CN109105736A - Utilize the fermented grain enzyme powder manufacturing method of the increase fermentation efficiency of the liquid strain bacteria culture fluid of inoculation solidification Bacillus strain - Google Patents

Utilize the fermented grain enzyme powder manufacturing method of the increase fermentation efficiency of the liquid strain bacteria culture fluid of inoculation solidification Bacillus strain Download PDF

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CN109105736A
CN109105736A CN201710693470.4A CN201710693470A CN109105736A CN 109105736 A CN109105736 A CN 109105736A CN 201710693470 A CN201710693470 A CN 201710693470A CN 109105736 A CN109105736 A CN 109105736A
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cereal
weight
soybean
parts
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CN109105736B (en
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慎镛喆
朴哲
尹荣晟
金利洙
金雄镛
金爱香
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Amicogen Inc
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Abstract

Fermented grain enzyme powder the present invention relates to the manufacturing method of the fermented grain enzyme powder d of the increase fermentation efficiency of liquid strain bacteria culture fluid using inoculation solidification Bacillus strain a kind of and to be manufactured by the method.It can provide balanced absorption enhancement fermented grain through the invention and supplement the kind bacterium substrate rice bran of beneficiating ingredient, wheat bran, yeast extract, powdered soybean, the fermented grain enzyme of the various physiologically active ingredients such as the tunning and digestive ferment that are generated when the various necessary nutritional ingredients and solidification fermentation of bacillus that contain in soybean separation protein white powder end, and by solidification fermentation of bacillus cereal and its by-product, supplement diversified pharmacological property ingredient and physiological activator.And beneficial bacterium in enteron aisle activates function of intestinal canal, and the ingredients such as high concentration amylase and protease that when fermentation generates effectively help to absorb the nutritional ingredient contained in cereal and food, and the effective thrombus help blood circulation of fibrinolytic enzyme.

Description

It is fermented and is imitated using the increase of the liquid strain bacteria culture fluid of inoculation solidification Bacillus strain The fermented grain enzyme powder manufacturing method of rate
Technical field
The present invention relates to a kind of increase fermentation efficiencies of liquid strain bacteria culture fluid using inoculation solidification Bacillus strain Fermented grain enzyme powder manufacturing method, specifically include (a) by rice bran, wheat bran, powdered soybean, soybean separation protein white powder end with And the kind bacterium that yeast extract powder is constituted manufactures liquid strain bacterium culture solution with inoculation solidification Bacillus strain in substrate Stage；(b) mixing rice bran in cereal, wheat bran, powdered soybean, soybean separation protein white powder end and yeast extract powder are constituted Culture substrate after carry out stage of boiling；(c) the liquid strain bacterium that (a) stage by described in manufactures is added to institute with culture solution State the stage fermented in the cereal of (b) stage boiling；(d) fermentation material that (c) stage fermentation described in drying is formed, and powder The stage of endization；Fermented grain enzyme powder manufacturing method.
Background technique
Enzyme is the key protein for acting on internal metabolic activity, is a kind of object of catalyst action as chemical reaction Matter.Being specifically divided into can not voluntarily generate in vivo, the enzyme food for needing to absorb in vitro, voluntarily generate in vivo and facilitate digestion Digestive ferment, and act on the metabolic enzyme of other metabolic activities in addition to digestion.Wherein, enzyme food is made for digesting and assimilating With decomposition Excretion, anti-inflammatory, antibacterial effect, detoxicating, killing bacteria effect, blood purification effect and cell reactivation etc. are raw Reason aspect.(currently used for enzyme treatment, p.29-41,2013)
Enzyme is generally divided into the digestion of intervention human body, excretion, digestive ferment (amylase, the albumen of the parts such as nutritional ingredient intake Enzyme, cellulase), and as sustain life and the nutritional ingredient absorbed intervene metabolic metabolic enzyme (nerve ending, Autonomic nerve and the adjusting of various hormone etc.), mainly by being generated in the incubation of fermentative microorganism.Fibrin is solidifying Poly- platelet-shaped is at clot, and curing shrinkage is an impediment to blood circulation to clot gradually.Fibrinous fibrin is decomposed in body Catabolic enzyme increases, and the cohesion of blood platelet can be effectively suppressed, and keeps blood flow unimpeded.
Food containing enzyme is the function to strengthen these enzymes, largely added in edible raw material the edible microorganismus manually cultivated or The general designation deserved enzyme component or process these enzyme components contained in extraction food.The food containing enzyme contains, and passes through food The process of itself enzyme and the fermented soy of microorganism generates various physiological activators, and the generation for passing through nutritional ingredient is made With and beneficial bacterium proliferation function generate facilitate digest and assimilate various microelements and physiological activator. (Huh, S.H.and Kim, M.H.The modern health and health food, 1997.Hongikjea Press.Korea, p.35-36)
And bacillus is solidified in addition to generating spore there are also the feature of the bacillus and lactic acid bacteria that generate lactic acid, make Used for acid resistance and heat resistance lactic acid bacteria, and generate it is various to the advantageous ingredient of human body and various physiological activators, Digestive ferment promotes intracorporal physiological activity and effectively facilitates metabolism, is conducive to indigestion, irritable colon syndrome, Constipation reduces cholesterol etc..And the ingredient digested and nutritional ingredient will be difficult to by fermentation in cereal effectively decomposed as people The nutritional ingredient that body easily absorbs.
In recent years, it mixes brown rice and several Major Cereals manufactures the manufacturing method widespread of various fermented foods.Greatly Contain main nutrient composition in partial husk, but because shell is harder, coarse, decladding is edible under normal circumstances.By husk Rice bran in ingredient, wheat bran use as kind of a bacterium substrate, can effectively absorb the beneficiating ingredient in husk and can take in hair simultaneously The beneficiating ingredient generated during ferment.
Summary of the invention
[project for needing to solve],
For this purpose, related personnel of the present invention is exploitation using the fermented grain powder for being the food containing enzyme, by case Research, picks out outstanding bacterial strain, manufactures fermented grain powder using the bacterial strain, and propose the application.
