials used rice of the present invention is Indica rice, 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 that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment, all technology realized based on foregoing of the present invention all belong to scope of the present invention.
embodiment 1
(1) monascus ruber is inoculated in solid slant culture base, in 32 DEG C 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, be eluted in 100ml physiological saline with physiological saline by thalline and spore, 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 DEG C, secondary seed medium is: rice meal 3%, SODIUMNITRATE 0.5%, potassium primary phosphate 0.25%, magnesium sulfate heptahydrate 0.1%, lactic acid adjusts pH5.5 ~ 6;
(3) the secondary liquid seed after cultivation 48 h is inoculated in fermention medium by the inoculum size of 6-10%, at 300ml triangular flask, liquid amount is 100ml, in 32 DEG C, 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 adjusts pH5.5 ~ 6;
(4) gradation in the fermented liquid after cultivation 24h is added the transforming agent A of 100 μ L 100mg/ml;
(5) during the fermentation, sample determination look valency, Citrinin, superoxide-dismutase (SOD) and free radical is taken out at 12h, 36h, 60h, 84h, 108h, 132h, 144h, 156h, 168h, 192h respectively.Result is see Fig. 2.
embodiment 2
Step (1)-(3) are identical with (1)-(3) in the method for embodiment 1.
(4) gradation in the fermented liquid after cultivation 24h is added the transforming agent B of 100 μ l 2mg/ml;
(5) during the fermentation, sample determination look valency, Citrinin, superoxide-dismutase (SOD) and free radical is taken out at 12h, 36h, 60h, 84h, 108h, 132h, 144h, 156h, 168h, 192h respectively.Result is see Fig. 3.
embodiment 3
Step (1)-(3) are identical with (1)-(3) in the method for embodiment 1.Result is see Fig. 4.
(4) gradation in the fermented liquid after cultivation 24h is added 100ul 5mg/ml transforming agent C.
(5) during the fermentation, respectively take out one bottle at 12h, 36h, 60h, 84h, 108h, 132h, 144h, 156h, 168h, 192h respectively and measure look valency, Citrinin, superoxide-dismutase (SOD) and free radical.Result is see Fig. 4.
embodiment 4
Step (1)-(3) are identical with (1)-(3) in the method for embodiment 1.
(4) gradation in the fermented liquid after cultivation 24h is added 100ul 3.72mg/ml transforming agent D.
(5) during the fermentation, respectively take out one bottle at 12h, 36h, 60h, 84h, 108h, 132h, 144h, 156h, 168h, 192h respectively and measure look valency, Citrinin, superoxide-dismutase (SOD) and free radical.Result is see Fig. 5.
comparative example
Except not adding 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 measures 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, the fermented liquid look valency of different transforming agent and the detected value (see table 1) of Citrinin is added by contrast, clearly can see that four kinds of transforming agents all increase to monascorubin, wherein add transforming agent A, B, the effect of C is all fine, look valency improves about 50% nearly, but the look valency adding D has fallen a bit.In addition, several transforming agent on the impact of citrinin content all clearly, have dropped 82.1%, 95.32%, 93.36%, 91.69% respectively.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 enzyme activity and free radical, we can see the fermented liquid not adding transforming agent clearly, the content of free radical is between 0-84h, slow increase, a peak value is reached to 84h, and SOD enzyme activity becomes downtrending at 0-60h, slowly rise afterwards, this illustrates the rising of generation induction of SOD enzyme activity of free radical.See the variation tendency of Citrinin again, 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, level when 168h drops to beginning, if this illustrates under the condition not adding transforming agent, after spending we Extending culture time to 168h, SOD enzyme activity has and raises by a small margin, Citrinin can decline under the effect of red colouring agent for food, also used as a Chinese medicine self, so when production pigment, the content of Citrinin is unstable, mainly because fermentation period problem.In liquid state fermentation process at ordinary times, general fermentation time at about 144h, Citrinin on the occasion of peak so the Citrinin of monascorubin is in the past all higher.And we at any time can stop fermenting and not affect the content of Citrinin after adding transforming agent.From the above mentioned, describe the generation of Citrinin 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 to find in Fig. 2 that also free radical reaches a peak value before Citrinin produces, but when generation Citrinin, SOD enzyme activity reaches peak value, free radical is but in low ebb, and Citrinin will well below the content of control group Citrinin after testing.And by Fig. 3, Fig. 4, we demonstrate further Citrinin produce to lean on free radical excite this conclusion, because we see, in both of the figures, all peak value is not produced at 0-108h free radical, so lacked the exciton producing Citrinin, Citrinin certain output lacking very after testing.And in Figure 5, transforming agent D is a kind of SOD enzyme activity inhibitor, adding of D greatly inhibits SOD enzyme activity, thus loses the ability of scavenging free radicals, makes free radical basic maintenance between 84-156h constant, not fluctuation, maintain a higher level, this explanation ought during the fermentation, if if SOD is in a very low activity always, the generation of Citrinin can be reduced equally, but also affect the accumulation of pigment simultaneously.
