CN105211752A - A kind of red yeast rice being rich in GABA and preparation method thereof - Google Patents
A kind of red yeast rice being rich in GABA and preparation method thereof Download PDFInfo
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Abstract
The present invention discloses a kind of red yeast rice being rich in GABA and preparation method thereof, filter out from 60 kinds of dissimilar paddy rice (comprising rice, red rice and black rice) catalysis germinate after GABA (GABA) content of brown rice up to kind " Nanton84 " (black rice) of 24.82mg/100g, be 4.0 times of check variety " good fortune rice No. 8 ".Be that substrate carries out fermentation test with the sprouted unpolished rice of " Nanton84 ", from 49 strain monascus strain of separated and collected, filter out high yield GABA and do not produce the monascus strain CICC of citrinin? 5008, GABA content in its tunning red yeast rice up to 22.43mg/100g, than control strain CICC? 5004 is high by 93.9%.The red yeast rice preparation method being rich in GABA of the present invention is simple, and in red yeast rice obtained under the fermentation condition of optimum, GABA content is up to 26.54mg/100g, and does not detect citrinin.
Description
Technical field
The present invention relates to a kind of red yeast rice and preparation method thereof, particularly relate to a kind of red yeast rice being rich in GABA and preparation method thereof.
Background technology
Red colouring agent for food, also used as a Chinese medicine, namely red yeast rice, be raw material with cooked rice, bred thereon and the goods obtained by Monascus.Sweat rice becomes red, is therefore referred to as red yeast rice or red rice.Red colouring agent for food, also used as a Chinese medicine is a kind of wholefood of medicine-food two-purpose, it appear at the Song dynasty that tenth century at least can be traced back to for by China.Traditionally, red colouring agent for food, also used as a Chinese medicine is widely used in food service industry, is mainly used in wine brewing, and make preserved red beancurd, be used as the preservative agent etc. of food color and meat, it both can improve food color, can give again food distinctive local flavor.In addition, red colouring agent for food, also used as a Chinese medicine also has the drug effect of health care, and the main pharmacodynamics of red colouring agent for food, also used as a Chinese medicine is summarized as by " Chinese medicine voluminous dictionary ", and " promoting blood circulation and removing blood stasis, reinforcing spleen to promote digestion, treatment dyspepsia is glutted, red white diarrhea and traumatic injury." found more and more along with the pharmacological active substance of red colouring agent for food, also used as a Chinese medicine, Chinese scholars is found by research, and red colouring agent for food, also used as a Chinese medicine not only has the effect reducing cholesterolemia, in addition the effect such as hypoglycemic, hypotensive, anti-cancer.Japan carries out in the animal experiment of feed interpolation " red colouring agent for food, also used as a Chinese medicine culture " in state-run health research place, finds that the feed adding 0.12%-0.13% red colouring agent for food, also used as a Chinese medicine culture can make the blood pressure suffering from congenital vascular hypertension mouse be down to below 180mmHg by more than 200mmHg.By analysis, hypotensive active ingredient is GABA (γ-aminobutyricacid, GABA) and Glucosamine, and wherein GABA plays very important effect.
The physiological function of GABA is mainly manifested in and reduces blood pressure, trophic nerve, improve liver kidney function and improve the aspects such as brain vigor.Nineteen fifty, research group Late Cambrian GABA in mammal brain extract of Flory and Robert, nowadays, physician has proved that GABA is the inhibitory transmitter substance of central nervous system, it is one of most important neurotransmitter in brain tissue, its effect reduces neuronal activity, prevents nerve cell overheated.GABA can make it to activate in conjunction with brain acceptor antianxity, then act synergistically with other material, stops the information relevant to anxiety to arrive at brain and indicate maincenter, and therefore, GABA can fundamentally calmness nerve, thus plays effect antianxity.GABA can be used for improving cerebral blood circulation as medicine, increases oxygen supply, improves brain cell metabolism, also contribute to treating produce because of headstroke, head injury and cerebral arteriovenous malformation sequelae etc. headache, tinnitus and the sequelae such as clouding of consciousness.