The purpose of the application be provide including,
(a) the kind bacterium being made of rice bran, wheat bran, powdered soybean, soybean separation protein white powder end and yeast extract powder The stage of liquid strain bacterium culture solution is manufactured with inoculation solidification Bacillus strain in substrate；
(b) mixing rice bran in cereal, wheat bran, powdered soybean, soybean separation protein white powder end and yeast extract powder structure At culture substrate after carry out stage of boiling；
(c) the liquid strain bacterium that (a) stage by described in manufactures with culture solution be added in the cereal of (b) stage boiling into The stage of row fermentation；
(d) fermentation material that (c) stage fermentation described in drying is formed, and the stage of powdered；Fermented grain enzyme powder Manufacturing method.
And further object is, provide including, manufactured by the method, by amylase, protease, Optionally more than one enzymatic activity increase is characterized in the group that lipase, fibrinolytic enzyme and cellulase are constituted Fermented grain enzyme powder.
[solution]
To achieve the object of the present invention, provide including,
(a) the kind bacterium being made of rice bran, wheat bran, powdered soybean, soybean separation protein white powder end and yeast extract powder The stage of liquid strain bacterium culture solution is manufactured with inoculation solidification Bacillus strain in substrate；
(b) mixing rice bran in cereal, wheat bran, powdered soybean, soybean separation protein white powder end and yeast extract powder structure At culture substrate after carry out stage of boiling；
(c) the liquid strain bacterium that (a) stage by described in manufactures with culture solution be added in the cereal of (b) stage boiling into The stage of row fermentation；
(d) fermentation material that (c) stage fermentation described in drying is formed, and the stage of powdered；Fermented grain enzyme powder Manufacturing method.
And provide and include, manufactured by the method, by amylase, protease, lipase, fibrinolytic enzyme with And the fermented grain enzyme powder that optionally more than one enzymatic activity increase is characterized in the group of cellulase composition.
The present invention described further below.
The present invention provides,
(a) the kind bacterium being made of rice bran, wheat bran, powdered soybean, soybean separation protein white powder end and yeast extract powder The stage of liquid strain bacterium culture solution is manufactured with inoculation solidification Bacillus strain in substrate；
(b) mixing rice bran in cereal, wheat bran, powdered soybean, soybean separation protein white powder end and yeast extract powder structure At culture substrate after carry out stage of boiling；
(c) the liquid strain bacterium that (a) stage by described in manufactures with culture solution be added in the cereal of (b) stage boiling into The stage of row fermentation；
(d) fermentation material that (c) stage fermentation described in drying is formed, and the stage of powdered；Fermented grain enzyme powder Manufacturing method.
In the present invention, (a) stage is, by rice bran, wheat bran, powdered soybean, soybean separation protein white powder end and yeast The kind bacterium that extract powder is constituted manufactures the stage of liquid strain bacterium culture solution with inoculation solidification Bacillus strain in substrate.
Described kind of bacterium substrate includes powdered soybean, rice bran, wheat bran, soybean protein isolate, yeast extract.Specifically, Described kind of bacterium is relatively pure 100 parts by weight of water purification, 1~5 parts by weight of powdered soybean, 1~5 parts by weight of rice bran, wheat bran 1 with substrate ~5 parts by weight, 1~5 parts by weight of soybean protein isolate, 0.1~3 parts by weight of yeast extract powder.
Inoculation solidification bacillus in described kind of bacterium substrate.
The bacterial strain of the preferred inoculation solidification bacillus is to entrust the bacterial strain entrusted in number KCTC13284BP.Institute Stating bacterial strain is, in traditional fermented food bacterial strain, selectes starch, albumen, fat and fibrin decomposition ability are fitst water Bacterial strain.
The inoculation is, using the preceding culture solution for cultivating the bacterial strain, to freeze to dry using separated bacterial strain or use The bacterial strain of forms powdered such as dry or culture furthermore.The inoculum concentration of the solidification Bacillus strain is, relatively described 100 parts by weight of mixture are inoculated with 5~20 parts by weight, preferred to be inoculated with 10 parts by weight.
The liquid strain bacterium culture solution is specifically, Liquid Culture.The cultivation temperature is 30 DEG C~40 DEG C, culture Time is 10~30 hours.
If following embodiment proves, cereal is inoculated in substrate by kind of bacterium and cultivates acquisition, passes through the excellent of the bacterial strain In the fermented grain enzyme that the activity of elegant amylolytic enzyme, lipolytic enzyme and protein decomposition enzyme cellulase obtains Protein is degraded, improves digestibility, it is beneficial to facilitate sanguimotor fibrinolytic enzyme active effect etc. Activity.
(b) stage is mixing rice bran in cereal, wheat bran, powdered soybean, and soybean separation protein white powder end and yeast mention The stage of boiling is carried out after the culture substrate for taking object powder to constitute.
The cereal that (b) stage uses is brown rice, soybean, barley, its in blended grain (wheat, corn, Semen Coicis) One.The cereal is sorted by appropriate, after cleaning process, using clear water soaking process.Cereal is impregnated according to type clear water 4~24 hours.According to the actual characteristic of cereal, clear water soaking time can be adjusted in the range.Specifically, brown rice 14~16 hours, soybean 5~9 hours, barley 3~5 hours, blended grain 14~16 hours.
Water temperature is impregnated specifically, 20 DEG C~35 DEG C, preferred 25 DEG C~30 DEG C.The reason for this is that according to temperature difference when impregnating Generate moisture content difference.The cereal of the cleaning and immersion be the form of crushing, the form of powder or without crushing Engineering directly uses.
This culture used in (b) stage is with substrate, by powdered soybean, rice bran, and wheat bran, soybean protein isolate, Yeast extract powder is formed.Affiliated mixture is specifically, relatively described 100 parts by weight of cereal, 5~10 weight of powdered soybean Part, 1~5 parts by weight of rice bran, 1~5 parts by weight of wheat bran, 1~5 parts by weight of soybean protein isolate, 1~5 weight of yeast extract powder Measure part.
