CN114586988A - Extraction process and application of soluble dietary fiber of holboellia latifolia peel with blood sugar reducing function - Google Patents
Extraction process and application of soluble dietary fiber of holboellia latifolia peel with blood sugar reducing function Download PDFInfo
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- CN114586988A CN114586988A CN202210324295.2A CN202210324295A CN114586988A CN 114586988 A CN114586988 A CN 114586988A CN 202210324295 A CN202210324295 A CN 202210324295A CN 114586988 A CN114586988 A CN 114586988A
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- enzymolysis
- peel
- dietary fiber
- soluble dietary
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Abstract
The invention relates to the technical field of soluble dietary fiber processing and preparation, and discloses an extraction process and application of soluble dietary fiber of holboellia latifolia peel with a blood sugar reducing function. The process method is simple to operate, convenient, reasonable, stable and quantifiable to use, and has important application value. The soluble dietary fiber obtained by the method can obviously reduce the activity of alpha-glucosidase and effectively control the rise of postprandial blood sugar through in-vitro blood sugar reduction activity evaluation, and the inhibition effect can cause the glucose absorption obstruction of the intestinal tract so as to generate more ideal blood sugar reduction effect. Has certain effects of reducing blood sugar and blood fat, and preventing colon cancer, cardiovascular diseases and the like. The method shows that the akebia fruit peel soluble dietary fiber has good application prospect in the aspect of developing related hypoglycemic functional foods or medicines.
Description
Technical Field
The invention relates to the technical field of soluble dietary fiber processing and preparation, and particularly relates to an extraction process and application of soluble dietary fiber of holboellia latifolia peels with a blood sugar reducing function.
Background
Akebia trifoliata (Akebia trifoliate kiodz) is a liana of the family MuTonidae, and the fruit is named as August melon, commonly named as wild banana, also named as August fried, cow rolling melon, sheep mouth, cow cashew nut, Akebia quinata, etc. The August melon is distributed in the southern part, Yangtze river basin and northwest region of China and in the southeast Asia and Japan. The August melon and fruit is edible and rich in sugar, multiple vitamins, various amino acids, polyphenol, flavone and the like required by human body; in particular, the content of free essential amino acids is high. The holboellia latifolia has the health-care medicinal effects of resisting aging, maintaining beauty and keeping young, enhancing immunity, resisting tumors, reducing blood pressure, inducing diuresis and the like, and is a typical medicine-food dual-purpose plant. The August melon is generally ripe in 8-9 months every year, has thick peel, is generally purple red, has milky white pulp, and can extract oil from seeds. The specific gravity of the peel is about 45%, and the peel becomes a main byproduct in the daily eating and processing process and is not developed and utilized.
Dietary fiber is a generic term for polysaccharide polymers that are not digested and absorbed by the small intestine of the human body and is known as the seventh major nutrient. Dietary fiber is classified into Soluble Dietary Fiber (SDF) and Insoluble Dietary Fiber (IDF) according to water solubility, and the two have different efficacies, and SDF is superior to IDF in physiological function. At present, the methods for extracting dietary fiber mainly comprise a chemical method, an enzymatic hydrolysis method, a physical method and a fermentation method. In recent years, soluble dietary fibers have received increasing attention, and a great deal of research has shown that the physiological functions of SDF are mainly: can be used for preventing hyperlipidemia, diabetes, and cardiovascular diseases. In addition, SDF can be fermented by beneficial microorganisms in intestinal tract, promotes the growth of intestinal probiotics, and has the effects of scavenging free radicals, resisting inflammation and resisting oxidation. The August melon and fruit peel is rich in dietary fiber, but few researches on the extraction method and physiological activity of the soluble dietary fiber are reported, so that a great amount of August melon and fruit peel is directly discarded to cause certain waste.
Disclosure of Invention
The invention aims to provide an extraction process and application of the soluble dietary fiber of the skin of the holboellia latifolia bunge with the function of reducing blood sugar, so as to make up the blank of the optimization of the extraction process and the research on physiological activity of the soluble dietary fiber of the skin of the holboellia latifolia bunge at present and improve the development and utilization rate of the holboellia latifolia.