Brown rice is a kind of health-care food ignored by people, it is containing the mineral matter, vitamin etc. of high-load, especially various bioactivators is contained as GABA etc., several times even higher of content of these active materials in brown rice polished rice often, therefore, be developed to functional health care food and be there is huge market value and meaning.Moreover, after brown rice germination, its nutritive value is further enhanced, and is also easy to picked-up simultaneously and digests and assimilates.Just in the ascendant to the research of sprouted unpolished rice in the world, researcher is devoted to optimize germination condition mostly makes it produce the physiological activator of more high-load.At present, be that the functional product of raw material appears on the market more and more with sprouted unpolished rice, pursuing today of nutrition and health diet, the appearance of these products greatly meets people for pursuit that is happy and grade life.
The ability that there is not yet in existing pertinent literature with regard to accumulating GABA carries out large-scale monascus strain screening operation, the present invention screens excellent, high yield, safety monascus strain and has important practical significance from the product such as preserved red beancurd, red yeast rice, can be used to produce the functional food or batching that are rich in GABA, as the goods such as health food drink, jam, flavoring of various strengthening GABA.Long-term these goods edible, have multiple physiological hygiene function to human body, thus have broad application prospects.Moreover, also the health-care efficacy of Monascus as beneficial bacterium can be enriched further.The Monascus Strains of the numerous New function of separation and purification from microorganism natural resources, and be used for producing GABA functional food ingredient, this is a new trial.Be conducive to further development and utilization microbial function food, drug resource, therefore there is profound significance.
Summary of the invention
The object of the present invention is to provide a kind of red yeast rice being rich in GABA and preparation method thereof.
Technical scheme provided by the present invention is:
Be rich in a red yeast rice of GABA, it is characterized in that: in described red yeast rice, the content of GABA can reach 26.54mg/100g, and does not detect citrinin.
Described red monascus CICC5008, this bacterial strain is purchased from Chinese industrial Microbiological Culture Collection administrative center (CICC), and preserving number is CICC5008.
A kind of preparation method being rich in the red yeast rice of GABA, described red yeast rice is with black rice " Nanton84 " (Zhang Mingwei, the anti-oxidant active component with reducing blood lipid of black rice and the mechanism of action thereof, 2013, South China Normal University) sprouted unpolished rice be substrate, with red monascus CICC5008 for starting strain, ferment according to its optimal conditions of fermentation.The preparation method of described red yeast rice comprises the following steps:
(1) preparation of sprouted unpolished rice culture medium: sprouted unpolished rice clear water is soaked three hours, steams 20min after draining in food steamer, every fermentation flask takes 50g sprouted unpolished rice, after good seal, for subsequent use after autoclaving 20min at 121 DEG C;
(2) preparation of monascus ruber seed liquid: by the monascus strain CICC5008 of high yield GABA on PDA flat board 28 DEG C after activation culture 5-7 days, the card punch that diameter is 5mm is used to get bacterium block, then 5 bacterium cakes are respectively added in the 250mL triangular flask that 50mL seed liquor culture medium is housed, in 30 DEG C, under 150rpm condition, after shaken cultivation 72h, namely obtain seed liquor; Described seed liquor culture medium is glucose 50g, peptone 5g, yeast extract 1g, KH
2pO
41g, FeSO
47H
2o0.01g, MgSO
47H
2o0.5g, adds water and is settled to 1000mL.
(3) inoculation and fermented and cultured: it is 1 × 10 that the seed liquor after cultivating 72h is diluted to spore concentration
7individual/mL, the seed liquor of dilution being added to by 10% inoculum concentration is equipped with in the plastics fermentation flask of sprouted unpolished rice culture medium, stirs with aseptic bamboo let, and is placed in 28 DEG C of constant incubators and cultivates 11d, at Initial stage of culture 2-3d, sprouted unpolished rice fermented and cultured basal part takes on a red color slightly; When being cultured to 4d, plastics fermentation flask is taken out, sprouted unpolished rice is clapped loose, make Monascus can utilize sprouted unpolished rice raw material more fully; When being cultured to 7d, Monascus anka Nakazawa et sato filament growth is vigorous; During 8d, moved to by plastics fermentation flask in 28 DEG C of constant incubators, continue to be cultured to 11d, sprouted unpolished rice fermentation completely, obtains red yeast rice.
