Summary of the invention
The object of the present invention is to provide one to be rich in Co-Q10 red colouring agent for food, also used as a Chinese medicine and preparation method thereof, the present invention utilizes two bacterial strain mixed fungus fermentations, and cultivates and prepare red colouring agent for food, also used as a Chinese medicine in conjunction with temperature control, can greatly improve the content of Co-Q10 in red colouring agent for food, also used as a Chinese medicine, and method is simple, is easy to control.
For achieving the above object, the present invention adopts following technical scheme:
One is rich in Co-Q10 red colouring agent for food, also used as a Chinese medicine, and the content of its Co-Q10 is 400~550mg/kg, not containing citrinin.
Described red colouring agent for food, also used as a Chinese medicine be by the feathering Monascus purchased from Japanese technology assessment research institute's living resources center (
monascus pilosus, IFO4521) and Monascus ruber (
monascus rubber, NBRC32318) and mixed culture fermentation gained.
The preparation method who is rich in Co-Q10 red colouring agent for food, also used as a Chinese medicine comprises the following steps:
1) seed liquor preparation: single bacterium colony of the feathering Monascus having activated and Monascus ruber is switched to 30 ℃ of dull and stereotyped PDA culture mediums and cultivates 7~8 days, again with the Tween-20 sterilized water of 2wt% respectively by the spore growing on PDA flat board wash-out gently, prepare feathering Monascus and Monascus ruber pityrosporion ovale suspension, the concentration that makes pityrosporion ovale suspension is 10
5~10
6individual/mL, two kinds of pityrosporion ovale suspensions are connected to respectively seed culture medium, and inoculum concentration is 15wt%, and under 30 ℃, 150rpm, shaking table is cultivated 2~4 days, then two kinds of seed liquor equal-volumes are mixed, for subsequent use;
2) bent meal preparation: will drain after polished rice and long-grained nonglutinous rice immersion, making its moisture is 35%~40%, add sucrose, Paprika and linoleic acid, mix thoroughly, adopt meal braizing machine steamed rice, steam pressure is 0.2MPa, steamed rice 18min, steamed rice discharging opening is installed air blast cooling, and rice temperature is inoculated after being down to 36~38 ℃;
3) inoculation: to step 2) access seed liquor prepared by step 1) in the bent meal prepared, inoculum concentration is 10~15wt%, fully mixes rear dress square-bottomed bamboo basket thoroughly and shakeouts, and covers clean gunnysack, sends into bent room heat-insulated cultivation;
4) sectional temperature-controlled cultivation: postvaccinal bent meal is put in the bent room after sterilization, and bent room temperature was controlled at 30~32 ℃ in first 4 days, temperature is controlled at 28 ℃ of cultivations 1~3 day subsequently, finally cultivates 7~9 days at 25 ℃, cultivates altogether 14~16 days;
5) dry: the red colouring agent for food, also used as a Chinese medicine fermenting is poured in drying pool, maintained the temperature at hot-air seasoning at 38~40 ℃.
Seed culture medium in step 1) is by glycerine 50mL, glucose 30g, yeast extract 10g, peptone 8g, potassium nitrate 2g, ammonium dihydrogen phosphate (ADP) 1g, epsom salt 1g, ground rice 20g, add water and be settled to 1L, then regulate pH to 5.8~6.0,121 ℃ of sterilizings 20 minutes with NaOH.
Step 2) in the weight ratio of polished rice and long-grained nonglutinous rice be 1:3~1:5.
Step 2) in dosage of sucrose be 3~5wt%, Paprika consumption is 0.1~0.2wt%, linoleic acid 0.02~0.04wt%.
remarkable advantage of the present invention is:
(1) the present invention adopts the Monascus that does not produce citrinin as fermentation strain, by two bacterial strain mixed fungus fermentations, and cultivates in conjunction with temperature control, can greatly improve the content of Co-Q10 in red colouring agent for food, also used as a Chinese medicine.
(2) in preparation method of the present invention, add Paprika and linoleic acid, can significant stimulation Monascus self metabolism synthesizing coenzyme Q 10, also promote the synthetic of Lovastatin and GABA simultaneously.
(3) the prepared red colouring agent for food, also used as a Chinese medicine of the present invention is not only rich in Co-Q10, and contains other biological active component (Lovastatin, GABA), can be used as raw material and is applied in food, medicine and cosmetics processing, and purposes is very extensive.
(4) preparation method of the present invention adopts solid-state pure-blood ferment, and technique is simple, and easily operation, can realize industrialized production.
