CN109757652A - A kind of highland barley products and its preparation method and application - Google Patents
A kind of highland barley products and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of preparation methods of highland barley products, it includes following content: (1) highland barley flour；(2) complex enzyme；(3) water；(4) it digests；(5) take solid content up to highland barley products；Wherein, the complex enzyme two or more combination in alpha-amylase, beta amylase, Pullulanase, carbohydrase.Above-mentioned highland barley products can be prepared the natural prodcuts for improving diabetes by the present invention.The research of the invention finds that the fasting blood-glucose and postprandial blood sugar of diabetic can be reduced using highland barley products of the present invention, or prevent the raising of diabetic's postprandial blood sugar.
Description
Technical field
The present invention relates to a kind of novel highland barley product, and its preparation method and application.
Background technique
Highland barley is Qinghai-Tibet a kind of important cereal, dietary fiber, protein and microelement with higher, sugar and
The lower feature of fat content is the good merchantable brand in cereal crops.Instantly the research and development of highland barley is utilized and relates generally to highland barley
The brew of wine, the processing of highland barley Flour product, forage crops and abstraction function factor etc..Has highland barley seedling on domestic and international market
The products such as powder, highland barley wheat element powder and naked barley malt, add them among various food, can be enhanced food sense organ and
Nutritional quality.In recent years, studies in China scholar to extraction of highland barley starch, protein content and the physiological function factor etc. into
A large amount of research is gone.Wang Hengliang is prepared for highland barley Beta-dextran crude product, and has studied the mechanism of action of its reducing blood lipid.Chen Dongfang
Et al. have studied highland barley extractive to the adjustment effect of rat fat.Zhao Yanqiao et al. has studied cellulase and extracts highland barley starch
Technique.Lv Yuandi is prepared for acetylated modification starch by being modified to highland barley starch, and grinds to its digestic property
Study carefully.
Summary of the invention
The present invention is intended to provide a kind of healthy highland barley products of diabetic preferably.
Specifically, the present invention provides a kind of preparation methods of highland barley products, it includes following content:
(1) highland barley flour；(2) complex enzyme；(3) water；(4) it digests；5) take solid content up to highland barley products；
Wherein, the complex enzyme in alpha-amylase, beta amylase, Pullulanase, carbohydrase two or more
Combination.
The mode that solid content is separated with liquid can also take the methods of common filtering, centrifugation.
Solid content after separation can be dried, and the drying of solid content can be dried using natural drying, sunning, normal pressure, be subtracted
Pressure drying, freeze-drying.
From the point of view of the hydrolysis to highland barley starch, the present invention mainly chooses alpha-amylase, Pullulanase, carbohydrase three
Kind enzyme is used in mixed way, as complex enzyme.
Carbohydrase, carbohydrase are also known as glucoamylase, and carbohydrase is a kind of customary title, and scientific name is α-Isosorbide-5-Nitrae-Portugal
Grape glycosylhydrolase.
Pullulanase (pullulanase) is a kind of starch debranching enzymes, because it can specificity hydrolysis pulullan
(pullulan, the polymer that maltotriose is got up with α -1,6 glucosides key connections) and gain the name.
The present invention has found that, using above-mentioned three kinds of complex enzymes, ratio is in alpha-amylase: Pullulanase in an experiment: saccharification
Within the scope of this, Starch Hydrolysis degree can reach 47% or more for 1~3:1 of enzyme~3:1~3, it might even be possible to up to 50% or more.Root
According to the screening to different ratio, finally found that, alpha-amylase: Pullulanase: the ratio of carbohydrase enzyme activity (U) be 1:2:2 this
Under proportion, starch hydrolysis is most strong.
The present invention tests discovery, when the additive amount of the complex enzyme is more than 150U/g highland barley flour, is further added by dosage, water
Xie Du increase also tends to be steady.It is found in the specific embodiment of the invention, when complex enzyme additive amount is 200U/g highland barley flour, water
Solution effect is stronger compared with other proportions.
In the specific embodiment of the invention, hydrolysis temperature can be 55~70 DEG C, such as 55,60,65,70 DEG C.Certainly, it tests
When showing that hydrolysis temperature is 60 DEG C, hydrolysis is stronger compared with other proportions.
In the specific embodiment of the invention, enzymolysis time can be 2~4h, such as can choose 2h, 2.5h, 3h, 3.5h, 4h
Deng.
Wherein, solid-liquid ratio is 1:7~13, is further selected from 1:13.The solid-liquid ratio refers to raw material highland barley flour and final enzyme
The ratio of the water in liquid is solved, ratio unit is mass/volume, can be g/mL, Kg/L etc..
Certainly, the present invention also provides a kind of highland barley products, in the product, the content of starch of highland barley is in 40%w/w or less；
It is further selected from 30~40%.Above-mentioned content of starch can rationally be calculated by the degree of hydrolysis of starch.The present invention one specific
In embodiment, the content of starch is about 65% in the highland barley flour that does not digest.
Diabetes are a kind of metabolic diseases, and the number with diabetes all increases with year, anxious due to caused by diabetes, slow
Property complication harm it is big, patient death rate is higher.Diabetes are one kind as caused by Islet dysfunction with hyperglycemia
The lifelong disease being characterized there is no the method for radical cure at present.It is mostly clinically chemical synthesis for treating the drug of diabetes
Western medicine be often accompanied by a variety of toxic side effects although short-term hypoglycemic is significant.
The research of the invention finds that the fasting blood-glucose and postprandial blood sugar of diabetes can be reduced using highland barley products of the present invention.
Based on above-mentioned discovery, above-mentioned highland barley products can be prepared the natural prodcuts for improving diabetes by the present invention.Specifically,
It is the product for reducing diabetes glucose.More specifically, being the product for the fasting blood-glucose or/and postprandial blood sugar for reducing diabetes.
Based on above-mentioned effect, characteristic in conjunction with highland barley as food, the present invention also provides a kind of food or health care product, it
Based on aforementioned highland barley products.