Described red yeast rice is specifically prepared in accordance with the following steps:
1) screening of GABA sprouted unpolished rice raw material is rich in
2) screening of high yield GABA monascus strain
3) optimization of red yeast rice zymotechnique
What step 1) was concrete is: each 10g of brown rice of precise 60 kinds of Different Types of Rice (comprising rice, red rice and black rice), be placed in conical flask, drain after cleaning 3 times with pure water, then 1% hypochlorite solution is added by brown rice submergence sterilization 5min, drain with after sterile water wash 3 times afterwards, continue to soak in the pure water of pH5.0, thermostat water bath conical flask being placed in 30 DEG C is incubated 6h, immersion terminates rear taking-up brown rice, spread in the culture dish through sterilizing in advance, cover sterilized and use 0.5mmol/LCaCl
2after the gauze of solution wets, be placed in 30 DEG C of constant incubator vernalization 24h.Period observes the moisture situation of gauze every 4h, and deficiency as moisture in gauze, uses splendid attire 0.5mmol/LCaCl in time
2the watering can of solution sprays in right amount.Take out after vernalization 24h, carefully throw off the gauze covered on it, germination completes.
All sprouted unpolished rices are placed in 800W micro-wave oven passivation 30s after germination terminates, are then placed in 50 DEG C of baking ovens and dry 12h, obtain dry sprouted unpolished rice.Finally pulverize with high speed disintegrator, obtain germination brown rice powder, splendid attire, in valve bag, posts label, is placed in 4 DEG C of refrigerators for subsequent use.Extract the GABA of sprouted unpolished rice in accordance with the following methods and after deriving, carry out HPLC detection.Result shows: the GABA content after black rice " Nanton84 " germinates in brown rice is the highest, and reaching 24.82mg/100g, is 4.0 times of check variety " good fortune rice No. 8 ".
The extraction of sprouted unpolished rice GABA and deriving method: get the ground sample of 5g and be placed in conical flask, add 60% ethanol 30ml, 55 DEG C of water bath with thermostatic control 6h, period shakes up 30s every 30min, centrifugal 15min under 3000rpm condition after terminating, get supernatant concentration to 5ml, finally obtain treating derivative sample with 0.45 μm of filtering with microporous membrane, detected sample is obtained again after deriving further, derivative method is: by 1%2, the acetonitrile solution of 4-dinitrofluorobenzene (FDNB) is as derivating agent, and sample thief liquid 1mL is placed in the brown measuring bottle of 10mL, adds 0.5mol/LNaHCO
3(pH9.0) solution and 1%2, the each 1mL of 4-dinitrofluorobenzene acetonitrile solution, take out after being placed in 60 DEG C of water bath with thermostatic control dark place heating 1h, 10mL is settled to pH7.0 phosphate buffer after cooling, fully shake up rear use 0.45 μm of filtering with microporous membrane, finally be transferred in clean sample cell for subsequent use, get 10 μ L sample introductions when needing, detect under 360nm wavelength.
Step 2) concrete be: adopt solid state fermentation mode, using the GABA content in monascus ruber tunning and citrinin as index, respectively the tunning red yeast rice of 49 strain Monascus Strains is placed in after 50 DEG C of baking ovens dry and spend the night, be ground into powder, according to 1) in method extract GABA and derivative after, the HPLC carrying out GABA content detects.Meanwhile, the tunning of each bacterial strain is carried out to the HPLC detection of citrinin content.Result shows: the GABA content in the tunning of monascus strain CICC5008 is the highest and do not produce citrinin, and the GABA content in its red yeast rice is up to 22.43mg/100g, higher by 93.9% than control strain CICC5004 (purchased from CICC).