In order to achieve the purpose, the invention adopts the following technical scheme: a process for extracting soluble dietary fiber from August melon and fruit peel with blood sugar lowering function comprises subjecting August melon and fruit peel to enzymolysis with papain, alpha-amylase and cellulase, concentrating, precipitating with ethanol, drying, and performing high temperature enzyme deactivation after single enzymolysis.
On the other hand, the technical scheme also provides the application of the soluble dietary fiber of the holboellia latifolia peel in preparing hypoglycemic drugs or hypoglycemic functional foods.
The principle and the advantages of the scheme are as follows: the August melon peels occupy most of the proportion of fruits, and a large amount of peels are discarded and wasted in the process of researching and developing the seeds and the pulp of the August melon. It is guessed by preliminary judgment that the pericarp may contain a large amount of polysaccharides. According to the view point of recycling the product and not wasting natural resources, the scheme initially explores the August melon and fruit peel, finds that the total sugar content is extremely high, and further explores the extraction of the soluble dietary fibers in the August melon and fruit peel. In practical application, the technical scheme establishes a process for extracting the soluble dietary fiber from the skin of the holboellia latifolia fruits, the holboellia latifolia fruits are used as raw materials, the biological enzyme method is used for extracting the soluble dietary fiber, the main principle is that the papain is used for deproteinization, the alpha-amylase is used for starch removal, the cellulose is decomposed by the cellulase, the macromolecular structure is hydrolyzed into a micromolecular state so as to expose more SDFs, the SDFs in the raw materials are extracted by extracting agents such as buffer solution and the like, the solid-liquid separation is carried out, the ethanol solution is used for precipitation, and then the SDFs are collected. The process method is simple to operate, convenient, reasonable, stable and quantifiable to use, and has important application value. In the process of technical research and development, the August melon and fruit peel powder is insoluble in water, has high viscosity, and has great waste in the extraction process of soluble dietary fiber, so that the yield is not ideal. Therefore, the enzymolysis condition of the holboellia latifolia peel is a big research and development difficulty of the scheme, the inventor comprehensively optimizes the types of enzymes, the enzymolysis pH, the enzymolysis temperature, the enzymolysis time and the addition amount of the enzymes, three enzymes are adopted for carrying out enzymolysis treatment in sequence, and in order to avoid interaction and influence among the enzymes, reaction conditions of the three enzymes are respectively provided for enzyme reaction and enzyme inactivation. The reaction conditions of the three enzymes are different, so that a great deal of time is spent in the extraction process, for example: the pH values of the three enzyme reactions are greatly influenced by the environment and are difficult to adjust to ideal values. According to the scheme, after one enzyme reaction is finished, enzyme of the enzymolysis liquid is inactivated in a water bath at 100 ℃ for 10min so as to prevent the enzyme from influencing the subsequent steps or generating cross action with other enzymes. However, in the development period, the inventor finds that the enzymolysis temperature, the enzymolysis time and the addition amount of enzyme have synergistic effect and influence mutually, so that the yield of the dietary fiber can be improved, the quality of the obtained dietary fiber can be influenced to a certain extent, and unexpected technical effects are achieved. After the extraction process is optimized, the August melon and fruit peel dietary fiber extracted by the method can remarkably reduce the activity of alpha-glucosidase and regulate the mode of gastrointestinal hormone after meal to effectively control the rise of the blood sugar after the meal through in-vitro blood sugar reduction activity evaluation, and the inhibition effect can possibly cause the absorption of glucose by intestinal tracts to be blocked, thereby generating a relatively ideal blood sugar reduction effect. The method shows that the akebia fruit peel soluble dietary fiber has good application prospect in the aspect of developing related hypoglycemic functional foods or medicines.
The invention selects the holboellia latifolia peel as the raw material, thereby avoiding the agricultureThe product is wasted, the dietary fiber is rich, and the utilization rate of agricultural product resources is fully developed. The obtained soluble dietary fiber has good functional properties such as good oil holding capacity, water holding capacity, swelling property, and oxidation resistance, and can adsorb cholesterol and NO2-And metal ions. But also can inhibit harmful bacteria in the intestinal tract, promote the proliferation of beneficial bacteria in the intestinal tract, and has certain effects of reducing blood sugar and blood fat, preventing colon cancer, cardiovascular diseases and the like.