Certainly, in food or health care product, the dosage of above-mentioned highland barley products can be adjusted according to different needs.This
It invents in a specific embodiment, in the food or health care product, the content of highland barley products is 40% or more；It is also possible to
50%, 60% etc..
The food or health care product have certain reduction to act on fasting blood-glucose and postprandial blood sugar, are particularly suitable for patient of diabetes
Person.
" highland barley flour " that chinese raw materials of the present invention uses is the crushed material of highland barley.Grinding mode can there are many, such as grind
Broken, mechanical crushing etc..
" enzymatic hydrolysis " refers to, by the decomposition of enzymatic.
" water " selects different ranks according to the professional standard of different product in actual use.
Detailed description of the invention
Fig. 1 preparation process
Influence of Fig. 2 difference amylase to highland barley Starch Hydrolysis degree
Influence of Fig. 3 alpha-amylase starch enzyme concentration to degree of hydrolysis
Influence of Fig. 4 enzymolysis time to α-amylasehydrolysis starch
Influence of Fig. 5 temperature to α-amylasehydrolysis starch
Influence of Fig. 6 Pullulanase enzyme concentration to Starch Hydrolysis degree
Influence of Fig. 7 enzymolysis time to Pullulanase hydrolysis starch
Influence of Fig. 8 temperature to Pullulanase hydrolysis starch
Influence of Fig. 9 carbohydrase enzyme concentration to Starch Hydrolysis degree
Influence of Figure 10 enzymolysis time to Glucoamylase hydrolysis starch
Influence of Figure 11 temperature to Glucoamylase hydrolysis starch
Influence of Figure 12 complex enzyme enzyme concentration to Starch Hydrolysis degree
Influence of Figure 13 complex enzyme zymohydrolysis temperature to Starch Hydrolysis degree
Influence of Figure 14 complex enzyme zymohydrolysis time to Starch Hydrolysis degree
Influence of Figure 15 complex enzyme solid-liquid ratio to Starch Hydrolysis degree
Influence of Figure 16 sugar highland barley flour to diabetic mice fasting blood-glucose
Influence of Figure 17 low sugar highland barley flour to diabetic mice postprandial blood sugar
Specific embodiment
The methodological study of 1 highland barley products of embodiment preparation
1.1 test material
Highland barley (place of production Qinghai)；High-temperatureα-amylase (40000U/mL) Jiangsu Ruiyang Biological Technology Co., Ltd.；β-starch
Enzyme (700,000 U/mL) Jiangsu Ruiyang Biological Technology Co., Ltd.；The limited public affairs of the sharp positive biotechnology in the Jiangsu carbohydrase (100000U/mL)
Department；Pullulanase (1500U/mL) Jiangsu Ruiyang Biological Technology Co., Ltd.；The marine Qin on 3,5 dinitrosalicylic acids (analysis is pure)
Chemical reagent Co., Ltd；Glucose (analysis is pure) Tianjin Yong great chemical reagent Co., Ltd；Sodium hydroxide (analysis is pure) day
Jinshi City Hedong District red rock chemical reagent work；Glycerine (analysis is pure) Tianjin Fu Yu Fine Chemical Co., Ltd；Dehydrated alcohol (analysis
It is pure) Tianjin Fu Yu Fine Chemical Co., Ltd.PH meter (PHSJ-4A) Shanghai INESA Scientific Instrument Co., Ltd.；Constant temperature
Oscillator (TH2-82) Changzhou Guohua Electric Appliance Co., Ltd.；Electronic balance (ESJ110-48) Shenyang Longteng Electronic Co., Ltd.；Electricity
The hot thermostat water bath Shanghai (HH-6) is than bright Instrument Ltd.；Eddy mixer (MVS-1) Beijing Orient opens object science equipment
Co., Ltd；Electric drying oven with forced convection Shanghai Yiheng Scientific Instruments Co., Ltd；Ultraviolet specrophotometer (UV-2600) day island proper
Saliva Co., Ltd.
1.2 test methods and research contents
1.2.1 low sugar highland barley flour preparation process flow and key points for operation (referring to Fig. 1)
(enzymatic hydrolysis) → centrifugation → removal supernatant is vibrated in highland barley coarse powder → addition enzymolysis liquid → be uniformly mixed → water bath with thermostatic control
→ low sugar highland barley flour
It prepares enzymolysis liquid: measuring 0.1mL alpha-amylase enzyme solution, 5.3mL Pullulanase enzyme solution and 0.08mL carbohydrase enzyme solution
It is settled to after mixing in 10mL volumetric flask, the complex enzyme liquid in volumetric flask contains 400U/mL alpha-amylase at this time, and 800U/mL is general
Shandong orchid enzyme, 800U/mL carbohydrase.
Addition enzyme solution: weighing 1g highland barley coarse powder and be put into test tube, and the complex enzyme liquid that 1mL is prepared is added, adds 12mL water,
Glass bar stirs evenly.Notice that highland barley coarse powder is not soluble in water, therefore to be sufficiently stirred, keeps highland barley corase meal and enzymolysis liquid sufficiently mixed
It closes.
Test tube: being put into 60 DEG C of constant temperature oscillation water-bath by enzymatic hydrolysis, with 140r/min constant temperature oscillation 3h, make highland barley coarse powder with
Enzymolysis liquid mixing.Sufficiently oscillation can prevent the sedimentation of highland barley coarse powder to be gathered in test tube bottom, connect highland barley flour sufficiently with enzymolysis liquid
Touching.
Centrifuge separation: it removes test tube and mixture is separated by solid-liquid separation, be transferred in 50mL centrifuge tube, then exist
It is centrifuged 10min under 2800r/min, is centrifuged again into centrifuge tube in a certain amount of water of addition, the supernatant obtained twice is removed.
Drying is taken out in remaining solid part, obtains low sugar highland barley flour.