The unimpeded system stage can also cultivate bacterial strain from cereal, but by the miscellaneous bacteria being heat-treated in the cereal that can go out and destroy paddy Object cell wall, gelatinization and protein deformation, are supplied to the active procreation enviroment of microorganism, can reduce strain inoculated amount And cost.The boiling is carried out using the various methods for deserving industry publicity, such as using steam or superheated steam. Specific point of 2 stages carry out boiling.1st stage was the pre-cooked stage, including 60 DEG C~110 DEG C 10~60 points of temperature pre-cooked The process of clock, preferably 80 DEG C~120 DEG C temperature pre-cooked 20~40 minutes.2nd stage be this boiling stage, 90 DEG C~140 DEG C temperature boiling 10~60 minutes, preferably 100 DEG C~120 DEG C temperature boiling 30~50 minutes.
According to the swelling state of cereal in digestion process, adjusts moisture content and carry out boiling.It is preferred that the stage remains former thus The cereal of state is the 50~90% of overall weight, more preferably the 60~80% of overall weight.This stage is by maintaining paddy Object original state, forming space between cereal ensures aeration.
The cooling stage of additional boiling cereal after boiling.Natural cooling can be carried out after boiling, or improves cooling For speed to prevent overheat, equilibrium is cooling.Cooling temperature is specially 35 DEG C~50 DEG C, preferably 40 DEG C~45 DEG C.Temperature is lower than described When range, cereal is really up to the mark to cause mixing difficult.
(c) stage is, the liquid strain bacterium that (a) stage by described in manufactures is added to described (b) stage with culture solution and steams The stage fermented in the cereal boiled.
The additive amount of the liquid strain bacterium culture solution is 100 parts by weight of cereal for including in the relatively described mixture, is connect 5~20 parts by weight of kind, preferably 10 parts by weight.
The cultural method of the fermentation does not limit to specifically, and Liquid Culture slot, rotary drum-type fermentation machine, pallet can be used Fermentation machine.Be conducive to cereal or the fermentation of its mixture in addition to the fermentation machine, be not limited to the present patent application, and according to Production scale may be selected device appropriate and use.The cultivation temperature is 20 DEG C~50 DEG C or 30 DEG C~45 DEG C, and culture humidity is 40%~90%, preferably 50%~85%.Incubation time is 1~72 hour, 12~72 hours, 36~50 hours or 48 Hour.
(d) stage is the fermentation material that (c) stage fermentation described in drying is formed, and the stage of powdered.
The drying stage is that can be carried out by the various methods of the sector publicity, but need to pay attention to excessive temperature environment Lower drying causes the raw bacterium in fermented grain object to be gone out and cuts reduction enzymatic activity.Specifically, the bacterial strain in the application is not dead, maintain It is dried in the environment of enzymatic activity.The drying temperature is 30 DEG C~60 DEG C, preferably 35 DEG C~45 DEG C.
Drying time is 12~48 hours, preferably 20~28 hours.The moisture content for the fermentation material dried at this time is 3 ~20%, preferably 3~7%.This is product enzyme potency and saves upper optimal moisture content.
The crushing process is, according to the use purpose of fermented grain object, crushes and grinds for all size that is, including crushing It is broken, it mills, grinds, polish, crush, grind or use the food material powdered or while being reduced into fine particle Other means.Specifically, using beater grinder.
It further include by the stage of powder random molding after the powdered process.It, will after the fermentation material for crushing drying fermentation Powder is made particle, ball, pastille, beverage etc. any one.
The powdered or molding the words stage after, the stage including packaging can be added.
The present invention is made by the method, is provided and is characterized by, by amylase, protease, lipase, fibrin point The fermented grain enzyme powder that optionally more than one enzymatic activity increase is characterized in the group that solution enzyme and cellulase are constituted.
The present invention provides a kind of thrombus food molding including the fermented grain enzyme powder.The food molding As long as object drinking food product, there is no particular restriction.
Specifically, the fermented grain enzyme powder is food containing enzyme.Food containing enzyme in the present invention refers to, to strengthen enzyme function Can, edible microorganismus is cultivated in edible raw material, makes to contain a large amount of organized enzymes in material；Or it extracts and contains from food Enzyme part；Or the food containing enzyme for processing this based food.
Food molding of the invention may include the molding of healthy food.By bacterial strain or fermented grain of the invention Powder term food perhaps healthy food when can directly add fermented grain powder of the invention or with other food or food Product ingredient is used together, or can suitably be used according to common method.The combined amount of bacterial strain or fermented grain object of the invention It can purpose (prevention, healthy or therapeutic prescription etc.) is appropriate to be determined according to using.The food molding is edible molding When, there is no particular restriction.Food molding is for example including meat, sausage, bread, cake, chocolate, carbohydrate, jellies, and zero Food class, Biscuits (cookies, biscuit etc.), Pizza, noodles (instant noodles etc.), chewing gum class, the junket agriculture product of ice cream etc., Various soup classes, catsup, condiment, gravy, baste, beverage, tea, nutritional agents, alcoholic beverage and vitamin synthetics etc..
Food or healthy food molding of the invention is identical as general beverage, can contain various fragrance, natural carbon water The ingredients such as compound.The natural carbohydrate is the monosaccharides such as glucose and fructose, and maltose and sucrose etc. are double The glycitols such as the polysaccharides such as carbohydrate, dextrin and cyclodextrin, xylitol and D-sorbite and erythritol.Sweetener can It uses, the natural sweeteners such as thaumatin and qualities of stevia extract or the synthetic sweeteners such as saccharin and sweetener.It is described The addition ratio of natural carbohydrate is, the food or healthy food molding in the present invention it is everyAddition 0.01~0.20g, specifically about 0.04~0.10g.