Preferably, as an improvement, the holboellia latifolia peel is pretreated before enzymolysis treatment, wherein the pretreatment operation is to wash the holboellia latifolia peel, dry the holboellia latifolia peel for 24-48 hours at 50-70 ℃, crush and screen the holboellia latifolia peel, and soak and extract the holboellia latifolia peel in a phosphate buffer solution.
Among this technical scheme, august melon and fruit skin washs before the enzymolysis treatment, can get rid of its surface impurity, avoids its surface material to influence follow-up enzymolysis process, adopts phosphoric acid buffer solution to soak and can leach the SDF in the august melon and fruit skin to the liquid phase, can avoid causing destruction to dietary fiber and other nutrients in the august melon and fruit skin through low temperature stoving.
Preferably, as an improvement, the feed-liquid ratio of the holboellia latifolia peel powder to the phosphate buffer is 1: 10-30.
According to the technical scheme, in the process of optimizing the enzymolysis conditions of the August melon and fruit peels, the extraction rate of the soluble dietary fibers of the August melon and fruit peels under different material-liquid ratio conditions is detected, the result shows that the material-liquid ratio is 1:10-30, the extraction rate of the soluble dietary fibers can reach more than 6.4%, and researches show that the material-liquid ratio and the extraction rate are in a nonlinear relation, and when the material-liquid ratio is 1:20, the extraction rate of the soluble dietary fibers reaches the highest and is 14.52%.
Preferably, as an improvement, the papain enzymolysis treatment conditions are that the enzymolysis pH is 6-7, the enzymolysis temperature is 60-70 ℃, the enzymolysis time is 30-60min, and the enzyme addition amount is 500-1000U/g.
In the technical scheme, in the process of optimizing the enzymolysis conditions of the holboellia latifolia peels, the processing conditions of the papain are optimized, the enzymolysis effect is good under the conditions, and protein impurities in the holboellia latifolia peels can be effectively removed.
Preferably, as an improvement, the enzymolysis conditions of the alpha-amylase are that the enzymolysis pH is 6.5-7, the enzymolysis temperature is 85-90 ℃, the enzymolysis time is 30-60min, and the enzyme addition amount is 20-40U/g.
In the technical scheme, in the process of optimizing the enzymolysis conditions of the holboellia latifolia peels, the alpha-amylase enzymolysis treatment conditions are optimized, the enzymolysis effect is good under the conditions, and starch impurities in the holboellia latifolia peels can be effectively removed.
Preferably, as an improvement, the enzymolysis conditions of the cellulase are enzymolysis pH 4-8, enzymolysis temperature 40-60 ℃, enzymolysis time 40-120min and enzyme addition amount 80-720U/g.
In the technical scheme, in the process of optimizing the enzymolysis conditions of the August melon and fruit peels, the enzymolysis treatment conditions of the cellulase are optimized, and the result shows that the enzymolysis pH, the enzymolysis temperature, the enzymolysis time and the enzyme addition amount have great influence on the enzyme treatment effect, and in the condition range, the extraction rate of the soluble dietary fibers can reach about 10 percent, so that the extraction requirement of the soluble dietary fibers can be met.
Preferably, as an improvement, the enzymolysis treatment condition of the cellulase is that the ratio of the feed to the liquid is 1:21, the enzymolysis pH is 5, the enzymolysis temperature is 48 ℃, the enzymolysis time is 95min, and the enzyme addition amount is 500U/g.
In the technical scheme, single-factor tests and response surface researches show that the enzymolysis pH, the enzymolysis temperature, the enzymolysis time and the enzyme addition amount have nonlinear influence on the enzyme treatment effect, the enzymolysis pH, the enzymolysis temperature, the enzymolysis time and the enzyme addition amount mutually influence each other, when the feed liquid ratio is 1:21, the enzymolysis pH is 5, the enzymolysis temperature is 48 ℃, the enzymolysis time is 95min, and the enzyme addition amount is 500U/g, the optimal extraction condition is realized, the yield of the soluble dietary fibers can reach 15.47%, and the extraction process is proved to be accurate and reliable.
Preferably, as an improvement, the concentration process is to carry out rotary evaporation concentration after the august melon and fruit peel enzymolysis liquid is subjected to centrifugal filtration, extract the mixture by using hot water at 70 ℃, repeat the centrifugation twice, collect and combine supernate; the rotary evaporation temperature is 55-65 ℃, and the volume ratio of the solution before and after rotary evaporation is 4:1-5: 1.