1.2.3 the measurement of main indicator Starch Hydrolysis degree
Solution is prepared: glucose standard liquid and DNS developing solution are prepared with reference to pertinent literature.
Glucose standard curve production: the drafting of glucose standard curve is surveyed using 3,5 dinitrosalicylic acid DNS colorimetric methods
It is fixed.
Total sugar content measurement: total sugar content bibliography measures, and total reducing sugar (with glucose meter) content calculates public in highland barley flour
Formula is as follows:
X=C*V2/V1
In formula: X is total sugar content (g)；C is the glucose mass concentration (mg/mL) being calculated by standard curve；V1For
Test sample volume (mL)；V2Constant volume (mL) is diluted for sample.
Sample reduced sugar hypoglycemic assay: 1g highland barley coarse powder precisely is weighed, 1g highland barley coarse powder is put into 25mL test tube
A certain amount of enzyme solution, moisturizing to 10mL is added.One group of test tube separately is taken, enzyme solution is not added, by above-mentioned same operation, is put into thermostatted water
In bath oscillation pot, constant temperature oscillation is for a period of time under certain temperature.Test tube is taken out from water bath with thermostatic control oscillation pot, in test tube
Mixture is transferred in 50mL centrifuge tube, is centrifuged 10min under the conditions of 2800r/min.The supernatant of enzymolysis sample is denoted as enzyme
Liquid is solved, the sample supernatant for not adding enzyme solution is denoted as non-enzymolysis liquid.Supernatant after taking centrifugation surveys light absorption value, according to 540nm wave
Standard curve under long calculates concentration, and calculates the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid.
The measurement of highland barley Starch Hydrolysis degree: the degree of hydrolysis of highland barley starch calculates as follows.
Starch Hydrolysis degree=(sample enzymolysis liquid content of reducing sugar-non-enzymolysis liquid control sample content of reducing sugar)/total reducing sugar contains
Amount
1.2.4 main research
1.2.4.1 being suitable for the single factor experiment of amylase screening
The amylase of alpha-amylase, beta amylase, Pullulanase, carbohydrase as enzymatic hydrolysis highland barley starch is selected, with starch
Degree of hydrolysis be index, screening enzymatic hydrolysis highland barley starch most suitable hydrolase.Take 1g highland barley coarse powder that a certain amount of enzyme solution, starch is added
The additive amount of enzyme is 15,30,45,60U/g highland barley coarse powder, and moisturizing to 10mL, enzymolysis time 3h, 65 DEG C of temperature carry out enzymatic hydrolysis examination
It tests, measures the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid, calculate Starch Hydrolysis degree.
1.2.4.2 enzymolyzing alpha-amylase condition single factor experiment
The temperature and enzymolysis time for controlling reaction system, the enzyme additive amount for studying alpha-amylase simultaneously do not form sediment to enzymatic hydrolysis highland barley
The influence of powder.Choosing alpha-amylase enzyme concentration is 10,50,100,150,200,250U/g highland barley coarse powder, enzymolysis time 3h, temperature
65 DEG C of progress proteolysis assays measure the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid, calculate Starch Hydrolysis degree.
The enzyme action time and enzyme additive amount of reaction system are controlled, research temperature does not simultaneously form sediment to enzymolyzing alpha-amylase highland barley
The influence of powder.35,45,55,65,75 DEG C of hydrolysis temperature, enzymolysis time 3h are chosen, alpha-amylase enzyme concentration 150U/g highland barley coarse powder
Proteolysis assay is carried out, the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid is measured, calculates Starch Hydrolysis degree.
The temperature and enzyme additive amount of reaction system are controlled, studies enzymolysis time not simultaneously to enzymolyzing alpha-amylase highland barley starch
Influence.Enzymolysis time 1,2,3,4,5h are chosen, 65 DEG C of temperature, alpha-amylase enzyme concentration 150U/g highland barley coarse powder carries out enzymatic hydrolysis examination
It tests, measures the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid, calculate Starch Hydrolysis degree.
1.2.4.3 Pullulanase enzymatic hydrolysis condition single factor experiment
The temperature and enzymolysis time of reaction system are controlled, research Pullulanase enzyme additive amount is not simultaneously to enzymatic hydrolysis highland barley starch
Influence.Choosing Pullulanase enzyme concentration is 10,50,100,150,200,250U/g highland barley coarse powder, enzymolysis time 3h, temperature 65
DEG C proteolysis assay is carried out, measures the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid, calculate Starch Hydrolysis degree.
The enzyme action time and enzyme additive amount of reaction system are controlled, research temperature does not simultaneously form sediment to Pullulanase enzymatic hydrolysis highland barley
The influence of powder.35,45,55,65,75 DEG C of hydrolysis temperature, enzymolysis time 3h are chosen, Pullulanase enzyme concentration 150U/g highland barley coarse powder
Proteolysis assay is carried out, the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid is measured, calculates Starch Hydrolysis degree.
The temperature and enzyme additive amount of reaction system are controlled, is formed sediment when studying enzyme action time difference to Pullulanase enzymatic hydrolysis highland barley
The influence of powder.Enzymolysis time 1,2,3,4,5h are chosen, 65 DEG C of temperature, Pullulanase enzyme concentration 150U/g highland barley coarse powder is digested
Test measures the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid, calculates Starch Hydrolysis degree.
1.2.4.4 carbohydrase enzymatic hydrolysis condition single factor experiment
The temperature and enzyme action time for controlling reaction system, the enzyme additive amount for studying carbohydrase simultaneously do not form sediment to enzymatic hydrolysis highland barley
The influence of powder.Choosing carbohydrase enzyme concentration is 10,50,100,150,200,250U/g highland barley coarse powder, enzymolysis time 3h, temperature 65
DEG C proteolysis assay is carried out, measures the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid, calculate Starch Hydrolysis degree.
