CN109805270A - A method of low albumen millet is produced using two-way solid state fermentation - Google Patents
A method of low albumen millet is produced using two-way solid state fermentation Download PDFInfo
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- CN109805270A CN109805270A CN201910080624.1A CN201910080624A CN109805270A CN 109805270 A CN109805270 A CN 109805270A CN 201910080624 A CN201910080624 A CN 201910080624A CN 109805270 A CN109805270 A CN 109805270A
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- cordyceps militaris
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Abstract
The invention discloses a kind of methods for producing low albumen millet using two-way solid state fermentation, belong to technical field of biological fermentation.The present invention uses Cordyceps militaris spawn, reduces millet protein content by two-way solid-state fermentation technology and generates more functional components.The protein content of obtained millet is obviously fewer than protein contained by unprocessed millet, 11.2~13.4g containing protein in former every 100g millet；Millet protein content after fermentation has dropped 32% compared to original, and due to generating a large amount of primary and secondary metabolite and enzyme when Cordyceps militaris spawn fermentation, change the ingredient of millet, impart the functional component abundant such as millet polysaccharide, amino acid, nucleosides material and mannitol, it is edible for nephropathy patient, the wasting of resources is avoided, economic value is improved.The technology can be used for cereal processing industry, be a kind of fermentation process of great developmental research value.The method disclosed in the present has a good application prospect in the field that medical edible fungal improves cereal.
Description
Technical field
The present invention relates to a kind of methods for producing low albumen millet using two-way solid state fermentation, belong to biofermentation technique neck
Domain.
Background technique
Nephrosis is a kind of general designation for seriously endangering human health common disease, mainly includes different types of ephritis, acute kidney
Failure, kidney stone, renal cyst etc..One of them is most basic to be characterized in High-grade Proteinuria, refers to nephrotic's Urine proteins
Discharge rate > 3.5g/d.Under normal physiological conditions, glomerular filtration membrane has molecular barriers and electrostatic barrier, causes in crude urine
Protein content increases, and when returning uptake far more than proximal convoluted tubule, forms High-grade Proteinuria.On this basis, all increase kidneys are small
Pressure and the factor (such as high-protein diet) for causing high perfusion, height to filter can aggravate the discharge of Urine proteins in ball.Patient easily produces
The complication such as raw infection, Gao Ning, microelement deficiencies, endocrine disturbance and immunologic hypofunction.Therefore nephropathy patient is not suitable for
The edible food containing high protein.
Millet is the staple food crop in NORTH CHINA area, protein rich in, fat and vitamin, it is civil often
For boiling milled congee.Millet nutritive value of cooking gruel is abundant, has the laudatory title of " Dai Cantang ", has the benefits of calming the nerves.Not due to millet
It needs to refine, so it remains many vitamin and inorganic salts, several times up to rice of the vitamin B1 in millet, inorganic salts
Content is also above rice.Millet not only can be edible, can also be used as medicine.Chinese medicine thinks millet sweet-salty, cool in nature, there is heat-clearing, clear thirsty,
Enriching yin, tonifying spleen kidney and stomach, diuresis are harnessed the river and are rushed down and other effects.Millet protein rich in, such as glutelin, alcohol albumen, ball
Albumen etc., and content is higher than rice, fatty 1.7g, carbohydrate 76.1g in every 100g, all not less than paddy such as paddy and wheats
Object.Secondly, the quality of protein is also superior to wheat, rice and corn in millet.It can be seen that nephropathy patient is not suitable for table egg
The millet of Bai Hanliang high.Therefore the content for reducing protein in millet is to improve nephropathy patient to be not suitable for this case that edible millet
A kind of approach.But protein content in millet should be reduced, guarantees the method for German millet nutrition value again, yet there are no phase
Close report.Therefore realize that this target seems very necessary by method efficiently, green, environmentally friendly.
Summary of the invention
In order to solve the above technical problems, the present invention uses two-way solid state fermentation, using millet as solid substrate fermentation pupa worm
On the one hand grass provides the nutritional need of growth for Cordyceps militaris, and will not introduce other reagents and generate damage, another party to Cordyceps militaris
Face, the secondary metabolite that Cordyceps militaris generates change the original functional component of Zymocyte, and playing reduces protein and increasing
Add the effect of other functional components, improves millet nutrition abundant and function to a certain extent.