Outside the content, food of the invention or healthy food molding may include various nutritional agents, vitamin, electrolysis Matter, flavoring agent, colorant, pectic acid and its salt, alginic acid and its salt, organic acid, protective colloid thickener, PH tune Save agent, stabilizer, preservative, glycerol, alcohol, the carbonating agent etc. for soda.In addition, food of the invention or Healthy food molding may include manufacturing fruit juice, the pulp of fruit drink and vegetable beverage.These ingredients can it is independent or It is used in mixed way.The addition ratio of these additives is every 100 parts by weight of food or healthy food molding in the present invention It is selected in the range of 0.01~0.20 parts by weight of addition.
Specifically, the thrombus molding, to treatment or prevention myocardial infarction, arteries and veins thrombus, apoplexy, cerebral infarction Plug, cerebral thrombosis or cerebral embolism disease have remarkable result.
The present invention staff manufactures the fermented grain enzyme powder that enzyme content increases, and specific enzyme content is as follows.
The other liquid strain bacterium culture solution of cereal is measured in the embodiment of the present invention and adds the cereal hair of culture substrate The enzymatic activity at ferment enzyme powder end.It is known that the activity of alpha-amylase is 3000-15000U/g, the activity of protease is its result 3000- 15000U/g, the activity of lipase are 5-30U/g, and the activity of fibrinolytic enzyme is 50-200U/g.This result It compares with the cereal of no addition culture substrate, enzymatic activity increased significantly.
[The effect of invention]
It can provide balanced absorption enhancement fermented grain through the invention and supplement the kind bacterium substrate rice bran of beneficiating ingredient, wheat Chaff, yeast extract, powdered soybean, the various necessary nutritional ingredients that contain and solidification gemma in soybean separation protein white powder end The fermented grain enzyme of the various physiologically active ingredients such as the tunning and digestive ferment that are generated when bacillus fermentation, and pass through solidification Fermentation of bacillus cereal and its by-product supplement diversified pharmacological property ingredient and physiological activator.And in enteron aisle Beneficial bacterium activate function of intestinal canal, the ingredients such as high concentration amylase and protease that when fermentation generates effectively help to absorb The nutritional ingredient contained in cereal and food, and the effective thrombus of fibrinolytic enzyme helps blood circulation.
Detailed description of the invention
Fig. 1 is the flow diagram of fermenting enzyme preparation method of powder of the invention.
Specific embodiment
The present invention described further below.Embodiment is only example of the invention, it is not limited to the following contents.
<embodiment 1>
<embodiment 1-1>
The measuring method of amylase activity
Substances are held in RO water (reverse osmosis water) with the concentration of 5g/100ml, in 30 DEG C of temperature, 200rpm's Shake culture 30 minutes in rated speed.Night will be tested with the rated speed rotation progress center of circle separation in 3 minutes of 12,000rpm Supernatant is obtained, and filters the supernatant with the filter of 0.45um.Use the appropriate dilute filtration liquid of reverse osmosis water.For sky examination It tests, filtered fluid is heated 30 minutes with 100 DEG C of temperature and is deactivated.1% soluble starch 5ml is put into teat glass, Mcllvaine buffer 13ml, 0.1% calcium chloride 1ml are prepared in advance.By substrate solution with 37 DEG C temperature preincubation 10 minutes. The test solution or empty test solution for adding 1ml, react 30 minutes at 37 DEG C.Prepare the iodine solution of 10ml in 15ml test tube, and adds The test of 200ul/sky tests reaction solution, is contrasting fluid with distilled water, tests absorbance in wavelength 660nm.
* the definition of amylase potency Unit/g: ferment is reacted 30 minutes with the potency contained in test body 1g, is decomposed The value of 1mg starch.
<embodiment 1-2>
The measuring method of proteinase activity
Substances are held in RO water (reverse osmosis water) with the concentration of 5g/100ml, in 30 DEG C of temperature, 200rpm's Shake culture 30 minutes in rated speed.Night will be tested with the rated speed rotation progress center of circle separation in 3 minutes of 12,000rpm Supernatant is obtained, and filters the supernatant with the filter of 0.45um.Use the appropriate dilute filtration liquid of reverse osmosis water.In test tube Prepare 0.6% casein 1ml, with 37 DEG C temperature preincubation 3 minutes.The test solution or empty test solution for adding 1ml, react at 37 DEG C 10 minutes.After being put into 0.4M trichloroacetic acid 2ml, after 37 DEG C of temperature are hatched 25 minutes, filtered with the filter of 0.45um.Make The filtered fluid of 1ml is taken with the conical tube of 15ml.0.4M sodium carbonate 5ml and Folin reagent 1ml is added in the liquid After liquid relief, hatch 20 minutes in 37 DEG C of temperature, and tests absorbance in wavelength 660nm.After adding test solution 1ml, in 37 DEG C of temperature Hatching 15 minutes.After adding 0.4M trichloroacetic acid 2ml in empty test solution, after adding the mixing of 0.6% casein at once, in 37 DEG C of temperature Degree hatching 25 minutes.It is filtered with the filter of 0.45um.The filtered fluid of 1ml is taken using the conical tube of 15ml.The liquid After adding 0.4M sodium carbonate 5ml and Folin reagent 1ml liquid relief in body, hatch 20 minutes in 37 DEG C of temperature, and in wavelength 660nm tests absorbance.
* the definition of protease potency Unit/g: keep ferment anti-using the potency contained in standardized product conversion test body 1g The value that the amount for the l-tyrosine that should be generated is calculated.
<embodiment 1-3>
The measuring method of lipase active
After diluting 5g substances with glycine buffer, 100ml is added with water to using the solution.In advance in titration apparatus In dropper in be put into 0.05N sodium hydroxide solution, adjust the scale of instrument, and temperature is adjusted to 30 DEG C, pH value is adjusted to 7.0. It takes 15.0ml substrate emulsion to be put into the reaction vessel of titration apparatus, and is put into stirring magnet.Drop is set on the reaction vessel After determining instrument, the test solution of 1.0ml is added, and starts titration apparatus.It is carried out using 0.05N sodium hydroxide solution adjustment pH value 7.0 Reaction, and formulate the titration curve per minute of the ml of 0.05N sodium hydroxide solution consumption at this time.
