CN104195179B - A kind of production method of food safety monascorubin - Google Patents
A kind of production method of food safety monascorubin Download PDFInfo
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- CN104195179B CN104195179B CN201410348938.2A CN201410348938A CN104195179B CN 104195179 B CN104195179 B CN 104195179B CN 201410348938 A CN201410348938 A CN 201410348938A CN 104195179 B CN104195179 B CN 104195179B
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Abstract
The invention discloses the production method of a kind of food safety monascorubin, inclined-plane seed culture is carried out including by monascus ruber, liquid seeds is cultivated, fermentation culture, wherein in fermentation culture process add transforming agent, described transforming agent be selected from one or more: carotene, vitamin C, fulvic acid, EDTA-2Na.The advantages such as in the monascorubin that the inventive method is produced, citrinin content is greatly lowered, and has and has safety high, easy and simple to handle, simple, efficient.
Description
The divisional application of patent application that the application is the application number submitted on October 29th, 2012 is 201210419781.9, denomination of invention is " production method of a kind of food safety monascorubin ".
Technical field
The invention belongs to biological engineering and technical field, be specifically related to the production method of a kind of food safety monascorubin.
Background technology
Monascorubin in state-owned long history, pigment have safety high, to thermally-stabilised, good to substance stain ability, tone is scarlet, steady quality and cheap advantage, be synthetic food color and other natural pigment do not caned than.But, the process that monascorubin produces having citrinin and supervenes, citrinin is a kind of mycotoxin, has nephrotoxicity, and toxicity is obvious, can cause the symptoms such as the kidney enlargement of laboratory animal, hydrouria, tubular ectasia and epithelial cell degeneration necrosis.Therefore, produce copper production technology and obviously reduce the safety of natural monascorubin.
At present, the method reducing citrinin is concentrated mainly on fermentation technology optimization, and induction mutation of bacterium screens, three aspects of genetic engineering.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 fermentation by Hassan etc. under different ventilations and stirring condition, find the increase along with ventilation or the raising of mixing speed, the Biomass of thalline and the yield 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 related gene to the synthesis of Monas cuspurpureus Went citrinin and enzyme, so be conducive to the deep route of synthesis understanding Monas cuspurpureus Went citrinin, provide the foundation theory and technology means for fundamentally controlling citrinin content in Monas cuspurpureus Went.But, controlling citrinin from the means of gene and inevitably the gene of Monas cuspurpureus Went is transformed, this just makes the natural pigment of safety originally become now than more sensitive transgenic pigment.It addition, also have some investigators to utilize chemical method to slough citrinin, for instance adopt H2O2Carry out detoxification, although by process can detoxification completely, but also can affect the quality of monascorubin in various degree.Monascus cell release active oxygen can be stimulated if found, and in intracellular reactive oxygen species generation scavenging system, produce the transforming agent of compound enzyme, and don't affect pigment Monas cuspurpureus Went quality and safety, such that it is able to fundamentally solve the bottleneck problem that pigment Monas cuspurpureus Went citrinin pollutes.
Summary of the invention
In view of the deficiencies in the prior art, the present invention SOD enzyme activity change by comparing in monascus ruber cell in Active oxygen release and intracellular reactive oxygen species generation scavenging system, the relation that the generation of research active oxygen synthesizes with pigment, citrinin, obtain transforming agent suppress citrinin to generate or promote the mechanism of the nontoxic conversion of citrinin, thus providing the production method of food safety monascorubin that a kind of citrinin content is low.
The object of the present invention is achieved like this:
A kind of production method of food safety monascorubin, inclined-plane seed culture is carried out including by monascus ruber, liquid seeds is cultivated, fermentation culture, wherein in fermentation culture process add transforming agent, described transforming agent be selected from one or more: carotene, vitamin C, fulvic acid, EDTA-2Na.
Preferably, the production method of described food safety monascorubin, wherein after fermentation culture 24h-96h, add described transforming agent in the gradation in fermentation liquid of the ratio of 0.2-100mg/L.
It is further preferred that the step of described inclined-plane seed culture is: monascus ruber to be inoculated in solid slant culture base, in 32 DEG C of constant temperature culture 5 days, obtain 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: take the inclined-plane seed Guan Yizhi after cultivation, with normal saline, thalline and spore are eluted in 100mL normal saline, being inoculated into secondary liquid seed culture medium by the inoculum concentration of 10%, at 250ml triangular flask, liquid amount is 100ml, add 20 beades, 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%, sodium nitrate 0.5%, potassium dihydrogen phosphate 0.25%, Magnesium sulfate heptahydrate 0.1%.