The first purpose of the invention is to provide a kind of methods for producing low albumen millet using two-way solid state fermentation, including
Following steps:
(1) prepared by seed liquor: being forwarded in liquid seed culture medium after Cordyceps militaris spawn is activated, cultivates at 22~30 DEG C
2~5d prepares seed liquor；
(2) two-way solid state fermentation: the seed liquor for taking step (1) to be prepared uniformly is sprinkled upon millet solid-state fermentation culture medium
Surface, in 22~30 DEG C of 20~25d of fermentation；
(3) the complete millet solid-state fermentation culture medium sterilizing of step (2) fermentation is removed into Cordyceps militaris spawn, cleans and dries
To low albumen millet.
Further, the seed culture medium is calculated as by mass content: glucose 4~6%, peptone 1~3%, ferment
Female medicinal extract 2~4%, potassium dihydrogen phosphate 0.05~0.15%, magnesium sulfate 0.03~0.08%, initial pH value are 6~9.
Further, the millet solid-state fermentation culture medium is prepared via a method which to obtain: millet is cleaned and is impregnated, leaching
It is dried after washing, 15~30min is steamed after drying and obtains millet solid-state fermentation culture medium.
Further, the Cordyceps militaris is preserved in China General Microbiological culture presevation administrative center, and deposit number is
CGMCC NO.3.17859。
Further, in step (1), the culture of the seed liquor is cultivated under the conditions of being protected from light.
Further, in step (1), the revolving speed of the seed liquor culture is 200~400rpm.
Further, in step (2), inoculum concentration is 2~5% (w/w).
Further, in step (2), the two-way solid state fermentation is fermented under the conditions of being protected from light.
Further, in step (3), the sterilizing is 15~25min of sterilizing under the conditions of 115~130 DEG C.
A second object of the present invention is to provide the low albumen millets that the method is prepared.
The beneficial effects of the present invention are:
The present invention provides a kind of methods suitable for industrializing improved production cereal, and optimize corresponding fermentation item
Part.The protein content of obtained millet is obviously fewer than protein contained by unprocessed millet, and crude protein content is 11.2%
11.2~13.4g containing protein in~13.4%, i.e., every 100g；Millet protein content after fermentation, which is compared, originally to be had dropped
7.6g containing albumen~9.1g in 32%, i.e., every 100g, and produced due to generating a large amount of primary and secondary metabolism when Cordyceps militaris spawn fermentation
Object and enzyme change the ingredient of millet, and it is rich to impart millet polysaccharide, amino acid, nucleosides material and mannitol etc.
Rich effect ingredient, it is edible for nephropathy patient, the wasting of resources is avoided, economic value is improved.The technology can be used for cereal
Processing industry is a kind of fermentation process of great developmental research value.
Specific embodiment
The present invention is further explained in the light of specific embodiments, so that those skilled in the art can be preferably
Understand the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
Protein detection: Kjeldahl nitrogen determination total nitrogen is 1. used.1g sample is weighed in digest tube, is successively weighed respectively
1g K is added2SO4, 0.5g CuSO4, the 20mL concentrated sulfuric acid carries out digestion process after mixing evenly, until liquid in pipe becomes pale green
Until color transparency liquid, constant volume uses Kjeldahl nitrogen determination after shaking up in 100mL volumetric flask after liquid to be processed cools down completely.
Total nitrogen calculates:
In formula: x is nitrogen content in sample, g/100g；V1 is sample titration consumption hydrochloric acid standard solution volume, mL；V2 is
Blank titration consumes hydrochloric acid standard solution volume, mL；V3 is the volume of digestive juice when measuring sample, mL；C is normal hydrochloric acid titration
Solution concentration, mol/L；14.008 be every mole of nitrogen atomic mass, g/mol；M is the quality of sample, g；F is nitrogen transformation ratio,
6.25。
2. measuring nonprotein nitrogen using trichloroacetic acid method.1g sample to be tested powder is weighed, the oscillation of 100mL distilled water is added
The solution of trichloroacetic acid of 20mL 10% is added after 30min, is filtered after standing 25min~35min after mixing, three chloroethenes of filter residue
Acid elution 3 times, collection filtrate, which is transferred in Rotary Evaporators, to be concentrated, and sample is added in the digest tube of graphite resolution instrument and is digested, is disappeared
Kjeldahl nitrogen determination is used after change.Blank control is 1mL distilled water, and processing method is same as above sample.Protein content/g=total nitrogen
Content-non-protein nitrogen content.