In the technical scheme, the enzymatic hydrolysate after enzymolysis is concentrated and purified after being washed and purified, and is convenient for subsequent drying and powdering treatment. And the concentration mode is rotary evaporation, and the excessive loss of nutrients in the enzymolysis liquid can be avoided by controlling the temperature of the rotary evaporation. The soluble dietary fiber in the filter residue can be dissolved in water by repeatedly centrifuging twice after being dissolved by hot water, so that the loss caused by filtration is greatly reduced.
Preferably, as an improvement, the alcohol precipitation process is carried out under the condition that the concentration of the ethanol is 95%, the addition amount of the ethanol is 4 times of the volume of the solution after rotary evaporation, the alcohol precipitation temperature is 4 ℃, the alcohol precipitation time is 12-36 hours, the precipitate is filtered and collected after alcohol precipitation, and then the precipitate is freeze-dried to obtain the soluble dietary fiber of the holy fruit peel.
In the technical scheme, the purpose of alcohol precipitation is mainly to further purify and remove impurities, the conditions are proper conditions verified by practice, and after alcohol precipitation, through freeze drying treatment, the drying requirement is met, meanwhile, nutrient substances in the precipitate can be kept as far as possible, and high-temperature sensitive components are prevented from being damaged.
Drawings
FIG. 1 is a process flow chart of the invention for extracting the August melon peel SDF.
Fig. 2 is a graph showing the effect of feed liquid ratio on the extraction of soluble dietary fiber from the skin of the holboellia latifolia fruits.
Fig. 3 is a graph showing the effect of enzymolysis temperature on the extraction of soluble dietary fiber from the skin of the holboellia latifolia fruits.
FIG. 4 is a graph showing the effect of enzyme addition on the extraction of soluble dietary fiber from the skin of August melon.
FIG. 5 is a graph showing the effect of enzymolysis time on the extraction of soluble dietary fiber from the skin of August melon.
FIG. 6 is a graph showing the effect of enzymatic hydrolysis pH on the extraction of soluble dietary fiber from Elaeagnus augustata peel.
FIG. 7 shows the enzyme digestion temperature (X)2) Material to liquid ratio (X)1) Response surface plot and contour plot of the interaction effect on SDF extraction rate.
FIG. 8 shows the enzyme addition amount (X)3) Andratio of material to liquid (X)1) Response surface plot and contour plot of the effect of interaction on SDF extraction rate.
FIG. 9 shows the enzymolysis time (X)4) Material to liquid ratio (X)1) Response surface plot and contour plot of the effect of interaction on SDF extraction rate.
FIG. 10 shows the amount of enzyme added (X)3) And the temperature of enzymolysis (X)2) Response surface plot and contour plot of the effect of interaction on SDF extraction rate.
FIG. 11 shows the enzymolysis time (X)4) And the temperature of enzymolysis (X)2) Response surface plot and contour plot of the effect of interaction on SDF extraction rate.
FIG. 12 shows the enzymolysis time (X)4) And enzyme addition amount (X)3) Response surface plot and contour plot of the effect of interaction on SDF extraction rate.
FIG. 13 is a graph showing the results of comparison of the inhibition rates of Alcalifornia augustata SDF and the positive drug acarbose in the activity of alpha-glucosidase.
Detailed Description
The following is a detailed description of the embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents and the like used are commercially available.
Example 1
As shown in figure 1, the extraction process of the soluble dietary fiber of the holboellia latifolia peel with the blood sugar reducing function comprises the following steps:
step one, crushing and extracting: the August melon and fruit peel is the peel of fresh mature August melon, after washing and naturally drying the moisture, oven dry 24 hours under the condition of 50 duC, pulverize, get August melon and fruit peel powder after 60 mesh; mixing the August melon and fruit peel powder with a phosphate buffer solution for extraction, wherein the feed-liquid ratio of the August melon and fruit peel powder to the phosphate buffer solution is 1:20 (g/mL).
Step two, papain enzymolysis: adjusting pH of the buffer solution extract of Bayue melon peel to 7, adding 600U/g papain, heating in a water bath at 70 deg.C for 30min, heating to 100 deg.C, inactivating enzyme at 100 deg.C for 10min, and naturally cooling at room temperature.