The specific embodiment
embodiment 1
A kind of preparation method who is rich in Co-Q10 red colouring agent for food, also used as a Chinese medicine comprises the following steps:
(1) seed liquor preparation: single bacterium colony of the feathering Monascus (IFO4521) having activated and Monascus ruber (NBRC32318) is switched to 30 ℃ of dull and stereotyped PDA culture mediums and cultivates 7 days, again with the Tween-20 sterilized water of 2wt% respectively by the spore growing on PDA flat board wash-out gently, prepare feathering Monascus and Monascus ruber pityrosporion ovale suspension, the concentration of pityrosporion ovale suspension is 2.4 × 10
5individual/mL, two kinds of pityrosporion ovale suspensions are connected to respectively seed culture medium, and inoculum concentration is 15wt%, and under 30 ℃, 150rpm, shaking table is cultivated 2 days, then two kinds of seed liquor equal-volumes are mixed, for subsequent use;
Seed culture medium is by glycerine 50mL, glucose 30g, yeast extract 10g, peptone 8g, potassium nitrate 2g, ammonium dihydrogen phosphate (ADP) 1g, epsom salt 1g, ground rice 20g, add water and be settled to 1L, then regulate pH to 5.8 with NaOH, with the triangular flask packing of 500mL, 121 ℃ of sterilizings 20 minutes;
(2) bent meal preparation: drain after 1kg polished rice and 3kg long-grained nonglutinous rice are soaked to 10 hours under 25 ℃ of water temperatures, making its moisture is 35%, after adding sucrose 120g, Paprika 4g and linoleic acid 1.2g, mix thoroughly, adopt meal braizing machine steamed rice, steam pressure is 0.2MPa, steamed rice 18min, steamed rice discharging opening is installed air blast cooling, and rice temperature is inoculated after being down to 36 ℃;
(3) inoculation: to by accessing 400g seed liquor in the bent meal after cooling, fully mix rear dress square-bottomed bamboo basket thoroughly and shakeout, cover clean gunnysack, send into bent room heat-insulated cultivation;
(4) sectional temperature-controlled cultivation: postvaccinal bent meal is put in the bent room after sterilization, and bent room temperature was controlled at 32 ℃ in first 4 days, temperature is controlled at 28 ℃ of cultivations 1 day subsequently, finally cultivates 9 days at 25 ℃, cultivates altogether 14 days;
(5) dry: the red colouring agent for food, also used as a Chinese medicine fermenting is poured in drying pool, maintained the temperature at hot-air seasoning at 38 ℃.
in red colouring agent for food, also used as a Chinese medicine, Co-Q10 content HPLC assay method is as follows:
Chromatographic condition: chromatographic column is Shim-Pack VP-ODS C18(5 μ m, 250 × 4.6nm, Japanese Shimadzu), mobile phase is methyl alcohol-ethanol (volume ratio is 1:1), and flow velocity is 1.0mL/min, and 35 ℃ of column temperatures detect wavelength 275nm; Sampling volume 10 μ L.
Sample treatment: red colouring agent for food, also used as a Chinese medicine was pulverized 40 mesh sieves, accurately takes 0.5g as sample, adds the ultrasonic extraction of methyl alcohol, with the centrifugal 10min of 5000r/min, gets supernatant through 0.22 μ m filtering with microporous membrane, to be checked.
Result: in red colouring agent for food, also used as a Chinese medicine, Co-Q10 content is with dry weight basis 400mg/kg.
in red colouring agent for food, also used as a Chinese medicine, Lovastatin content HPLC assay method is as follows:
Chromatographic condition: chromatographic column is Shim-Pack VP-ODS C18(5 μ m, 250 × 4.6nm, Japanese Shimadzu), mobile phase is methanol-water (volume ratio is 83:17), and flow velocity is 1.0mL/min, and 35 ℃ of column temperatures detect wavelength 238nm; Sampling volume 10 μ L.
Sample treatment: red colouring agent for food, also used as a Chinese medicine is crossed 40 mesh sieves after pulverizing, and accurately takes 2g as sample, adds the ultrasonic extraction of isopropyl alcohol, with the centrifugal 10min of 5000r/min, gets supernatant through 0.22 μ m filtering with microporous membrane, to be checked.
Result: in red colouring agent for food, also used as a Chinese medicine, Lovastatin content is with dry weight basis 3.34g/kg.
in red colouring agent for food, also used as a Chinese medicine, alpha-aminobutyric acid content HPLC assay method is as follows:
Chromatographic condition: chromatographic column is Shim-Pack VP-ODS C18(5 μ m, 250 × 4.6nm, Japan's Shimadzu), mobile phase A is methyl alcohol, Mobile phase B is oxolane-methyl alcohol-0.05mol/L NaOH sodium (volume ratio 5:75:420), mobile phase A: Mobile phase B=20:80(v/v), flow velocity is 1.0mL/min, 35 ℃ of column temperatures, detect wavelength 275nm; Sampling volume 10 μ L.