Polysaccharide detection method: Thick many candies are 1. extracted using water extraction and alcohol precipitation method.2. with total in Phenol-sulphate acid method measurement Thick many candies
Sugared content.It is 0,0.02,0.04,0.06,0.08,0.1mg/mL titer that glucose is made into concentration respectively.1mL titer point
5% phenol solution 1mL, the concentrated sulfuric acid 5mL now matched is not added, shakes up 30 DEG C of placement 30min, light absorption value is measured at 490nm.With
Glucose content is abscissa, and light absorption value is that ordinate draws standard curve, obtains equation of linear regression.1mL sample liquid is drawn,
Light absorption value is measured according to above-mentioned steps, and substitutes into the glucose content (mg/mL) in standard curve calculating reaction system.Total reducing sugar contains
Measure=concentration of glucose/1000 (μ g/mg) × extension rate × extracting liquid volume/extract quality.3. with 3,5- dinitro water
Poplar acid colorimetric method measures content of reducing sugar in Thick many candies.Be separately added into 6 test tubes 1mg/mL glucose standards solution 0,
0.10,0.20,0.30,0.40,0.50mL mend distilled water to 0.5mL, DNS reagent 1.5mL are then added, mixes well.It is placed in
5min is boiled in boiling water bath, then rapid flowing water is cooling, and 4mL distilled water is added into test tube respectively and mixes.It is surveyed at 540nm
Determine light absorption value.Using concentration of glucose as abscissa, light absorption value is that ordinate draws standard curve, obtains its equation of linear regression.
1mL sample solution is taken, measures light absorption value by above-mentioned steps, substitutes into the concentration of glucose (mg/ in standard curve calculating reaction system
mL).Content of reducing sugar (μ g/mg)=concentration of glucose/1000 × extension rate × extracting liquid volume/extract quality.Polysaccharide
Content (μ g/mg)=total sugar content-content of reducing sugar.
Amino acid detection method: it is measured using ninhydrin method.0,0.2,0.4,0.6,0.8,1.0mL standard are drawn respectively
For amino acid solution in 6 test tubes, distilled water complements to 1mL, adds 1mL acetic acid-sodium acetate buffer solution, 1mL ninhydrin
Developing solution, the ascorbic acid solution of 0.1mL 0.1% mix well rear tube sealing, 15min are heated in 100 DEG C of water-baths, after taking-up
It is cooling with flowing water rapidly, 5min is stood, the dilution of 60% ethyl alcohol of 3mL is added, sufficiently shakes up, its absorbance is surveyed at 570nm, with
Light absorption value is ordinate, and amino acid content is that abscissa draws standard curve, obtains regression equation.Accurately weigh what drying and crushing was crossed
Then plus 5mL sample powder 1g adds acetic acid mechanical shaking extraction 5h with the solid-liquid ratio of 1: 20 (g/mL) in 90 DEG C of water-bath,
10% solution of trichloroacetic acid stands 30min after vibrating 1min, and 4000r/min is centrifuged 10min, and supernatant is settled to through filtering
50mL is up to sample to be tested.Wherein filtrate 1mL is taken, measures light absorption value under 570nm by above-mentioned steps, standard curve is brought into and calculates ammonia
Base acid solutions.Amino acid content is finally calculated by formula: amino acid content/%=(the amino acid solution concentration of measurement ×
Extension rate × measurement amino acid solution volume)/sample quality × 100.