What step 3) was concrete is: by experiment of single factor and Plackett-Burman experiment, have studied the soak time of sprouted unpolished rice, steaming rice time, dress rice amount, cultivation temperature, soak pH, the ventilative factor such as hole count and cultivation cycle, on the impact of GABA content in red yeast rice, determine cultivation temperature, cultivation cycle, ventilative hole count be three main affecting factors.On this basis, adopt Box-Behnken response surface optimization method to carry out the optimization of red yeast rice zymotechnique, determine that best zymotechnique is: ventilative hole count 5, cultivate 11 days at 25 DEG C.In the red yeast rice prepared under Optimal technique process, GABA content, up to 26.54mg/100g, improves 11.8% than before process optimization.
The invention has the advantages that: to be rich in the optimum sprouted unpolished rice of GABA for substrate, with high yield GABA and the optimum monascus strain not producing citrinin ferment, prepare and be rich in GABA and the red yeast rice not detecting citrinin.
Detailed description of the invention
Below in conjunction with specific embodiment, set forth the present invention further.Embodiment is only not used in for illustration of the present invention and limits the scope of the invention.
Embodiment 1
(1) separation of monascus strain and collection: the sample such as commercially available red yeast rice, preserved red beancurd of separate sources is placed in culture dish, add sterilized water extremely just submergence rice, vibrate 1min gently, and with pipette, extract 1ml bacteria suspension on PDA flat board, this bacterial concentration is designated as 10
-1, another absorption 1ml bacteria suspension adds 9ml sterilized water and is made into 10
-2gradient, then be made into 10 respectively
-3, 10
-4, 10
-5, 10
-6with 10
-7, the bacteria suspension of totally seven concentration gradients.Cultivate under all flat boards being placed in 28 DEG C of constant temperatures, observe bacterium colony after three days, choose and present micro-red or red bacterium colony and be forwarded to new flat board, continue after line to cultivate, purifying like this, until flat board grows single bacterium colony, is finally separated and obtains 36 strain monascus strains.In addition, have purchased totally 13 monascus strains such as CICC5008 and CICC5004 from DSMZs such as Chinese Research for Industrial Microbial Germ preservation administrative centers (CICC).Therefore, be altogether separated, have collected 49 monascus strains, be all incubated on PDA slant medium.
(2) screening of monascus strain: adopt solid state fermentation mode, using the GABA content in monascus ruber tunning and citrinin as index, respectively the tunning red yeast rice of 49 strain monascus strains is placed in after 50 DEG C of baking ovens dry and spend the night, be ground into powder, after also deriving according to the method extraction GABA in (5) of embodiment 2, the HPLC carrying out GABA content detects.Meanwhile, the tunning of each bacterial strain is carried out to the HPLC detection of citrinin content.Result shows: the GABA content in monascus strain CICC5008 tunning is the highest and do not produce citrinin, and the GABA content in its red yeast rice is up to 22.43mg/100g, higher than control strain CICC5004 by 93.9%.
Embodiment 2
(1) preparation of sprouted unpolished rice: each 10g of brown rice of precise 60 kinds of Different Types of Rice (comprising rice, red rice and black rice), be placed in conical flask, drain after cleaning 3 times with pure water, then 1% hypochlorite solution is added by brown rice submergence sterilization 5min according to the percentage of brown rice quality, drain with after sterile water wash 3 times afterwards, continue to soak in the pure water of pH5.0, thermostat water bath conical flask being placed in 30 DEG C is incubated 6h, immersion terminates rear taking-up brown rice, spread in the culture dish through sterilizing in advance, cover sterilized and use 0.5mmol/LCaCl
2after the gauze of solution wets, be placed in 30 DEG C of constant incubator vernalization 24h.Period observes the moisture situation of gauze every 4h, and deficiency as moisture in gauze, uses splendid attire 0.5mmol/LCaCl in time
2the watering can of solution sprays in right amount.Take out after vernalization 24h, carefully throw off the gauze covered on it, germination completes, and obtains sprouted unpolished rice.