Step three, carrying out enzymolysis by alpha-amylase: adjusting the pH of the enzymatic hydrolysate obtained in the step two to 6.5, adding 30U/g alpha-amylase, putting the enzymatic hydrolysate into a water bath kettle with the temperature of 85 ℃ for heating for 60min, heating the water bath kettle to 100 ℃, keeping the temperature of 100 ℃ for 10min, inactivating the enzyme, and then naturally cooling at room temperature.
Step four, enzymolysis of cellulase: adjusting the pH value of the enzymolysis liquid obtained in the third step to 5, adding 400U/g cellulase, putting the enzymolysis liquid into a water bath kettle with the temperature of 50 ℃ for heating for 60min, heating the water bath kettle to 100 ℃, keeping the temperature of 100 ℃ for 10min for inactivating enzyme, and then naturally cooling at room temperature.
Step five, filtering and concentrating: centrifuging the holboellia latifolia peel enzymolysis liquid in the step four for 10min at 4500r/min, extracting with hot water at 70 ℃, repeatedly centrifuging twice, collecting and combining supernate, setting the temperature of a rotary evaporator to be 60 ℃, and concentrating, wherein the volume ratio of the solution before and after concentration is 5: 1.
step six, alcohol precipitation: adding 95% ethanol into the concentrated solution obtained in the fifth step, wherein the volume ratio of the concentrated solution to the ethanol is 1: and 4, standing and precipitating for 24 hours at the temperature of 4 ℃. And then the vacuum pump is used for pumping filtration, the residue is washed twice by 78 percent ethanol and twice by 95 percent ethanol in the process of filtering the ethanol precipitation solution, and the precipitate is collected.
Step seven, freeze drying: freeze-drying the precipitate obtained in the sixth step in a vacuum freeze dryer at the freeze-drying temperature of-60 ℃ to-80 ℃ for 24 hours until the precipitate is completely dried into powder; and calculating the yield of the soluble dietary fiber of the holboellia latifolia peel.
The first experimental example: influence of feed liquid ratio on extraction of soluble dietary fiber from holboellia latifolia peels
1. Experimental methods
Taking 2g of the holboellia latifolia peel, respectively setting the material-liquid ratios of 1:10, 1:15, 1:20, 1:25 and 1:30(g/mL) under the same conditions as in example 1, respectively exploring the change of the soluble dietary fiber yield of the holboellia latifolia peel in different material-liquid ratios, and carrying out three times of repeated tests on each group.
2. Results of the experiment
As shown in the figure 2, the feed liquid ratio has a significant effect on the SDF extraction rate of the Bayue melon peel (P <0.05), the SDF yield is 6.44, 10.52, 14.52, 13.00 and 11.97%, and the extraction amount of the soluble dietary fiber of the Bayue melon peel is significantly higher than that of the soluble dietary fiber when the feed liquid ratio is 1:20(g/mL) than that of the Bayue melon peel when the feed liquid ratio is 1:10, 1:15, 1:25 and 1:30 (g/mL). Therefore, in the extraction condition, 1:20(g/mL) is the optimal feed-liquid ratio for extracting the soluble dietary fiber from the Elaeagnus augustata peel.
Experiment example two: influence of enzymolysis temperature of cellulase on extraction of soluble dietary fiber of holboellia latifolia peels
1. Experimental methods
Taking 2g of the holboellia latifolia peel, wherein the enzymolysis temperatures are respectively 40, 45, 50, 55 and 60 ℃, the other conditions are fixed under the same conditions as in example 1, the change conditions of the different enzymolysis temperatures on the yield of the soluble dietary fibers of the holboellia latifolia peel are respectively explored, and each group is subjected to three repeated tests.
2. Results of the experiment
As shown in the figure 3, the enzymolysis temperature has a significant influence on the SDF extraction rate of the August melon and fruit peel (P is less than 0.05), and the SDF yield is 10.11, 13.24, 14.99, 12.17 and 9.47%, respectively, and when the enzymolysis temperature is 50 ℃, the extraction amount of the soluble dietary fiber of the August melon and fruit peel is significantly higher than that of the soluble dietary fiber at the enzymolysis temperature of 40, 45, 55 and 60 ℃. Therefore, in the extraction condition, the 50 ℃ is the optimal enzymolysis temperature for extracting the soluble dietary fiber from the holboellia latifolia peel.