It is further preferred that the step of described fermentation culture is: be inoculated in fermentation medium by liquid two stage seed by the inoculum concentration of 6-10%, 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 the gradation in fermentation liquid of the ratio of 0.2-100mg/L；The formula of described fermentation medium is: rice meal 9%, sodium nitrate 0.5%, potassium dihydrogen 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 sweat first, distinguish gradation during the fermentation and add transforming agent, have detected different fermentations time cell and produce the production of secondary metabolite pigment, citrinin and intracellular superoxide dismutase and free radical, illustrate these transforming factors and cause the change of desmoenzyme work and the reason of cell secondary metabolite citrinin reduction thereof.By contrasting the detected value of fermentation liquid color valency and the citrinin adding different transforming factor, comparing three kinds of transforming factor valencys of checking colors of discovery has raising in various degree, wherein adds transforming factor B and C, and color valency has been respectively increased about 50%.The impact of citrinin content it is obvious that citrinin have dropped 94.77%, 94.26%, 96.23% respectively, has been reached desirable effect by several transforming factors, it is achieved that the directed conversion of food safety monascorubin.Through to the mensuration of the free radical of SOD enzyme activity and generation in monascus cell, relatively discovery with the addition of transforming agent A, the fermentation liquid SOD enzyme activity of B and C is of a relatively high, free radical significantly increases, this significantly reduces phenomenon and is consistent with citrinin, the interpolation of transforming agent is described, stimulate monascus cell release active oxygen, promote that cell interior produces more free radical, serve the effect of similar exciton, change the oxidoreduction enzyme system in born of the same parents, thus showing as, incubation produces more SOD enzyme, the synthetic quantity having promoted citrinin reduces, owing to transforming agent is food additive, safety non-toxic, and improve the color valency of pigment Monas cuspurpureus Went in various degree, ensure that the quality of monascorubin and safety, thus fundamentally solving the bottleneck problem that color red song citrinin pollutes.
The method of existing reduction citrinin has gene knockout, and acid-alkali treatment etc., compared with these methods, the present invention and the monascorubin produced of monascorubin production method in citrinin content be greatly lowered, have and have safety 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 Monas cuspurpureus Went cell.
Fig. 3. in embodiment 2, transforming agent B is to the influence curve figure of SOD, free radical and citrinin in Monas cuspurpureus Went cell.
Fig. 4. in embodiment 3, transforming agent C is to the influence curve figure of SOD, free radical and citrinin in Monas cuspurpureus Went cell.
Fig. 5. in embodiment 4, transforming agent D is to the influence curve figure of SOD, free radical and citrinin in Monas cuspurpureus Went cell.
It is embodied as example
The raw materials used rice of the present invention is Indica rice, and strain is the strain that laboratory filters out from Monas cuspurpureus Went, and transforming agent A is carotene, and transforming agent B is vitamin C, and transforming agent C is fulvic acid, and transforming agent D is EDTA-2Na.The foregoing of the present invention is described in further detail by form more by the following examples, but this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to below example, and all technology realized based on foregoing of the present invention belong to the 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) take one, inclined-plane seed after cultivation, with normal saline, thalline and spore are eluted in 100ml normal saline, are inoculated into secondary liquid seed culture medium by the inoculum concentration of 10%, at 250ml triangular flask, liquid amount is 100ml, adds 20 beades, in 200rpm, cultivate 24h-48 hour for 32 DEG C, secondary seed medium is: rice meal 3%, sodium nitrate 0.5%, potassium dihydrogen phosphate 0.25%, Magnesium sulfate heptahydrate 0.1%, lactic acid adjusts pH5.5 ~ 6；
(3) the secondary liquid seed after cultivation 48h is inoculated in fermentation medium by the inoculum concentration of 6-10%, at 300ml triangular flask, liquid amount is 100ml, in 32 DEG C, 200rpm cultivates 6d, and fermentation medium is: rice meal 9%, sodium nitrate 0.5%, potassium dihydrogen phosphate 0.25%, Magnesium sulfate heptahydrate 0.1%, lactic acid adjusts pH5.5 ~ 6；
(4) gradation in the fermentation liquid after cultivation 24h is added the transforming agent A of 100 μ L100mg/ml；
(5) during the fermentation, sample determination color valency, citrinin, superoxide dismutase (SOD) and free radical are taken out at 12h, 36h, 60h, 84h, 108h, 132h, 144h, 156h, 168h, 192h respectively.Result is referring to Fig. 2.
Embodiment 2
Step (1)-(3) are identical with (1)-(3) in the method for embodiment 1.
(4) gradation in the fermentation liquid after cultivation 24h is added the transforming agent B of 100 μ l2mg/ml；
(5) during the fermentation, sample determination color valency, citrinin, superoxide dismutase (SOD) and free radical are taken out at 12h, 36h, 60h, 84h, 108h, 132h, 144h, 156h, 168h, 192h respectively.Result is referring to Fig. 3.
Embodiment 3
Step (1)-(3) are identical with (1)-(3) in the method for embodiment 1.Result is referring to Fig. 4.
(4) gradation in the fermentation liquid after cultivation 24h is added 100ul5mg/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 color valency, citrinin, superoxide dismutase (SOD) and free radical.Result is referring to Fig. 4.
Embodiment 4
Step (1)-(3) are identical with (1)-(3) in the method for embodiment 1.
(4) gradation in the fermentation liquid after cultivation 24h is added 100ul3.72mg/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 color valency, citrinin, superoxide dismutase (SOD) and free radical.Result is referring to Fig. 5.