The enzyme action time and enzyme additive amount of reaction system are controlled, research temperature does not digest highland barley starch to carbohydrase simultaneously
Influence.35,45,55,65,75 DEG C of hydrolysis temperature, enzymolysis time 3h are chosen, carbohydrase enzyme concentration 200U/g highland barley coarse powder carries out
Proteolysis assay measures the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid, calculates Starch Hydrolysis degree.
The temperature and enzyme additive amount of reaction system are controlled, highland barley starch is digested to carbohydrase when studying enzyme action time difference
Influence.Enzymolysis time 1,2,3,4,5h are chosen, 65 DEG C of temperature, carbohydrase enzyme concentration 150U/g highland barley coarse powder carries out enzymatic hydrolysis examination
It tests, measures the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid, calculate Starch Hydrolysis degree.
1.2.4.5 the experiment of single factor of enzyme ratio screening
On the basis of single factor experiment, alpha-amylase, Pullulanase and carbohydrase are chosen, control enzyme concentration total amount is
200U/g highland barley coarse powder, handles time 3h by 65 DEG C of hydrolysis temperature, handles highland barley flour by different proportion.Research different composite enzyme is matched
Compare the influence of enzymatic hydrolysis highland barley starch.Alpha-amylase: Pullulanase: the ratio of carbohydrase be respectively 1:1:1,1:2:2,1:3:3,
2:1:2,2:2:3,2:3:1,3:1:3,3:2:1,3:3:2 carry out proteolysis assay, measure the reduction in enzymolysis liquid and non-enzymolysis liquid
Sugared content calculates Starch Hydrolysis degree.
1.2.4.6 complex enzyme zymohydrolysis condition single factor experiment
Take 1g highland barley coarse powder in test tube, enzyme ratio presses alpha-amylase: Pullulanase: carbohydrase=1:2:2 is added,
Complex enzyme enzyme concentration is set as 10,100,200,300,400,500U/g highland barley coarse powder, solid-liquid ratio 1:10 handles time 3h,
65 DEG C of progress proteolysis assays of temperature measure the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid, calculate Starch Hydrolysis degree, and research is multiple
Influence of the synthase enzyme concentration to enzymatic hydrolysis highland barley starch.
Take 1g highland barley coarse powder in test tube, enzyme ratio presses alpha-amylase: Pullulanase: carbohydrase=1:2:2 is added,
Complex enzyme zymohydrolysis temperature is set as 35,45,55,65,75 DEG C, solid-liquid ratio 1:10, handles time 3h, enzyme total amount is 200U/g
Highland barley coarse powder carries out proteolysis assay, measures the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid, calculates Starch Hydrolysis degree, and research is multiple
The not influence to complex enzyme zymohydrolysis highland barley starch simultaneously of synthase hydrolysis temperature.
Take 1g highland barley coarse powder in test tube, enzyme ratio presses alpha-amylase: Pullulanase: carbohydrase=1:2:2 is added,
The complex enzyme zymohydrolysis time is set as 1,2,3,4,5h, enzyme total amount 200U/g highland barley coarse powder, solid-liquid ratio 1:10,65 DEG C of temperature into
Row proteolysis assay measures the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid, calculates Starch Hydrolysis degree, when studying complex enzyme zymohydrolysis
Between to enzymatic hydrolysis highland barley starch influence.
Take 1g highland barley coarse powder in test tube, enzyme ratio presses alpha-amylase: Pullulanase: carbohydrase=1:2:2 is added,
The solid-liquid ratio of complex enzyme zymohydrolysis highland barley coarse powder is set as 1:5,1:10,1:15,1:20, enzyme total amount 200U/g highland barley coarse powder is located
Time 3h is managed, 65 DEG C of progress proteolysis assays of temperature measure the content of reducing sugar in enzymolysis liquid and non-enzymolysis liquid, calculate Starch Hydrolysis
Degree studies the solid-liquid ratio not influence to complex enzyme zymohydrolysis highland barley starch simultaneously.
1.2.4.7 the orthogonal test of complex enzyme zymohydrolysis technique
On the basis of single factor experiment, to comprehensively consider influence of each factor to enzymatic hydrolysis highland barley coarse powder hypoglycemic technique, really
Fixed optimal fabrication process condition.Temperature, enzyme additive amount, enzymolysis time and solid-liquid ratio are chosen as 4 factors, sets 3 water
Flat, the most suitable process conditions of experiment sieving complex enzyme formula design L9(34) orthogonal arrage, using Starch Hydrolysis degree as test result
Index.Orthogonal test factor level table is shown in Table 1.
1 complex enzyme process optimization orthogonal test of table
1.2.5 data processing
All data indicate that the difference of different factors and level is examined using T using average value X ± S.
2. result and analysis
The measurement result of 2.1 glucose standard curves and total sugar content
Glucose mass concentration (mg/mL) and absorbance (A) have certain linear relationship, using glucose mass concentration as
Abscissa, A are that ordinate draws standard curve.Regression equation are as follows: y=6.6743X+0.008, R2=0.9995.
After measured, the total sugar content in highland barley coarse powder is 0.7042 ± 0.0173g/g highland barley coarse powder.
2.2 amylase the selection results
2.2.1 influence of the different amylase to highland barley coarse powder hypoglycemic effect
Influence of the different amylase to starch degradation effect in highland barley coarse powder is as shown in Figure 2.
As shown in Fig. 2, alpha-amylase, carbohydrase and Pullulanase have preferable hydrolysis effect to highland barley coarse powder, with
The degree of hydrolysis of the increase of enzyme additive amount, highland barley starch is continuously increased；Beta amylase is when enzyme additive amount reaches 45U/g highland barley coarse powder
Starch Hydrolysis degree reaches highest, further increases enzyme amount, and the degree of hydrolysis of starch will not further increase.Therefore, alphalise starch is selected
The suitable amylase of enzyme, Pullulanase and carbohydrase as enzymatic hydrolysis highland barley coarse powder.