Experiment example three: influence of enzyme addition amount of cellulase on extraction of soluble dietary fiber of holboellia latifolia peels
1. Experimental methods
Taking 2g of the holboellia latifolia peel, wherein the enzyme addition amounts are respectively 80, 240, 400, 560 and 720(U/g), the other conditions are fixed under the same condition as the example 1, the change conditions of the different enzyme addition amounts on the yield of the soluble dietary fibers of the holboellia latifolia peel are respectively researched, and each group is subjected to three times of repeated tests.
2. Results of the experiment
As shown in FIG. 4, the SDF extraction rate of the August melon and fruit peel was significantly affected by the enzyme addition (P <0.05), and the SDF yields were 9.77, 12.62, 15.21, 14.89, and 13.57%, respectively, and the soluble dietary fiber extraction amount of the August melon and fruit peel was significantly higher than that of the August melon and fruit peel when the enzyme addition was 400 (U/g). Therefore, in the extraction conditions, 400(U/g) is the optimal enzyme addition amount for extracting the soluble dietary fiber from the holly bark.
Experimental example four: influence of enzymolysis time of cellulase on extraction of soluble dietary fiber of holboellia latifolia peels
1. The experimental method comprises the following steps:
taking 2g of the holboellia latifolia peel, carrying out enzymolysis for 40 min, 60min, 80 min, 100min and 120min respectively, fixing the enzymolysis conditions under the same conditions as in example 1, respectively exploring the change conditions of the different enzymolysis times on the yield of the soluble dietary fibers of the holboellia latifolia peel, and carrying out three repeated tests on each group.
2. Results of the experiment
The results are shown in fig. 5, the enzymolysis time has a significant influence on the extraction rate of the august peel SDF (P <0.05), and as a result, the SDF yields are 11.10, 12.50, 12.94, 15.51, and 14.17%, respectively, and when the enzymolysis time is 100min, the extraction amount of the soluble dietary fiber of the august peel is significantly higher than that of the soluble dietary fiber when the enzymolysis time is 40, 60, 80, and 120 min. Therefore, in the extraction condition, 100min is the optimal enzymolysis time for extracting the soluble dietary fiber from the holboellia latifolia peels.
Experimental example five: influence of enzymolysis pH of cellulase on extraction of soluble dietary fiber of holboellia latifolia peels
1. Experimental methods
Taking 2g of the holboellia latifolia peel, wherein the enzymolysis pH values are 4, 5, 6, 7 and 8 respectively, the other conditions are fixed under the same conditions as in example 1, the change conditions of the different enzymolysis pH values on the yield of the soluble dietary fibers of the holboellia latifolia peel are researched, and each group is subjected to three repeated tests.
2. Results of the experiment
As shown in fig. 6, the enzymatic hydrolysis pH has a significant effect (P <0.05) on the extraction rate of the august peel SDF, and as a result, the yields of SDF are 13.22, 15.56, 13.07, 11.64, and 9.24%, respectively, and when the enzymatic hydrolysis pH is 5, the extraction amount of the soluble dietary fiber of the august peel is significantly higher than that of the soluble dietary fiber when the enzymatic hydrolysis pH is 4, 6, 7, and 8. Therefore, in this extraction condition, pH 5 is the optimum pH for extraction of the soluble dietary fiber from the skin of the holboellia latifolia fruit. Considering the factors of difficult pH adjustment to proper pH due to external conditions, simple operation and efficiency improvement, the pH value is not optimized as a response surface factor any more, so that the proper enzymolysis pH value is selected to be 5.
Experimental example six: response surface experiment optimization method for extracting soluble dietary fiber from holboellia latifolia peels
1. Experimental methods
On the basis of a single-factor test, the optimal feed-liquid ratio, enzymolysis temperature, enzyme addition amount and enzymolysis time of cellulase on the SDF extraction of the holboellia latifolia peels are further optimized by using response surface experimental analysis. Experiments were designed according to the Box-Behnken principle: selecting 4 single factors of the feed-liquid ratio, the enzymolysis temperature, the enzyme addition amount and the enzymolysis time as independent variables, and taking the SDF extraction rate as a response value to carry out a four-factor three-level test, wherein the design of each factor and level is shown in table 1. And (3) performing multifactor multilevel response surface test analysis by using Design-Expert data analysis software, and calculating to obtain the optimal extraction process condition of the August melon and fruit peel SDF.