(* note: at titration 5 minutes, the reaction shown in recorder should be straight line.)
The potency of enzyme agent is sought according to following calculation formula.
R: ml number (ml/min) is consumed in the titration per minute of line interval
N: the normality of sodium hydroxide solution
1,000: the mmol of acid is converted to the coefficient of μm ol
W: what is contained in test solution 1ml tests the amount (g) of body
* the definition of potency: 1 enzyme unit (LU) is under the experimental condition, dissociates the enzyme of 1 μm of ol butyric acid from substrate per minute Quantity.
<embodiment 1-4>
The active measuring method of fibrinolytic enzyme
* test solution
It modulates sodium borate buffer liquid (50mM): dissolving sodium tetraborate 19.07g and sodium chloride in the distilled water of 900ml After 9.0g, adjusting pH value with hydrochloric acid is 8.5, and adds distilled water to 1,000ml.
Modulation solution of trichloroacetic acid (0.2M): trichloroacetic acid 32.678 is put into distilled water, and is adjusted in an amount of from 1, 000ml。
Fibrinogen solution (0.72%): it is put into 50mM sodium borate buffer liquid 10ml in conical flask, and is put into fiber After proteinogen (Sigma-aldrich, F8630) 96mg dissolution, is filtered and used using filter paper (Advantech No.6).(* It is modulated when use)
Modulation thrombin solution: fibrin ferment (Sigma-aldrich, T6634) is dissolved in 50mM sodium borate buffer liquid In, after 1,000U/mL Concentration Modulation, a small amount of plant division is freezed to take care of in microtest tube.When test, in 50mM sodium borate buffer Liquid dilutes 50 times of uses.
* method
It modulates test solution: diluting sample concentration to 0.67~1.33FU/g with 50mM sodium borate buffer liquid.Divide in 2 test tubes Sodium borate buffer liquid 1.4ml and fibrinogen solution 0.4ml is not taken, and is heated 5 minutes in 37 DEG C of thermostatic water baths Afterwards, fibrin ferment 0.1ml is put into be stirred.The solution is placed in 37 DEG C of thermostatic water baths after ten minutes, 1 test tube (AT) It is inside put into test solution 0.1ml, places 1 hour after stirring 5 seconds.(every 5 place after twenty minutes, stir 5 seconds) 2 test tube contents Addition 0.2M trichloroacetic acid stirring in page, is not put into the test tube (AB) of test solution after being put into test solution stirring, is placed in 37 DEG C of thermostatted waters 20 minutes in slot.The solution is put into microtest tube, with the center of circle 15,000x g separating treatment 5 minutes.In wavelength 275nm Measure the absorbance of supernatant 1ml.
* it calculates
Fibrinolytic enzyme activity (FU/g)=(AT-AB/0.01) × (1/60) × (1/0.1) × D
0.01:FU conversion coefficient (/ minute)
60: the reaction time (minute)
0.1: test solution additive amount (mL)
* the definition of potency: Unit 1 (1FU) is to have determined in test method, when the wavelength 275nm of sample, is increased per minute The quantity of the enzyme of 0.01 extinction.
<embodiment 1-5>
The measuring method of cellulase activity
* modulate test solution: final dilution 1ml is shown under conditions of this test method, and test solution, which is diluted with water, to be made 5 minutes Generation relative flow variation between interior 0.18~0.22.Add water after taking a certain amount of sample to be put into glass pulverizer.It will After this is moved to the measuring bottle of suitable capacity, the water dilution of appropriate amount is added, and using the graceful No.1 of water or similar filtering using preceding Paper is filtered.
* experimental implementation: the viscosimeter for correcting scale is sufficiently soaked in detergent in advance, and wash with water it is clean after, It is vertically mounted at 40 DEG C of glass flume.Substrate solution 20ml and acetate buffer (pH 4.5) 4mL is taken, 50ml is put into In the track conical flask of capacity, and prepare enzyme test that every 1 sample needs with 2, flask, flask is used in the test of substrate sky. It is placed on after enzyme test is closed the lid with flask in sink 15 minutes, after equilibrium temperature, test solution 1ml is taken to be added to the burning In bottle, start time of measuring, and be sufficiently mixed solution.That is, taking mixed liquor 10ml, the mobile jib for being added to viscosimeter waits 2 points Zhong Hou, links rubber tube in viscosimeter thin bar, and reaction solution is drawn to top scale.For the reaction time within 15 minutes (TR), reaction solution is drawn to top scale 4 repeatedly and is returned.It measures the time (dividing) until solution reaches top scale and is set to TR, and measure the time (second) from new by passing through lower part scale after high scale and be set to TT.Reaction solution is drawn to from newly High scale measures the time (dividing) until liquid surface reaches high scale immediately and is set to TR, measurement from high scale to Time (second) until the scale of lower part is simultaneously set to TT.Substrate solution 20ml and acetate are added in substrate sky test flask After buffer (pH 4.5) 4mL mixed liquor fills water 1ml mixing, mixed liquor 10ml is taken immediately, is added to the mobile jib of viscosimeter Afterwards, time T of the repeated measurement until from high scale to lower part scaleSAfter (second) 5 times, it is averaged and is set to TS.It is tried from water sky It tests with the prior 40 DEG C water 10ml balanced are taken in flask, the mobile jib of viscosimeter is added, repeated measurement reaches between two scales Time TWAfter (second) 5 times, it is averaged and is set to TW.According to the following formula, 4 delivery time (T are soughtT) and reaction time (TR) Respective corresponding relative flow (FR) and TNValue.
FR: each reaction time corresponding relative flow
TS: the average delivery time (second) tested according to substrate sky
TW: the average delivery time (second) tested according to water control
TT: according to the average delivery time (second) of enzyme reaction solution
TR: reaction by time (divide) (since addition test solution time, to start measurement delivery time (TT) until)
TN: reaction time (TR) (dividing)+test solution delivery time (TT) (dividing) 1/2
Calibration curve is formulated using 4 reaction time corresponding 4 relative flows.At this point, the line should be a straight line. The corresponding relative flow variation per minute of the variation of line, it is proportional to enzyme amount.Pass through the variation of the line of a series of testing site Than the benchmark that individual relative flow value more effectively becomes enzyme potency.When being measured 10 minutes and 5 minutes according to calibration curve FRValue.The difference of mobility should be 0.18~0.22.