The single factor experiment result of 2.3 single enzyme enzymatic hydrolysis conditions
2.3.1 enzymolyzing alpha-amylase condition experiment of single factor result
Influence of the alpha-amylase enzyme concentration to highland barley Starch Hydrolysis degree is as shown in Figure 3.
From the figure 3, it may be seen that with the variation of alpha-amylase enzyme additive amount, in enzymatic hydrolysis initial stage degree of hydrolysis as initial enzyme adds
The increase of dosage and increase.When alpha-amylase enzyme additive amount reaches 150U/g highland barley coarse powder, Starch Hydrolysis degree reaches maximum value
(47.05%).After enzyme additive amount is greater than 150U/g highland barley coarse powder, Starch Hydrolysis degree gradually tends towards stability, and almost no longer increases, very
To there is downward trend.
Influence of the enzymolyzing alpha-amylase time to highland barley Starch Hydrolysis degree is as shown in Figure 4.
As shown in figure 4, the degree of hydrolysis of highland barley starch constantly increases with the extension in enzymolyzing alpha-amylase reaction time.When
When enzymolysis time reaches 3h, degree of hydrolysis reaches maximum value (47.02%).The enzyme digestion reaction of subsequent alpha-amylase tends to balance, after
It renews the long enzyme digestion reaction time, there is no significantly changing for degree of hydrolysis.
Influence of the hydrolysis temperature to α-amylasehydrolysis highland barley starch is as shown in Figure 5.
As shown in Figure 5, enzymolysis temperature has a significant effect to the starch in α-amylasehydrolysis highland barley coarse powder.Work as temperature
When being increased to 65 DEG C, the degree of hydrolysis of highland barley starch reaches maximum value (45.78%), continues to increase with temperature, degree of hydrolysis is bright
Aobvious decline.
2.3.2 Pullulanase enzymatic hydrolysis condition experiment of single factor result
Influence of the Pullulanase enzyme concentration to highland barley Starch Hydrolysis degree is as shown in Figure 6.
It will be appreciated from fig. 6 that in the enzymatic hydrolysis initial stage, degree of hydrolysis increases with the increase of Pullulanase enzyme additive amount.When adding
When dosage reaches 150U/g highland barley coarse powder, Starch Hydrolysis degree reaches maximum value (34.72%).When enzyme additive amount is greater than 150U/g blueness
After highland barley coarse powder, Starch Hydrolysis degree gradually tends towards stability, or even downward trend occurs.
Influence of the Pullulanase enzymolysis time to highland barley Starch Hydrolysis degree is as shown in Figure 7.
As shown in Figure 7, with the extension of Pullulanase enzymolysis time, the degree of hydrolysis of highland barley starch constantly increases.Work as enzymatic hydrolysis
When time reaches 3h, degree of hydrolysis reaches maximum value (34.82%), continues to extend the enzyme digestion reaction time, there is no bright for degree of hydrolysis
Aobvious change.
Influence of the Pullulanase hydrolysis temperature to highland barley Starch Hydrolysis degree is as shown in Figure 8.
As shown in Figure 8, enzymolysis temperature also has a significant effect to the effect of Pullulanase hydrolysis starch.When temperature increases
When to 65 DEG C, the degree of hydrolysis of highland barley starch reaches maximum value (34.37%), continues to increase temperature, degree of hydrolysis is decreased obviously.Cause
This, it is best in the effect of 65 DEG C of Pullulanase enzymatic starch.
2.3.3 carbohydrase enzymatic hydrolysis condition experiment of single factor result
Influence of the carbohydrase enzyme concentration to highland barley Starch Hydrolysis degree is as shown in Figure 9.
As shown in Figure 9, with the increase of glucoamylase enzyme additive amount, the degree of hydrolysis of highland barley starch is continuously increased.Work as carbohydrase
When enzyme additive amount is 200U/g highland barley coarse powder, Starch Hydrolysis degree reaches maximum value (31.84%), when being more than this value, Starch Hydrolysis
There is no significant changes for degree.
Influence of the carbohydrase enzymolysis time to highland barley Starch Hydrolysis degree is as shown in Figure 10.
As shown in Figure 10, with the extension of enzyme digestion reaction time, the degree of hydrolysis of highland barley starch constantly increases.Work as enzymolysis time
When reaching 5h, degree of hydrolysis reaches maximum value (33.78%).Hereafter the enzyme digestion reaction of carbohydrase tends to balance, and continues to extend enzymatic hydrolysis
In the reaction time, there is no apparent change occurs for degree of hydrolysis.
Influence of the carbohydrase hydrolysis temperature to highland barley Starch Hydrolysis degree is as shown in figure 11.
As shown in Figure 11, the content of starch in 35~65 DEG C, carbohydrase enzymatic hydrolysis highland barley coarse powder constantly increases.When temperature liter
When height is to 65 DEG C, the degree of hydrolysis of highland barley starch reaches maximum value (34.58%), as temperature continues to increase, under degree of hydrolysis is obvious
Drop, when temperature reaches 75 DEG C, Starch Hydrolysis degree is only 1.38%, almost it is considered that carbohydrase does not have highland barley starch at this time
Effect.Therefore, best in 65 DEG C of carbohydrase enzymatic starch effects.
The test result of 2.4 complex enzyme zymohydrolysis conditions
2.4.1 the screening single factor experiment result of enzyme ratio
Complex enzyme is shown in Table 2 with the influence for comparing highland barley Starch Hydrolysis degree.
The influence with comparison Starch Hydrolysis degree of 2 complex enzyme of table
As shown in Table 2, when alpha-amylase: Pullulanase: when carbohydrase is 1:2:2, content of reducing sugar reaches in enzymolysis liquid
Maximum (containing 396.30mg reduced sugar in the enzymolysis liquid of every 1g highland barley coarse powder), Starch Hydrolysis degree reaches 56.28%, works as alphalise starch
Enzyme: Pullulanase: when carbohydrase is 3:1:3, enzymolysis liquid content of reducing sugar is minimum (345.83mg reduced sugar), and Starch Hydrolysis degree is
49.12%.