(2) preparation of monascus ruber seed liquid: by monascus strain activation culture (28 DEG C) after 5-7 days on PDA flat board, the card punch that diameter is 5mm is used to get bacterium block, then respectively add 5 bacterium cakes and (fill little bead) in the 250mL triangular flask that 50mL seed liquor culture medium is housed, in 30 DEG C, under 150rpm condition after shaken cultivation 72h, obtain seed liquor.
(3) preparation of sprouted unpolished rice culture medium: sprouted unpolished rice clear water is soaked three hours, steams 20min after draining in food steamer, every fermentation flask takes 50g sprouted unpolished rice, after good seal, for subsequent use after autoclaving 20min at 121 DEG C.
(4) inoculation and fermented and cultured: the seed liquor after cultivating 72h is diluted to spore concentration and is about 1 × 10
7individual/mL, to add to the seed liquor of dilution by 10% inoculum concentration and is equipped with in the plastics fermentation flask of sprouted unpolished rice culture medium, stir with aseptic bamboo let, and is placed in 28 DEG C of constant incubators and cultivates 11d.At Initial stage of culture 2-3d, sprouted unpolished rice fermented and cultured basal part takes on a red color slightly; When being cultured to 4d, plastics fermentation flask is taken out, sprouted unpolished rice is clapped loose, make Monascus can utilize sprouted unpolished rice raw material more fully; When being cultured to 7d, Monascus anka Nakazawa et sato filament growth is vigorous; During 8d, moved to by plastics fermentation flask in 28 DEG C of constant incubators, continue to be cultured to 11d, sprouted unpolished rice fermentation completely.
(5) be rich in the screening of GABA sprouted unpolished rice raw material: all sprouted unpolished rices are placed in 800W micro-wave oven passivation 30s, be then placed in 50 DEG C of baking ovens and dry 12h, obtain dry sprouted unpolished rice.Finally pulverize with high speed disintegrator, obtain germination brown rice powder, splendid attire, in valve bag, posts label, is placed in 4 DEG C of refrigerators for subsequent use.Get the ground sample of 5g and be placed in conical flask, add 60% ethanol 30mL, 55 DEG C of water bath with thermostatic control 6h, period shakes up 30s every 30min, centrifugal 15min under 3000rpm condition after terminating, get supernatant concentration to 5mL, finally obtain treating derivative sample with 0.45 μm of filtering with microporous membrane, then obtain detected sample after deriving further.Derivative method is: by the acetonitrile solution of 1%2,4-dinitrofluorobenzene (FDNB) as derivating agent, sample thief liquid 1mL is placed in the brown measuring bottle of 10mL, adds 0.5mol/LNaHCO
3(pH9.0) solution and 1%2, the each 1mL of 4-dinitrofluorobenzene acetonitrile solution, take out after being placed in 60 DEG C of water bath with thermostatic control dark place heating 1h, 10mL is settled to pH7.0 phosphate buffer after cooling, fully shake up rear use 0.45 μm of filtering with microporous membrane, finally be transferred in clean sample cell for subsequent use, get 10 μ L sample introductions when needing, detect under 360nm wavelength.Detect through HPLC, from 60 kinds of dissimilar paddy rice (comprising rice, red rice and black rice), filter out kind " Nanton84 " (black rice) of the GABA content after germinateing in brown rice up to 24.82mg/100g, be 4.0 times of check variety " good fortune rice No. 8 ".
(6) optimization of red yeast rice zymotechnique: by experiment of single factor and Plackett-Burman experiment, have studied the soak time of sprouted unpolished rice, steaming rice time, dress rice amount, cultivation temperature, soak pH, the ventilative factor such as hole count and cultivation cycle, on the impact of GABA content in red yeast rice, determine cultivation temperature, cultivation cycle, ventilative hole count be three main affecting factors.On this basis, adopt Box-Behnken response surface optimization method to carry out the optimization of red yeast rice zymotechnique, determine that best zymotechnique is: ventilative hole count 5, cultivate 11 days at 25 DEG C.In the red yeast rice prepared under Optimal technique process, GABA content, up to 26.54mg/100g, improves 11.8% than before process optimization.