Nucleosides material (mainly cordycepin) detection method: 1. the cordycepin standard reserving solution of 500mg/mL is diluted to
0,5,10,15,20,25 μ g/mL cordycepin titer, is measured, using chromatographic peak area as ordinate, concentration is using HPLC
Abscissa draws standard curve, obtains regression equation.2. accurately weighing sample dry powder 1g, it is settled to 10mL, is ultrasonically treated 30min,
13000r/min is centrifuged 5min, takes merging filtrate after supernatant 3 times processing, and filtrate crosses 0.22 μm of miillpore filter, takes 1mL filtrate fixed
Hold to 10mL volumetric flask, be measured using HPLC, as a result substitutes into standard curve and carry out calculating sample cordycepin concentration.3. chromatography
Condition is chromatographic column: Waters C18 (4.6mm × 150mm, 5 μm)；Mobile phase: 15% methanol+water；Flow velocity 1mL/min；Column
Temperature: 20 DEG C；Ultraviolet detection wavelength 260nm, 10 μ L sample introductions.Cordycepin content (μ g/mg)=cordycepin concentration × extracting liquid volume/
Extract quality.
Mannitol detection method: 1. precision weighs 5g sample in round-bottomed flask, adds the reflux of 50mL distilled water 2h, 4500r/
Min is centrifuged 20min, and residue, it is to be measured to be settled to 50mL wash with distilled water.2. precision weighs dry sterling mannitol 50mg,
Distilled water is settled to 50mL, and being made into concentration is 1.0mg/mL standard sample.Then respectively it is accurate draw standard sample 1.0,2.0,
4.0,5.0,6.0mL constant volume is in 100mL volumetric flask, and each 1mL that draws is placed in test tube, and sequentially adding concentration is 0.015mol/L
Sodium periodate solution 1mL, mixing are stored at room temperature 10min, then the L- sandlwood sugar juice 2mL of accurate addition 0.1%, precision after shaking up
The Nash reagent that 4mL is newly configured is added and mixes well, 15min is heated in 53 DEG C of thermostat water baths makes it sufficiently develop the color and wait
To cooling, blank control is sat with distilled water, surveys light absorption value in 413nm.Using mannitol concentration as abscissa, light absorption value is ordinate
Standard curve is drawn, regression equation is obtained.3. accurate pipette samples 1mL is handled by above-mentioned steps.Light absorption value is surveyed in 413nm.As a result
It substitutes into standard curve and calculates sample mannitol concentration.Mannitol content (mg/g)=mannitol concentration × extracting liquid volume/extraction
Amount of substance.
Embodiment 1: two-way solid state fermentation conditions optimization
Cordyceps militaris spawn is taken at the China General Microbiological culture presevation administrative center of Institute of Microorganism, Academia Sinica,
Deposit number is CGMCC NO.3.17859.
Using single factor test optimisation strategy, different carbon sources, nitrogen source, inorganic salts and temperature, pH and revolving speed are chosen, to Cordyceps militaris
The liquid seed culture medium and condition of culture and solid fermentation culture medium and solid state fermentation conditions of strain optimize.It obtains most suitable
Liquid seed culture medium formula is calculated as according to mass content: glucose 5%, peptone 2%, yeast extract 3%, potassium dihydrogen phosphate
0.1%, magnesium sulfate 0.05%, initial pH value is 6~9.Liquid culture condi are as follows: be protected from light, 22~30 DEG C of cultivation temperature, revolving speed
200~400rpm, 3~5d of cultivation cycle.Most suitable solid fermentation culture medium prescription are as follows: after Xian millet is cleaned plus clear water is in 20 DEG C of items
5h is impregnated under part, is eluted, and is put in steaming 20min or so sterilizing in food steamer after drying moisture.Solid state fermentation conditions are as follows: be protected from light, ferment
22~30 DEG C of temperature, 20~25d of fermentation period.Fermented product through composition detection, containing polysaccharide, amino acid, nucleosides material with
And the functional component abundant such as mannitol, polyoses content are 69.2%~77.1% (692~771 μ g/mg)；Amino acid content is
1.06%~1.28% (10.6~12.8mg/g)；Nucleosides material content is；0.023%~0.026% (235.2~264.7
μg/g)；Mannitol content is 3.58%~4.33% (35.8~43.3mg/g).And crude protein content be 11.2%~
11.2~13.4g containing protein in 13.4%, i.e., every 100g；Millet protein content after fermentation, which is compared, originally to be had dropped
7.6g containing albumen~9.1g in 32%, i.e., every 100g.