Sample treatment: red colouring agent for food, also used as a Chinese medicine is crossed 40 mesh sieves after pulverizing, and accurately takes 0.5g as sample, adds water and extracts in 85 ℃ of water-baths, and the centrifugal 10min of 5000r/min, gets supernatant through 0.22 μ m filtering with microporous membrane, to be checked.
Result: in red colouring agent for food, also used as a Chinese medicine, alpha-aminobutyric acid content is with dry weight basis 330g/kg.
in red colouring agent for food, also used as a Chinese medicine, citrinin content HPLC assay method is as follows:
Chromatographic condition: chromatographic column is Shim-Pack VP-ODS C18(5 μ m, 250 × 4.6nm, Japanese Shimadzu), mobile phase is second cyanogen-water (with chromatographically pure phosphoric acid tune pH to 2.5, volume ratio 35:65), and flow velocity is 1.0mL/min, 35 ℃ of column temperatures, detect wavelength 254nm; Sampling volume 10 μ L.
Sample treatment: red colouring agent for food, also used as a Chinese medicine is crossed 40 mesh sieves after pulverizing, accurately take 1g as sample, under magnetic agitation, slowly add 20mLTEF solution (toluene: ethyl acetate: formic acid=7:3:1, v/v/v), stir 30min, the centrifugal 10min of 5000r/min, gets supernatant through 0.22 μ m filtering with microporous membrane, to be checked.
Result: do not detect citrinin.
embodiment 2
A kind of preparation method who is rich in Co-Q10 red colouring agent for food, also used as a Chinese medicine comprises the following steps:
(1) seed liquor preparation: the feathering Monascus (IFO4521) having activated and the single bacterium colony of Monascus ruber (NBRC32318) are switched to 30 ℃ of cultivations of dull and stereotyped PDA culture medium 8 days, again with the Tween-20 sterilized water of 2wt% respectively by the spore growing on PDA flat board wash-out gently, prepare feathering Monascus and Monascus ruber pityrosporion ovale suspension, the concentration that makes pityrosporion ovale suspension is 5.6 × 10
5individual/mL, two kinds of pityrosporion ovale suspensions are connected to respectively seed culture medium, and inoculum concentration is 15wt%, and under 30 ℃, 150rpm, shaking table is cultivated 3 days, then two kinds of seed liquor equal-volumes are mixed, for subsequent use;
Seed culture medium is by glycerine 50mL, glucose 30g, yeast extract 10g, peptone 8g, potassium nitrate 2g, ammonium dihydrogen phosphate (ADP) 1g, epsom salt 1g, ground rice 20g, add water and be settled to 1L, then regulate pH to 5.9 with NaOH, with the triangular flask packing of 500mL, 121 ℃ of sterilizings 20 minutes;
(2) bent meal preparation: drain after 1kg polished rice and 5kg long-grained nonglutinous rice are soaked to 10 hours under 25 ℃ of water temperatures, making its moisture is 38%, after adding sucrose 300g, Paprika 12g and linoleic acid 1.2g, mix thoroughly, adopt meal braizing machine steamed rice, steam pressure is 0.2MPa, steamed rice 18min, inoculates after making bent meal temperature be down to 37 ℃ afterwards with air blast;
(3) inoculation: to by accessing 660g seed liquor in the bent meal after cooling, fully mix rear dress square-bottomed bamboo basket thoroughly and shakeout, cover clean gunnysack, send into bent room heat-insulated cultivation;
(4) sectional temperature-controlled cultivation: postvaccinal bent meal is put in the bent room after sterilization, and bent room temperature was controlled at 30 ℃ in first 4 days, temperature is controlled at 28 ℃ of cultivations 3 days subsequently, finally cultivates 9 days at 25 ℃, cultivates altogether 16 days;
(5) dry: the red colouring agent for food, also used as a Chinese medicine fermenting is poured in drying pool, maintained the temperature at hot-air seasoning at 39 ℃.
HPLC method with embodiment 1 detects, and in red colouring agent for food, also used as a Chinese medicine, the content of Co-Q10, Lovastatin and GABA is respectively 550mg/kg, 3.08g/kg and 294mg/kg, and citrinin does not detect.