FR10: the reaction time 10 minutes relative flows
FR5: the reaction time 10 minutes relative flows
1,000:g is scaled mg
W: the amount (mg) for the sample that test solution 1ml contains
* the definition of potency: 1 Cellulase Unit (CU) is under the experimental condition, 5 points in organic cellulose substrate The quantity of the enzyme of relative flow variation is generated in clock.
* device
Viscosimeter: the Canon-Fensk type viscosimeter or same type for having done scale correction of size -100 are used.
Glass flume: thermostatic water bath or same type using temperature for 40 DEG C ± 0.1 DEG C.
* reagent and test solution
Acetate buffer (pH 4.5): 0.4N acetate buffer is added in the 0.4N acetic acid of 400ml and is constantly stirred It mixes and adjusts pH value 4.5 ± 0.05.
Organic cellulose sodium: organic cellulose sodium (cellulose gum Hercules (Aqualon) type 7HF or identical is used Type)
Substrate solution: adjusting is stirred at low speed after filling the water of 200ml in mixing vessel.For the organic fibre for preventing 1g from drying It ties up plain sodium to be scattered to outside container, scattering in container slowly.Rubber stirring rod is only used, after scrubbing container inner wall with hot water, The high-speed stirred that closes the lid 1 minute.After moving to 500ml measuring bottle, add water increment to 500ml, and makes using preceding filtered through gauze With.
<embodiment 2>
The measuring method of total bacteria count
1) test operation: after uniformly mixing using sterilizing glass bar and sterilizing reagent spoon and test body, take it is a certain amount of (10~ The mixture that the dilution for 25g) being put into sterilization container, and measuring with 9 times mixes is as test solution.It is sterile to take test solution 1ml and 10 Times stage dilution liquid 1ml is respectively dropped into sterilizes culture dish 2 or more, and sterile plant division maintains the standard fine jade of 43~45 DEG C of temperature Rouge culture medium about 15ml, the mixing that slowly tilts are tested body and culture medium and are solidified.For the expanding for inhibiting bacterium colony, add again 3~5ml of standard agar culture medium overlapping.At this point, take test body after add culture medium until time max 20 mins. Put upside down after solidification culture dish, 35 ± 1 DEG C of temperature cultures 48 ± 2 hours (can be in 30 ± 1 DEG C of temperature or 35 ± 1 DEG C according to sample Temperature culture 72 ± 3 hours).Substantially to select, no bacterium colony is spread and 1 plate generates the flat of about 15~300 bacterium colonies It is principle that plate, which calculates clump count,.Taking the identical dilution 1ml for being not added with test fluid is comparison test solution, validation test operation Whether sterile.
2) clump count calculates: using clump count computer after culture immediately, calculates the clump count to generate.Special circumstances exist 5 DEG C of temperature calculate within 24 hours after saving.Substantially to select no bacterium colony diffusion (when whole 1/2 or less, having no serious problem) simultaneously And it is principle that 1 plate, which generates the plate calculating clump count of about 15~300 bacterium colonies,.Integral flat-plate generates 300 or more bacterium When falling, calculated according to intensive plate assay plate is closed on.When Integral flat-plate generates 15 bacterium colonies below, with dilute Release that multiple is minimum to be measured
<embodiment 1>
Selected bacterial strain
Brown rice, soybean, barley, optionally its steady 100g in blended grain (wheat, corn, Semen Coicis).Brown rice at this time, Soybean, barley blended grain (wheat, corn, Semen Coicis) use after wanting pre-cleaning to sort.Prepare the water that can be completely soaked cereal After amount, the cereal for impregnating 24 moves to pallet.And it puts into after culture substrate 3 and pallet is moved into 121 DEG C of thermophilic digestions in precooker After 40 minutes, 40~45 DEG C are cooled in precooker.The same bacterium that each cereal compares 10% component is inoculated in pallet respectively Several solidification bacillus (KCTC13284BP), bacillus amyloliquefaciens (KCTC 3002), bacillus natto (KCTC 3239), Lactobacillus fermenti (KCTC 13097).30~40 DEG C of temperature are maintained, after fermenting 48 hours in the environment of humidity 50~85%, 40 Fermentation is dried in~60 DEG C of temperature, finally maintains 3~7% moisture.
By the embodiment it has been confirmed that solidification bacillus is to amylase, the activity of protease, lipase is best.
[table 1]
The setting of bacterial strain
<embodiment 2>
The setting of kind bacterium substrate combination
To determine that kind of bacterium adapts to the kind bacteria culture fluid of best ferment effect when using cereal and implements.
[table 2]
Kind bacterium substrate composition
Kind bacterium substrate It combines (on the basis of 100 parts by weight of pure water)
Kind bacterium substrate 1 Powdered soybean 1%, rice bran 1%, wheat bran 1%, soybean protein isolate 1%, yeast extract 0.5%
Kind bacterium substrate 2 Powdered soybean 2%, rice bran 2%, wheat bran 2%, soybean protein isolate 1%, yeast extract 0.5%
Kind bacterium substrate 3 Powdered soybean 5%, rice bran 5%, wheat bran 5%, soybean protein isolate 1%, yeast extract 0.5%
For the combination of liquid strain bacteria culture fluid, manufacture kind bacterium is carried out after sterilization and cooling with substrate, by LB culture medium bottom (tryptose Peptone 1%, yeast extract 0.5%, sodium chloride 1%) in cultivate solidification bacillus by 10% component of culture solution inoculation, and At 37 DEG C with 180rpm, 15 hours confirmation total bacteria counts are cultivated.