Therefore, we confirm that the generation of Citrinin has close associating with the content of free radical and SOD enzyme activity in red colouring agent for food, also used as a Chinese medicine liquid state fermentation process, want to control Citrinin, must control the content of free radical, keep SOD enzyme activity.Transforming agent add the quality and security that ensure that monascorubin, thus fundamentally solve the bottleneck problem that the bent Citrinin of color red pollutes.
Claims (4)
1. the production method of a food safety monascorubin, comprise and monascus ruber is carried out inclined-plane seed culture, liquid seeds is cultivated, fermentation culture, it is characterized in that: after fermentation culture 24h, add transforming agent in ratio gradation in fermented liquid of 0.2-100mg/L, described transforming agent is EDTA-2Na.
2. the production method of food safety monascorubin according to claim 1, is characterized in that the step of described inclined-plane seed culture is: monascus ruber is inoculated in solid slant culture base, in 32 DEG C 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 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 DEG C-32 DEG C; 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 monascorubin according to claim 1, it is characterized in that the step of described fermentation culture is: be inoculated in fermention medium by liquid two stage seed by the inoculum size of 6-10%, at 250ml triangular flask, liquid amount is 100ml, in 32 DEG C, 200rpm cultivates 6d-8d; After fermentation culture 24h-96h, add described transforming agent in ratio gradation in fermented liquid of 0.2-100mg/L; 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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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104962483A (en) * | 2015-07-20 | 2015-10-07 | 光明乳业股份有限公司 | Monascus purpureus strain and application thereof |
| CN105062894A (en) * | 2015-07-20 | 2015-11-18 | 光明乳业股份有限公司 | Monascus purpureus strain and application thereof |
| CN107858231A (en) * | 2017-12-12 | 2018-03-30 | 湖北工业大学 | Citrinin yield and the method for preparing health-care red rice wine in a kind of reduction red yeast rice |
| CN108486164A (en) * | 2018-03-30 | 2018-09-04 | 福建省农业科学院农业工程技术研究所 | The production technology of low citrinin Monascus color |
| CN109371053A (en) * | 2018-12-24 | 2019-02-22 | 江西科技师范大学 | A kind of High-productive Monascus Pigment Strain construction method |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962483A (en) * | 2015-07-20 | 2015-10-07 | 光明乳业股份有限公司 | Monascus purpureus strain and application thereof |
| CN105062894A (en) * | 2015-07-20 | 2015-11-18 | 光明乳业股份有限公司 | Monascus purpureus strain and application thereof |
| CN105062894B (en) * | 2015-07-20 | 2019-01-29 | 光明乳业股份有限公司 | A kind of purple Monascus Strains and its application |
| CN104962483B (en) * | 2015-07-20 | 2019-01-29 | 光明乳业股份有限公司 | A kind of monascus purpureus bacterial strain and application thereof |
| CN107858231A (en) * | 2017-12-12 | 2018-03-30 | 湖北工业大学 | Citrinin yield and the method for preparing health-care red rice wine in a kind of reduction red yeast rice |
| CN107858231B (en) * | 2017-12-12 | 2021-02-02 | 湖北工业大学 | Method for reducing citrinin output in red yeast rice and preparing health red yeast rice wine |
| CN108486164A (en) * | 2018-03-30 | 2018-09-04 | 福建省农业科学院农业工程技术研究所 | The production technology of low citrinin Monascus color |
| CN109371053A (en) * | 2018-12-24 | 2019-02-22 | 江西科技师范大学 | A kind of High-productive Monascus Pigment Strain construction method |
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