In red yeast rice, the HPLC assay method of citrinin content is as follows:
The pre-treating method of testing sample: red colouring agent for food, also used as a Chinese medicine crosses 40 mesh sieves after pulverizing, accurately take 1g as sample, slowly add 20mLTEF solution (toluene: ethyl acetate: formic acid=7:3:1 under magnetic stirring, v/v/v), stir 30min, the centrifugal 10min of 5000r/min, gets supernatant after 0.22 μm of filtering with microporous membrane as the testing sample that HPLC detects.
Chromatographic condition: chromatographic column is Shim-PackVP-ODSC18(5 μm, 250 × 4.6nm, Japanese Shimadzu), mobile phase is: acetonitrile-water (volume ratio is 35:65, adjusts pH to 2.5 with chromatographically pure phosphoric acid), and flow velocity is 1.0mL/min, column temperature 35 DEG C, determined wavelength 254nm; Sampling volume 10 μ L.
Measurement result: the present invention adopts red monascus strain CICC5008 to carry out, in the standby red yeast rice of fermentation, not detecting citrinin.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (2)
1. be rich in a red yeast rice of GABA, it is characterized in that, with the sprouted unpolished rice of black rice " Nanton84 " for substrate, with red monascus CICC5008 for starting strain carries out fermentation gained.
2. be rich in the preparation method of GABA red yeast rice as claimed in claim 1, it is characterized in that: the preparation method of described red yeast rice comprises the following steps:
(1) preparation of sprouted unpolished rice culture medium: sprouted unpolished rice clear water is soaked three hours, steams 20min after draining in food steamer, every fermentation flask takes 50g sprouted unpolished rice, after good seal, for subsequent use after autoclaving 20min at 121 DEG C;
(2) preparation of monascus ruber seed liquid: by the monascus strain CICC5008 of high yield GABA on PDA flat board 28 DEG C after activation culture 5-7 days, the card punch that diameter is 5mm is used to get bacterium block, then 5 bacterium cakes are respectively added in the 250mL triangular flask that 50mL seed liquor culture medium is housed, in 30 DEG C, under 150rpm condition, after shaken cultivation 72h, namely obtain seed liquor;
(3) inoculation and fermented and cultured: it is 1 × 10 that the seed liquor after cultivating 72h is diluted to spore concentration
7individual/mL, the seed liquor of dilution being added to by 10% inoculum concentration is equipped with in the plastics fermentation flask of sprouted unpolished rice culture medium, stirs with aseptic bamboo let, and is placed in 28 DEG C of constant incubators and cultivates 11d, at Initial stage of culture 2-3d, sprouted unpolished rice fermented and cultured basal part takes on a red color slightly; When being cultured to 4d, plastics fermentation flask is taken out, sprouted unpolished rice is clapped loose, make Monascus can utilize sprouted unpolished rice raw material more fully; When being cultured to 7d, Monascus anka Nakazawa et sato filament growth is vigorous; During 8d, moved to by plastics fermentation flask in 28 DEG C of constant incubators, continue to be cultured to 11d, sprouted unpolished rice fermentation completely, obtains red yeast rice.
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| CN106509622A (en) * | 2016-11-04 | 2017-03-22 | 成都译山生物科技有限公司 | Monascus purpureus mycelium parasited black rice and preparation method and use thereof |
| CN111387409A (en) * | 2019-05-14 | 2020-07-10 | 江苏臻大天园健康科技有限公司 | Fermentation device and processing method of functional red yeast rice powder |
| CN115843964A (en) * | 2022-11-04 | 2023-03-28 | 浙江大学 | Preparation method of high-nutrition red yeast rice |
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Application publication date: 20160106 |