Prepare the brown rice of 100g, soybean, barley, blended grain (wheat, corn, Semen Coicis) first.Brown rice at this time, greatly Beans, barley blended grain (wheat, corn, Semen Coicis) use after wanting pre-cleaning to sort.Prepare the water that can be completely soaked cereal Afterwards, after being impregnated according to the other soaking time of cereal, cereal is moved into pallet.And move to pallet in precooker, at 100 DEG C Pre-cooked 15 minutes in temperature are cooled to 40~45 DEG C in 121 DEG C of temperature after boiling 30 minutes in precooker.It will be described The liquid strain bacterium of kind bacterium substrate 1,2,3 is respectively put into boiling each 10% component of cereal after cooling with culture solution and is connect After kind mixing, 30~40 DEG C of temperature are maintained, after fermenting 48 hours in the environment of humidity 50~85%, is dried in 40~60 DEG C of temperature Dry fermentation measures total bacteria count after finally maintaining 5~10% moisture and the crushing of 40 sieve pores.
Confirm the total bacteria count of liquid strain bacterium culture solution and each final fermented grain enzyme powder, discovery kind bacterium substrate 2, kind bacterium is most with the total bacteria count of substrate 3, and, setting kind bacterium substrate 2 is most economical applicable, efficient combination.(reference Table 3)
[table 3]
The not final powder total bacteria count of liquid strain bacteria culture fluid
<embodiment 3>
The setting of soaking time
For the other corresponding soaking time of optimal conditions of fermentation of cereal in the determination embodiment, prepare to soak completely first After the water for steeping cereal, the cereal of 100g impregnates 4 hours, 7 hours, 15 hours, 24 hours respectively.Cereal after immersion is moved To pallet, after putting into culture substrate, pallet is moved in precooker, 121 DEG C temperature boiling 40 minutes, and in precooker It is cooled to 40~45 DEG C.After cooling, the same bacterium number of cereal 10% component of comparison is inoculated in pre-cooked time other pallet respectively It cultivates bacterium and solidifies bacillus.30~40 DEG C of temperature are maintained, after fermenting 48 hours in the environment of humidity 50~85%, 40~60 Fermentation is dried in DEG C temperature, finally maintains 3~7% moisture.
By the embodiment it has been confirmed that soybeans soaking 7 hours, barley is impregnated 4 hours, and brown rice impregnates 15 hours, mixes When connected valleys object (corn, wheat, Semen Coicis) impregnates 15 hours, amylase, protease, the activity of lipase is best.(referring to table 4)
[table 4]
The setting of soaking time
<embodiment 4>
The setting of pre-cooked time
To determine the other corresponding digestion time of optimal conditions of fermentation of cereal, prepare the water that can be completely soaked cereal first Afterwards, the cereal of 100g impregnates respectively.Soybeans soaking 7 hours, barley was impregnated 4 hours, and brown rice impregnates 15 hours, and blended grain is (beautiful Rice, wheat, Semen Coicis) it impregnates and moves back to pallet for 15 hours.This culture is put into be moved back with substrate to precooker, respectively in 80 DEG C of height Middle benefit gas pre-cooked 10 minutes, 30 minutes, 60 minutes, pre-cooked 10 minutes in 100 DEG C of high temperature, 30 minutes, after sixty minutes, 121 After DEG C high temperature carries out formal boiling in 30 minutes, 40~45 DEG C are cooled in precooker.After cooling, the pre-cooked time is not held in the palm The culture bacterium solidification bacillus that cereal compares the same bacterium number of 10% component is inoculated in disk respectively.30~40 DEG C of temperature are maintained, After fermenting 48 hours in the environment of humidity 50~85%, fermentation is dried in 40~60 DEG C of temperature, finally maintains 3~7% water Point.
By the embodiment it has been confirmed that in 100 DEG C of temperature at pre-cooked 30 minutes, amylase, protease, lipase Activity be best.(referring to table 5)
[table 5]
The setting of pre-cooked time
<embodiment 5>
The setting of fermentation condition
It in fermentation temperature is respectively 30 DEG C using brown rice, 37 DEG C, 40 DEG C, be all to set the other optimal conditions of fermentation of cereal Respectively 50%, 60%, 70%, it ferments respectively 12 hours in the environment of 85%, 24 hours, 36 hours, 48 hours, 60 hours, Enzyme potency is measured after 72 hours.
By the embodiment, at 37 DEG C, when moisture ferments 48 hours in the case where 70%, amylase, protease, rouge The activity of fat enzyme is best.(referring to table 6, table 7, table 8)
[table 6]
The setting of fermentation temperature
[table 7]
The setting for humidity of fermenting
[table 8]
The setting of fermentation time
<embodiment 6>
The setting of fermentating drying condition
To set the other optimal conditions of fermentation of cereal, using brown rice, after soybean is fermented, drying temperature is respectively 30 DEG C, Confirm that enzyme potency and moisture contain under conditions of 40 DEG C, 50 DEG C, 60 DEG C, after being respectively dried 12 hours, 24 hours and 48 hours Amount.
By the embodiment, when 40 DEG C of drying are fermented 24 hours, optimum moisture content 5% can be met simultaneously and formed sediment Powder enzyme, protease, two conditions of lipase active.(referring to table 9)
[table 9]
<embodiment 7>
The setting of this culture substrate combination
Prepare the brown rice of 100g, soybean, barley, blended grain (wheat, corn, Semen Coicis).Brown rice at this time, soybean, greatly Wheat blended grain (wheat, corn, Semen Coicis) uses after wanting pre-cleaning to sort.After preparation can be completely soaked the water of cereal, root After being impregnated according to the other soaking time of cereal, cereal is moved into pallet.