Embodiment 2: the two-way solid state fermentation pilot scale production of hybrid seeds
To meet the needs of engineering production process is to a large amount of liquid seeds, carried out according to the result of study before laboratory
3t fermentor seed spreads cultivation test, and 3 parallel tests are arranged.Test result shows: fermentor carrier fluid amount 75%.Culture medium exists
It 121 DEG C, sterilizes under the conditions of 20min, is cooled to 22~30 DEG C, the shake-flask seed liquid of inoculation 7.0 ‰, 200~300r/min of revolving speed,
Ventilation ratio 1.5vvm, 22~30 DEG C of cultivation temperature, culture is inoculated with admittedly after reaching 40g/L to growth mid-log phase, that is, mycelium content
State fermentation medium.The seed growth period is shorter under this condition, the growth conditions of strain, and organoleptic feature and morphological feature are all very
Good, growth is vigorous, and mycelia is denseer, and culture process is stablized, and has reached ideal result.It is shown in Table 1.
1 liquid seeds of table spread cultivation test result
Embodiment 3: two-way solid state fermentation scale up test
To realize that engineering produces low albumen millet, according to the result of study and pilot scale production of hybrid seeds test knot before laboratory
Fruit carries out the solid state fermentation scale up test test of 750kg rank, 3 parallel tests is arranged.Test result shows: Xian millet is washed
After net plus clear water impregnates 5h at 20 °C, pulls out, drains away the water, and is packed into steamed rice vehicle, leads to steam 30min or so sterilizing,
Enter spreading-and-cooling machine, temperature is down to 22~30 DEG C, is inoculated with inoculation device.The long-grained nonglutinous rice culture medium for covering with Cordyceps militaris spawn is transported to
It ferments in fermentation vat.Medium pH, inoculum concentration, fermentation temperature, refers to afore-mentioned test result at fermentation time.In fermentation process,
Air blower is controlled by temperature sensor and carries out thermostatic control and oxygen replenishment, and each stirring is primary sooner or later daily.After fermentation,
Sterilizing, 60 DEG C of drying obtain low albumen millet.It is shown in Table 2.
2 solid state fermentation culture pilot plant test result of table
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
It encloses without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in the present invention
Protection scope within.Protection scope of the present invention is subject to claims.
Claims (10)
1. a kind of method for producing low albumen millet using two-way solid state fermentation, which comprises the steps of:
(1) seed liquor prepare: be forwarded in liquid seed culture medium after Cordyceps militaris spawn is activated, 22~30 DEG C cultivate 2~
5d prepares seed liquor；
(2) two-way solid state fermentation: the seed liquor for taking step (1) to be prepared uniformly is sprinkled upon millet solid-state fermentation culture medium surface,
In 22~30 DEG C of 20~25d of fermentation；
(3) the complete millet solid-state fermentation culture medium sterilizing of step (2) fermentation is removed into Cordyceps militaris spawn, clean drying obtains low
Albumen millet.
2. the method according to claim 1, wherein the seed culture medium is calculated as by mass content: grape
Sugared 4~6%, peptone 1~3%, yeast extract 2~4%, potassium dihydrogen phosphate 0.05~0.15%, magnesium sulfate 0.03~
0.08%, initial pH value is 6~9.
3. the method according to claim 1, wherein the millet solid-state fermentation culture medium is made by the following method
It is standby to obtain: millet being cleaned and is impregnated, is dried after elution, 15~30min is steamed after drying and obtains millet solid-state fermentation culture medium.
4. the method according to claim 1, wherein the Cordyceps militaris is preserved in China General Microbiological strain
Preservation administrative center, deposit number are CGMCC NO.3.17859.
5. the method according to claim 1, wherein the culture of the seed liquor is to be protected from light in step (1)
Under the conditions of cultivated.
6. the method according to claim 1, wherein in step (1), the revolving speed of the seed liquor culture is
200~400rpm.
7. the method according to claim 1, wherein inoculum concentration is 2~5% (w/w) in step (2).
8. the method according to claim 1, wherein the two-way solid state fermentation is to be protected from light in step (2)
Under the conditions of ferment.
9. the method according to claim 1, wherein the sterilizing is in 115~130 DEG C of items in step (3)
Sterilize 15~25min under part.
10. the low albumen millet that a kind of any one of claim 1~9 the method is prepared.
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