2.4.2 complex enzyme zymohydrolysis condition single factor experiment result
Influence of the complex enzyme enzyme concentration to highland barley Starch Hydrolysis degree is as shown in figure 12.
As shown in Figure 12, Starch Hydrolysis degree increases with the increase of the initial enzyme additive amount of complex enzyme.When additive amount reaches
When 200U/g highland barley coarse powder, Starch Hydrolysis degree reaches maximum value (56.28%), when enzyme additive amount is greater than 200U/g highland barley coarse powder
Afterwards, Starch Hydrolysis degree gradually tends towards stability, almost no longer further hydrolysis.
Influence of the complex enzyme zymohydrolysis temperature to highland barley Starch Hydrolysis degree is as shown in figure 13.
As shown in Figure 13, when temperature is increased to 65 DEG C, the degree of hydrolysis of highland barley starch reaches maximum value (56.65%), with
Temperature continue to increase, degree of hydrolysis is decreased obviously.
Influence of the complex enzyme zymohydrolysis time to highland barley Starch Hydrolysis degree is as shown in figure 14.
As shown in Figure 14, with the extension in complex enzyme zymohydrolysis reaction time, the degree of hydrolysis of highland barley starch constantly increases.Work as enzyme
When the solution time reaches 3h, degree of hydrolysis reaches the larger value (53.66%).The enzyme digestion reaction of subsequent complex enzyme tends to balance, after reneing
The long enzyme digestion reaction time, there is no significantly changing for degree of hydrolysis.
Influence of the complex enzyme solid-liquid ratio to highland barley Starch Hydrolysis degree is as shown in figure 15.
As shown in Figure 15, the degree of hydrolysis of highland barley starch reaches maximum value (47.40%) when solid-liquid ratio is 1:10, in feed liquid
When smaller than ratio and larger, the degree of hydrolysis of highland barley starch is all lower.
2.4.3 complex enzyme process optimization orthogonal experiments
Complex enzyme process optimization orthogonal test the results are shown in Table 3.
3 complex enzyme process optimization orthogonal test of table and range analysis result table
Seen from table 3, the best enzymolysis process conditional combination of highland barley starch is A3B1C3D2, i.e., suitable enzymatic hydrolysis condition is multiple
Synthase formula is alpha-amylase: Pullulanase: carbohydrase=1:2:2, when complex enzyme additive amount is 200U/g highland barley coarse powder, enzymatic hydrolysis
Between 3.5h, hydrolysis temperature be 60 DEG C, solid-liquid ratio 1:13.Four factors are B > D > C > A to the influence degree of enzymatic starch, i.e., warm
Degree > enzyme total amount > enzymolysis time > solid-liquid ratio.
Embodiment 2
1.1 test material
1.1.1 test material and animal
Highland barley (place of production Qinghai Province north)；High-temperatureα-amylase (40000U/mL), beta amylase (700000U/mL), saccharification
Enzyme (100000U/mL) and Pullulanase (1500U/mL) are provided by Jiangsu Ruiyang Biological Technology Co., Ltd..
SPF grades of BALB/c test mices, 120, male, 19 ± 2g of weight is ground by Chinese Academy of Agricultural Sciences Lanzhou animal doctor
Study carefully and is provided.18~23 DEG C of animal feeding room temperature.Experimental animal ad lib, feed and padding are the SPF grades of dedicated feedings of mouse
Material.Drink aqua sterilisa, 121 DEG C of sterilizing 20min of sterilized pot.
1.1.2 the preparation of tested material
Take highland barley several, surface watering wetting, placement makes highland barley grain feel like jelly afterwards for 24 hours, rolls repeatedly three times extremely through noodle press
Highland barley grain rupture shows money or valuables one carries unintentionally, and keeps not broken in flakes, is placed in 50 DEG C of baking oven drying.
0.5L alpha-amylase enzyme solution is measured, 0.8L Pullulanase enzyme solution and 0.4L carbohydrase enzyme solution mix and be settled to 1.5L
In volumetric flask, it is prepared into complex enzyme liquid (400U alpha-amylase/mL, 800U Pullulanase/mL, 800U carbohydrase/mL).
5kg highland barley coarse powder is weighed, 5L complex enzyme liquid and 12L water is added, stirs evenly, is put into 60 DEG C of constant temperature oscillations in batches
Device, 140r/min constant temperature oscillation 3h, mixes highland barley coarse powder with enzymolysis liquid.Mixed liquor is separated by solid-liquid separation after the completion of oscillation,
2800r/min is centrifuged 10min, removes supernatant, a certain amount of water is added into centrifuge tube and is centrifuged again, removes supernatant, solid
Partially desiccated both obtains low sugar highland barley flour.
By low sugar highland barley flour in different proportion (it is 40%, 60% that low sugar highland barley flour, which accounts for feed ratio) and common SPF mouse
Feed mixing；It separately takes highland barley several, does not do desaccharification processing, be processed into after crushing with normal diet mixing, highland barley accounting 60%
Finished feed, is placed in 50 DEG C of baking oven drying, and ultraviolet disinfection 0.5h is kept in dark place after package is tight.
1.2 laboratory apparatus and reagent
1.2.1 major experimental instrument
The single clean work station of SW-CJ-1D type (Purifying Equipment Co., Ltd., Suzhou)；HH-6 type electric-heated thermostatic water bath
(Shanghai is than bright Instrument Ltd.)；3-16KL type table model high speed centrifuge (German SIGMA company)；SM-303 type noodle press
(Shanghai Chuan Li Industrial Co., Ltd.)；GA-3 type blood glucose meter (Sinocare Biosensing Co., Ltd).
1.2.2 major experimental reagent
Alloxan (Hefei Bo Mei biotechnology Co., Ltd)；Ether (the limited public affairs of the wide promise chemistry science and technology in Shanghai
Department)；Physiological saline (upper Industrial Co., Ltd., seascape nation)；Glucose (upper Industrial Co., Ltd., seascape nation).