embodiment 3
A kind of preparation method who is rich in Co-Q10 red colouring agent for food, also used as a Chinese medicine comprises the following steps:
(1) seed liquor preparation: the feathering Monascus (IFO4521) having activated and the single bacterium colony of Monascus ruber (NBRC32318) are switched to 30 ℃ of cultivations of dull and stereotyped PDA culture medium 8 days, again with the Tween-20 sterilized water of 2wt% respectively by the spore growing on PDA flat board wash-out gently, prepare feathering Monascus and Monascus ruber pityrosporion ovale suspension, the concentration that makes pityrosporion ovale suspension is 1.8 × 10
5individual/mL, two kinds of pityrosporion ovale suspensions are connected to respectively seed culture medium, and inoculum concentration is 15wt%, and under 30 ℃, 150rpm, shaking table is cultivated 4 days, then two kinds of seed liquor equal-volumes are mixed, for subsequent use;
Seed culture medium is by glycerine 50mL, glucose 30g, yeast extract 10g, peptone 8g, potassium nitrate 2g, ammonium dihydrogen phosphate (ADP) 1g, epsom salt 1g, ground rice 20g, add water and be settled to 1L, then regulate pH to 6.0 with NaOH, with the triangular flask packing of 500mL, 121 ℃ of sterilizings 20 minutes;
(2) bent meal preparation: drain after 1kg polished rice and 4kg long-grained nonglutinous rice are soaked to 10 hours under 25 ℃ of water temperatures, making its moisture is 40%, after adding sucrose 200g, Paprika 10g and linoleic acid 2g, mix thoroughly, adopt meal braizing machine steamed rice, steam pressure is 0.2MPa, steamed rice 18min, inoculates after making bent meal temperature be down to 38 ℃ afterwards with air blast;
(3) inoculation: to by accessing 750g seed liquor in the bent meal after cooling, fully mix rear dress square-bottomed bamboo basket thoroughly and shakeout, cover clean gunnysack, send into bent room heat-insulated cultivation;
(4) sectional temperature-controlled cultivation: postvaccinal bent meal is put in the bent room after sterilization, and bent room temperature was controlled at 30 ℃ in first 4 days, temperature is controlled at 28 ℃ of cultivations 3 days subsequently, finally cultivates 8 days at 25 ℃, cultivates altogether 15 days;
(5) dry: the red colouring agent for food, also used as a Chinese medicine fermenting is poured in drying pool, maintained the temperature at hot-air seasoning at 40 ℃.
HPLC method with embodiment 1 detects, and in red colouring agent for food, also used as a Chinese medicine, the content of Co-Q10, Lovastatin and GABA is respectively 496mg/kg, 3.15g/kg and 288mg/kg, and citrinin does not detect.
embodiment 4
The present embodiment routinely technique only take long-grained nonglutinous rice as raw material, take feathering Monascus as fermentation strain through sectional temperature-controlled cultivation function in contrast.Its preparation method comprises the following steps:
(1) seed liquor preparation: the single bacterium colony of feathering Monascus (IFO4521) having activated is forwarded to 30 ℃ of dull and stereotyped PDA culture mediums and cultivates 8 days, again with the Tween-20 sterilized water of 2wt% respectively by the spore growing on PDA flat board wash-out gently, prepare feathering Monascus and Monascus ruber pityrosporion ovale suspension, the concentration that makes pityrosporion ovale suspension is 7.4 × 10
5individual/mL, is connected to seed culture medium by pityrosporion ovale suspension, and inoculum concentration is 15wt%, and under 30 ℃, 150rpm, shaking table is cultivated 3 days, obtains seed liquor for subsequent use;
Seed culture medium adopts PDA fluid nutrient medium, then regulates pH to 5.9 with NaOH, with the triangular flask packing of 500mL, and 121 ℃ of sterilizings 20 minutes.
(2) bent meal preparation: drain after 6kg long-grained nonglutinous rice is soaked to 10h under 25 ℃ of water temperatures, making its moisture is 35%, adopts meal braizing machine steamed rice, and steam pressure is 0.2MPa, and steamed rice 18min makes bent meal temperature be down to 36 ℃ with air blast afterwards.
(3) inoculation: to by accessing 600g seed liquor in the bent meal after cooling, fully mix rear dress square-bottomed bamboo basket thoroughly and shakeout, cover the gunnysack after sterilizing, send into bent room heat-insulated cultivation.
(4) constant temperature culture: bent meal after inoculation is put in the bent room after sterilization to incubation time 16 days at 30 ℃;
(5) dry: the red colouring agent for food, also used as a Chinese medicine fermenting is poured in drying pool, maintained the temperature at hot-air seasoning at 39 ℃.
HPLC method with embodiment 1 detects, and in red colouring agent for food, also used as a Chinese medicine, the content of Co-Q10, Lovastatin and GABA is respectively 46.34mg/kg, 2.05g/kg and 220mg/kg, and citrinin does not detect.
By relatively showing, in the red colouring agent for food, also used as a Chinese medicine that employing the present invention makes, the content of Co-Q10 is much higher than common process, and the content of Lovastatin and GABA is also significantly improved, visible, preparation method of the present invention is simple, and the red colouring agent for food, also used as a Chinese medicine quality making is high, has significant effect.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing 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.