Comparative example
Except being added without any transforming agent in fermentation culture process, other steps are all same as embodiment 1.
The mensuration of Monas cuspurpureus Went fermentation liquid color valency adopts spectrophotometer method；Citrinin content measures according to the bioassay standard (GB/T5009.222-2008) of citrinin in national standard Monas cuspurpureus Went series products, adopts RP-HPLC HPLC-fluorescence detector (RP-HPLC-FLD) detection.The mensuration of superoxide dismutase adopts superoxide dismutase；The method measuring employing azanol oxidation of free radical.
Different transforming agents is different to red koji fermentation process influence, by contrasting the detected value (see table 1) of fermentation liquid color valency and the citrinin adding different transforming agent, can clearly see that monascorubin is all increased by four kinds of transforming agents, wherein add transforming agent A, B, the effect of C is all fine, and color valency improves nearly about 50%, but any has dropped in the color valency adding D.It addition, several transforming agents on the impact of citrinin content all it is obvious that have dropped 82.1%, 95.32%, 93.36%, 91.69% respectively.Consider citrinin and color valency, it is determined that transforming agent B is optimum transforming agent.
Table 1. color valency and citrinin measurement result (fermentation 144h)
Through the mensuration (see figure 1) to SOD enzyme activity and free radical, we are not it is clear that add the fermentation liquid of transforming agent, the content of free radical is between 0-84h, it is slowly increased, a peak value is reached to 84h, and SOD enzyme activity becomes downward trend at 0-60h, slowly rising afterwards, this illustrates the generation rising induction of SOD enzyme activity of free radical.Variation tendency referring again to citrinin, very mild between 0-108h, generation almost without 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 during beginning, if this illustrates when being added without transforming agent, as spent we to after 168h Extending culture time, SOD enzyme activity has and raises by a small margin, citrinin can decline under the effect of Monas cuspurpureus Went self, so when producing pigment, the content of citrinin is unstable, it is primarily due to fermentation period problem.In liquid fermentation process at ordinary times, general fermentation time at about 144h, citrinin on the occasion of peak so the citrinin of conventional monascorubin is all higher.And we can at any time stop fermentation and not affect the content of citrinin after adding transforming agent.From the above mentioned, describing the generation of citrinin fully, free radical plays very important exciton effect.
Pass through Fig. 1, contrast all the other several width and add the detection figure of transforming agent, we have found that in Fig. 2 that also free radical has reached a peak value before citrinin produces, but when producing citrinin, SOD enzyme activity has reached peak value, free radical is but in low ebb, and citrinin will well below the content of matched group citrinin after testing.And by Fig. 3, Fig. 4, we demonstrate citrinin further and produce to lean on this conclusion that excites of free radical, because we have seen that, in both of the figures, peak value is not all produced at 0-108h free radical, so having lacked the exciton producing citrinin, the certain yield of citrinin lacking very after testing.And in Figure 5, transforming agent D is a kind of SOD enzyme activity inhibitor, the addition of D greatly inhibits SOD enzyme activity, thus losing the ability of scavenging free radicals, makes free radical be held essentially constant between 84-156h, not fluctuation, maintain a higher level, this illustrate ought during the fermentation, if if SOD is constantly in a very low activity, the generation of equally possible reduction citrinin, but also affect the accumulation of pigment simultaneously.
Therefore, we demonstrate that the generation of citrinin has close associating with the content of free radical and SOD enzyme activity in Monas cuspurpureus Went liquid fermentation process, wanted to control citrinin, the content of free radical must be controlled, keep SOD enzyme activity.The addition of transforming agent ensure that quality and the safety of monascorubin, thus fundamentally solving the bottleneck problem that color red song citrinin pollutes.
Claims (4)
1. a production method for food safety monascorubin, carries out inclined-plane seed culture including by monascus ruber, and liquid seeds is cultivated, fermentation culture, it is characterized in that: after fermentation culture 24h, adding transforming agent in the gradation in fermentation liquid of the ratio of 5mg/L, described transforming agent is fulvic acid.
2. the production method of food safety monascorubin according to claim 1, it is characterised 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: take the inclined-plane seed Guan Yizhi after cultivation, with normal saline, thalline and spore are eluted in 100mL normal saline, being inoculated into secondary liquid seed culture medium by the inoculum concentration of 10%, at 250ml triangular flask, liquid amount is 100ml, add 20 beades, 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%, sodium nitrate 0.5%, potassium dihydrogen 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 fermentation medium by liquid two stage seed by the inoculum concentration 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 the gradation in fermentation liquid of the ratio of 5mg/L；The formula of described fermentation medium is: rice meal 9%, sodium nitrate 0.5%, potassium dihydrogen phosphate 0.25%, Magnesium sulfate heptahydrate 0.1%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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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| 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 |
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| 红曲生产工艺中桔霉素的控制;曹岚等;《肉类研究》;20081230(第12期);44-45 * |
| 高色价低桔霉素红曲色素的提取研究;杨涛等;《食品科技》;20051120(第11期);42-44 * |
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