1.3 experimental methods and research contents
1.3.1 laundering period and preliminary experiment
Test mice carries out the preliminary experiment of 7d and laundering period by a definite date before carrying out test.Preliminary experiment particular content: right
Test mice feeds the feed mixed with low sugar highland barley flour, observes it and has no adverse reaction.
1.3.2 diabetic mouse model is established
After healthy male mice is carried out the 7d laundering period under test conditions, 24 mouse are randomly selected as blank control
Group and blank test sample group, fasting 3-5h measure blood glucose value as this experimental basis blood glucose value.Remaining mouse fasting can't help
Water 12h is injected to test group mouse peritoneal (with physiology with the alloxan solution of 2% newly configured according to every 130mg/kg
It is 2%) mouse ad lib that concentration, which is made, in salt water, is deprived of food but not water after 72h, and blood glucose is surveyed in mouse tail blood sampling.Take blood sugar concentration
10-25mmol/L mouse is as hyperglycemia model success mouse.
1.3.3 dosage is grouped
The successful hyperglycemia mouse of modeling is randomly divided into 4 groups, a model control group and low dose of low sugar highland barley flour are set
Amount group (sample accounts for feed ratio 40%), low sugar highland barley flour high dose group (sample accounts for feed ratio 60%), common highland barley group (sample
Product account for feed ratio 60%) three model experiment groups, optionally healthy adult mouse is grouped by the blood glucose level of fasting 3-5h, if one
A blank control group and a blank test sample group (low sugar highland barley flour accounts for feed ratio 60%).Every group 12.Test component
The given the test agent of different formulations is not given, and model control group and blank control group give normal diet, and blank test sample group is given
Low sugar highland barley flour high dose feed (sample accounts for feed ratio 60%) is given, 30d, the daily activity condition of observation mouse are continuously raised
And between record cast group mouse hair variation, food-intake, the moist degree of padding, every 7d weighs a mouse weight and food
Consumption.
1.3.4 resistance to sugar amount experiment
Hyperglycemia model mouse fasting 3-5h, test group give the tested feed of different formulations and various dose, model pair
Equivalent normal diet is awarded according to group, gives glucose 2.0g/kg through oral examination after 15-20min, and glucose 0,0.5,2h are given in measurement
Blood glucose value area under the curve variation.
1.3.5 index determining
1.3.5.1 the weight of animals detects
Test mice daily behavior situation is observed during test and is recorded, and every 7d weighs each test group mouse weight (g) simultaneously
Record.
1.3.5.2 feed consumption
Every 7d weighs the feed consumption (g) of primary each test group mouse during test.
1.3.5.3 animal blood glucose detects
After weighing, tail portion blood sampling surveys blood glucose value with blood glucose meter, and every 7d surveys each test group mouse blood sugar (mmol/L) and remembers
Record；Culling heart blood method is taken a blood sample after 30d, and part blood sample is used to detect mouse fasting blood sugar, remaining blood sample is centrifuged through 3500r/min
10min obtains serum sample, is placed in Liquid nitrogen storage in -70 DEG C of refrigerators.
1.3.5.4 the processing of Organs of Mice
The mouse of the dead after blood will be taken to be placed in superclean bench to dissect, take out liver, pancreas, kidney, spleen, pancreas,
It is placed in physiological saline, then takes out, be placed in filter paper and absorb water, weigh, be accurate to 1mg, calculate dirty body ratio.
1.3.55 data processing
Test data is for statistical analysis to data using SPSS 21.0 and 2007 software of Excel, and statistical result is used flat
Means standard deviation (3 ± s) indicates that Analysis of variance compares the difference between each group.
2 test results and analysis
Mouse basic condition is shown in Table 4 in 2.1 test mice basic condition experiment periods
Basic condition in 4 experiment mice experimental period of table
After modeling, mouse hair is sparse, lacks gloss and most of mouse has slight hair loss phenomenon, reacts more blunt, pad
Material is serious moist, and tested group of mouse hair of blank control group and blank is than thicker, pure white, and movement response is very fast, and padding is more dry；
During feeding, low sugar highland barley flour is low, high dose group and common highland barley group mouse hair situation, the state of mind and padding humidity
Degree is taken on a new look.
Influence of the 2.2 low sugar highland barley flours to mouse weight
The changes of weight value of each group mouse is shown in Table 5.
5 low sugar highland barley flour of table to mouse weight influence (n=8,)
Unit: g
By table 5 as it can be seen that the empty body weight of mouse is declined after modeling.Model group mouse body weight is below blank control group
Mouse weight (p < 0.05), and common manifestation is " three-many-one-little " symptom, illustrates that model mice models successfully.Model control group is small
Mouse weight is decreased obviously after a week in modeling, though there is increase in the later period, weight is consistently lower than 0 week weight；Low sugar highland barley flour is low
The phase is risen dosage group mouse weight after experiment, is illustrated during test, and low sugar highland barley flour is to blood glucose in diabetic mice
Level has a certain impact, and facilitates the increase of mouse weight；And the weight and model control group of common highland barley flour group mouse are poor
It is different unobvious, illustrate that common highland barley flour group processing is unobvious to diabetic mice ghost image sound.
2.3 each group mouse feed consumptions
Each group mouse feed consumption is shown in Table 6.
6 each group mouse feed consumption of table
Unit: g
By table 6 as it can be seen that the feed consumption of mouse is declined after modeling.Model group mouse feed consumption in 0 week after modeling
Amount is below blank control group mouse feed consumption.Model control group mouse feed consumption is on modeling is obvious after a week
It rises, though the later period is reduced, is consistently higher than the feed consumption of blank control group；Low sugar highland barley flour is low, high dose group mouse is raised
Material consumption persistently rises after a week in modeling, but feed consumption weekly is below model control group, illustrates in experimental period
Interior low sugar highland barley flour has a certain impact to blood glucose in diabetic mice, affects the feed consumption of mouse.2.4 low sugar highland barleys
Influence of the powder to diabetic mice fasting blood-glucose
Influence of the low sugar highland barley flour to mouse fasting blood-glucose is shown in Table 7 and Figure 16.