[table 10]
Culture is not combined with substrate
Substrate is used in culture It combines (on the basis of 100 parts by weight of pure water)
Culture substrate 1 Powdered soybean 5%, rice bran 5%, wheat bran 5%, soybean protein isolate 1%, yeast extract 1%
Culture substrate 2 Powdered soybean 10%, rice bran 5%, wheat bran 5%, soybean protein isolate 1%, yeast extract 1%
Culture substrate 3 Powdered soybean 10%, rice bran 5%, wheat bran 5%, soybean protein isolate 2%, yeast extract 1%
Culture substrate 4 Powdered soybean 10%, rice bran 5%, wheat bran 5%, soybean protein isolate 5%, yeast extract 2%
It is put into respectively in no added culture substrate and culture medium matrix bottom 1~4 and 100 parts by weight of blended grain is to score Amount.Pallet is moved into precooker, 100 DEG C high temperature pre-cooked 15 minutes, 121 DEG C after thermophilic digestion 30 minutes in precooker It is cooled to 40~45 DEG C.Culture is added to the component of cereal comparison 10% in saucer with 10% component of substrate.After mixing 30~40 DEG C of temperature are maintained, humidity 50~85% dries fermentation, most after the interior fermentation of fermenting cellar 48 hours in 40~60 DEG C of temperature 3~7% moisture is maintained eventually.
The total bacteria count and enzymatic activity ability discovery for confirming the other final fermented grain enzyme powder of culture substrate, and do not add The case where adding culture substrate, is compared, and the total bacteria count two when adding culture substrate is obviously relatively more, and enzyme activity sexuality also compares It is prominent.(referring to table 11)
[table 11]
Enzyme activity sexuality based on culture substrate
[the utilization possibility in industry]
The present invention can supplement various pharmacological property ingredients and life by the solidification bacillus of fermented cereal and its by-product Manage active material.And function of intestinal canal is activated by beneficial bacterium in enteron aisle, help to absorb the high concentration shallow lake generated when fermentation Powder enzyme, protease, the effect of the nutritional ingredient contained in the cereal such as lipase and food, fibrinolytic enzyme dissolve blood Bolt is conducive to sanguimotor effect, is that one kind has industry to invent using possibility.
[preservation information]
Preservation office name: South Korea's Culture Collection
Address: Jeollabuk-do, Korea
Classification naming: solidification bacillus

Claims (15)

1. the manufacturing method of fermented grain enzyme powder includes,
(a) the kind bacterium base being made of rice bran, wheat bran, powdered soybean, soybean separation protein white powder end and yeast extract powder The stage of inoculation solidification Bacillus strain manufacture liquid strain bacterium culture solution in bottom；
(b) what mixing rice bran in cereal, wheat bran, powdered soybean, soybean separation protein white powder end and yeast extract powder were constituted The stage of boiling is carried out after culture substrate；
(c) the liquid strain bacterium that (a) stage by described in manufactures is added in the cereal of (b) stage boiling with culture solution and is sent out The stage of ferment；
(d) fermentation material that (c) stage fermentation described in drying is formed, and the stage of powdered.
2. the manufacturing method of fermented grain enzyme food includes,
(a) the kind bacterium base being made of rice bran, wheat bran, powdered soybean, soybean separation protein white powder end and yeast extract powder The stage of inoculation solidification Bacillus strain manufacture liquid strain bacterium culture solution in bottom；
(b) what mixing rice bran in cereal, wheat bran, powdered soybean, soybean separation protein white powder end and yeast extract powder were constituted The stage of boiling is carried out after culture substrate；
(c) the liquid strain bacterium that (a) stage by described in manufactures is added in the cereal of (b) stage boiling with culture solution and is sent out The stage of ferment；
(d) fermentation material that (c) stage fermentation described in drying is formed, and the stage of powdered；
(e) powder in (d) stage by described in is converted into beverage, and fruit is refined, powder ball, the formative stages such as capsule.
3. manufacturing method according to claim 1 or 2, which is characterized in that the cereal in (b) stage is brown rice, big The one or more selected in the group that the blended grains such as wheat, soybean and corn, wheat, Semen Coicis are constituted.
4. manufacturing method according to claim 1 or 2, which is characterized in that the cereal immersion 3 in (b) stage by described in~ 16 hours.
5. manufacturing method according to claim 1 or 2, which is characterized in that the kind bacterium in (a) stage is phase with substrate To 100 parts by weight of pure water, 1~5 parts by weight of powdered soybean, 1~5 parts by weight of rice bran, 1~5 parts by weight of wheat bran, soybean separation protein White 1~5 parts by weight, 0.1~3 parts by weight of yeast extract powder.
6. manufacturing method according to claim 1 or 2, which is characterized in that the culture in (b) stage is phase with substrate To 100 parts by weight of cereal, 5~10 parts by weight of powdered soybean, 1~5 parts by weight of rice bran, 1~5 parts by weight of wheat bran, soybean separation protein White 1~5 parts by weight, 1~5 parts by weight of yeast extract powder.
7. manufacturing method according to claim 1 or 2, which is characterized in that the solidification bacillus bacterium in (a) stage It is entrusted in strain commission number KCTC13284BP.
8. manufacturing method according to claim 1 or 2, which is characterized in that the boiling in (b) stage is 70 DEG C~ Boiling 10~60 minutes in 140 DEG C of high temperature.
9. manufacturing method according to claim 1 or 2, which is characterized in that the boiling in (b) stage is 60 DEG C additional Pre-cooked 10~60 minutes in~110 DEG C of high temperature.
10. manufacturing method according to claim 1 or 2, which is characterized in that the fermentation in (c) stage is 30 DEG C~ It is carried out in 40 DEG C of temperature or 50%~85% humidity.
11. manufacturing method according to claim 1 or 2, which is characterized in that the drying in (d) stage is 35 DEG C~ It is dried 20~28 hours in 45 DEG C of temperature.
12. manufacturing method according to claim 1 or 2, which is characterized in that the liquid strain bacterium in (c) stage is trained Nutrient solution, 100 parts by weight of cereal in relatively described (b) stage, adds 5~20 parts by weight.
13. the cereal enzyme powder of method according to claim 1 manufacture, which is characterized in that fermented grain enzyme powder is The one or more selected in the group that amylase, protease, lipase, fibrinolytic enzyme and cellulase are constituted Enzymatic activity increase.
14. food products are used in the thrombus decomposition including fermented grain enzyme powder described in claim 13.
15. food products are used in the improvement digestion including fermented grain enzyme powder described in claim 13.
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