Influence (n=8, x ± s) of the 7 low sugar highland barley flour of table to diabetic mice fasting blood-glucose
Unit: mmol/L
Note: * indicates p < 0.05；* indicates p < 0.01
By table 7 as it can be seen that model group mouse is after injecting alloxan 3d, with blank control group ratio, mouse blood sugar value is obvious
It increases (p ﹤ 0.01), shows that mouse pancreas is impaired, model successfully.In entire experimental period, blank test sample group mouse blood sugar water
No significant change is put down, shows that low sugar highland barley flour has not significant impact the blood glucose of normal mouse.By Figure 16 and table 7 as it can be seen that low
Sugared highland barley flour is low, high dose group mouse more has compared with 0 week after modeling mouse blood sugar value in 1,3 week mouse blood sugar value of feed
It is reduced, and low sugar highland barley flour is low, high dose group mouse blood sugar value is lower than model control group (p < 0.05)；And common highland barley flour group
1,3 week mouse blood sugar value of feed does not have significant change, no significant difference (p > 0.05) compared with model control group mouse.The above knot
Fruit illustrates that low sugar highland barley flour has certain reduction effect to diabetic mice fasting blood-glucose.
Influence of the 2.5 low sugar highland barley flours to diabetic mice postprandial blood sugar
Influence of the low sugar highland barley flour to mouse postprandial blood sugar is shown in Table 8 and Figure 17.
Influence (n=8, x ± s) of the 8 low sugar highland barley flour of table to diabetic mice postprandial blood sugar
Unit: mmol/L
Note: * indicates p < 0.05；* indicates p < 0.01
By table 8 with Figure 17 as it can be seen that low sugar highland barley flour is low, postprandial plasma glucose level of high dose group mouse 4th week is lower than second week
Postprandial blood sugar (p < 0.05), and low sugar highland barley flour is low, high dose group it is lower than model control group mouse postprandial plasma glucose level (p <
0.05)；And 2,4 weeks mouse blood sugar values of common highland barley flour group feed do not have significant change, without aobvious compared with model control group mouse
It writes difference (p > 0.05), effect is unobvious, these results suggest that low sugar highland barley flour has a certain impact after the meal to diabetic mice.
2.6 sugar tolerance testing results
Sugar tolerance measuring the results are shown in Table 9.
9 sugar tolerance laboratory test results of table
Unit: mmol/L
Note: * indicates p < 0.05；* indicates p < 0.01
By table 9 as it can be seen that under the premise of modeling successfully, given the test agent dosage group compared with the control group, give glucose 0,
0.5,2h Area under the curve of blood glucose reduces (p < 0.05), can determine that given the test agent carbohydrate tolerance test result is positive, low sugar highland barley flour
There is certain reduction effect to blood glucose in diabetic mice.
The dirty body ratio of 2.7 each group mouse
Each group mice organs and body weight determination the results are shown in Table 10.
10 each group mice organs of table and body weight determination result
Unit: g
Note: * indicates p < 0.05；* indicates p < 0.01
Dirty body than be mice organs weight in wet base and mouse weight ratio, diabetes can be reflected to the damage feelings of each organ
Condition.After the test, it wins each group mice organs to weigh weight in wet base and calculate its dirty body ratio (percentage), by table 10 as it can be seen that glycosuria
Disease has different degrees of damage to mouse kidney, liver, spleen, and low sugar highland barley flour is low, high dose group and common highland barley group are equal
There are different degrees of recovery effects.The pancreas of model group mouse/body ratio is below blank control group (p < 0.01), low sugar highland barley flour
Low, high dose group and common highland barley group mice pancreatic/body illustrate low sugar highland barley than (p < 0.01) higher than model control group mouse
Powder and common highland barley flour have different degrees of recovery effects to diabetic mice pancreas, to influence blood glucose in diabetic mice
It is horizontal.
Claims (10)
1. a kind of preparation method of highland barley products, it is characterised in that: it includes following content:
(1) highland barley flour；(2) complex enzyme；(3) water；(4) it digests；(5) take solid content up to highland barley products；
Wherein, the complex enzyme two or more group in alpha-amylase, beta amylase, Pullulanase, carbohydrase
It closes.
2. preparation method according to claim 1, it is characterised in that: the complex enzyme is alpha-amylase: Pullulanase: sugar
The ratio for changing enzyme enzyme activity is 1~3:1~3:1~3；Further, alpha-amylase: Pullulanase: carbohydrase ratio is 1:2:2.
3. preparation method according to claim 1 or 2, it is characterised in that: the additive amount of the complex enzyme is with enzyme-activity unit
It is calculated as 150~250U/g highland barley flour；It is further selected from 200U/g highland barley flour.
4. preparation method according to claim 1, it is characterised in that: hydrolysis temperature is 55~70 DEG C；It is further selected from 60
℃。
5. preparation method according to claim 1, it is characterised in that: enzymolysis time is 2~4h, is further selected from 3.5h.
6. preparation method according to claim 1, it is characterised in that: solid-liquid ratio is 1:7~13, is further selected from 1:13.
7. a kind of highland barley products, it is characterised in that: in the product, the content of starch of highland barley is in 40%w/w or less；It is further selected from
30~40%.
8. highland barley products according to claim 7, it is characterised in that: the product is as described in claim 1~6 any one
Method be prepared.
9. purposes of the highland barley products described in claim 8 in the product that preparation improves diabetes.
10. a kind of food or health care product, it is characterised in that: it is based on the highland barley products of claim 7 or 8；Further
Ground, in the food or health care product, the content of highland barley products is 40% or more.
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