The yield of the August melon and fruit peel SDF/% ═ m1/m×100
In the formula: m is1For extracting SDF quality; and m is the mass of the sample.
TABLE 1Box-Behnken test design factors and levels
2. Results of the experiment
According to the single-factor test result, a response surface method is applied for optimization, X1 (material-liquid ratio), X2 (enzymolysis temperature), X3 (enzyme addition amount) and X4 (enzymolysis time) are used as independent variables, the SDF yield is used as a response value Y, the extraction process conditions are further optimized through Box-Behnken response surface design, and the test design and the result are shown in Table 2.
TABLE 2 response surface test design and results
Performing second multiple regression fitting analysis by using Box-Behnken to obtain SDF extraction rate (Y) and each extraction condition X1、X2、X3、X4Second order polynomial model in between. By utilizing the quadratic regression equation, a better mathematical model can be provided for optimizing the extraction conditions of the SDF, and the change of the SDF extraction rate is predicted. The quadratic polynomial regression equation is as follows:
Y＝15.16+0.77X1-0.81X2+1.29X3+0.11X4+0.31X1X2+0.57X1X3+0.73X1X4+0.29X2X3-0.27X2X4-0.74X3X4-2.10X1 2-1.05X2 2-1.40X3 2-1.26X4 2
in the formula: y is SDF extraction rate/%; x1、X2、X3、X4The coding values of 4 variables of feed liquid ratio, enzymolysis temperature, enzyme addition amount and enzymolysis time are respectively.
Analysis of variance was performed on the quadratic multiple regression equation, and the results are shown in table 3.
TABLE 3 analysis of the variance of the model regression equation and the results
The significance of the regression equation coefficient and the model first-order feed-liquid ratio X can be obtained by analyzing in Table 31Temperature of enzymolysis X2And the amount X of enzyme added3(P<0.01) has very significant influence on the yield of SDF, and the enzymolysis time X4(P>0.05) has no significant influence on the yield of SDF; model quadratic term X1 2、X2 2、X3 2And X4 2(P<0.01) has extremely obvious influence on the yield of the SDF; interaction item X1X4、X3X4(P<0.01) has a very significant influence on the SDF yield, X1X3(P<0.05) had a significant effect on SDF yield, X1X2、X2X3And X2X4(P>0.05) had no significant effect on SDF yield.
As can be seen from table 3, the regression model is highly significant (F is 38.80, P is less than 0.0001), and the mismatching term difference is insignificant (P is 0.1891 is greater than 0.05), indicating that the regression equation fits well to the experiment, indicating that the residuals are both caused by random errors and have little interference to the experiment. The results show that the model regression coefficient R2To 0.9749, the coefficient of determination R is corrected2Adj is 0.9498, indicating that 94.98% change in response value can be interpreted by the model, which can be used for theoretical prediction, and the coefficients of the quadratic terms in the model are all negative numbers, indicating that the paraboloid of the model is open downwards with a maximum point. Therefore, the model has a certain reference value for analyzing the SDF yield of the holboellia latifolia peels, and can predict the SDF yield. According to the F value, the influence of 4 factors on the SDF yield of the holboellia latifolia peel is sequentially the enzyme addition amount X3>Enzymolysis temperature X2>Ratio of material to liquid X1>Time of enzymolysis X4。
Quadratic multiple regression fitting was performed by the data in Table 3, enzyme addition X3Temperature of enzymolysis X2Material to liquid ratio X1And enzymesSolution time X4The four factors are analyzed in pairwise interaction, and a response surface diagram and a contour diagram are respectively made, and the results are shown in fig. 7-12.
The response surface is a stereo graph formed by the response values to the test factors, and the highest point of the response surface formed by the interaction of the two factors in the graph is also the central point of the smallest ellipse in the contour line, so that the extreme value of the response value (Y) exists in the selected range. Whether the influence of a certain factor on the response value (Y) is obvious or not can be observed through the change of the color of the curved surface from blue to red, and the larger the influence of the factor on the test result is, the steeper the surface curved surface is. In addition, the shape of the contour line reflects the significance degree of interaction between every two factors, the circular contour line shows that the interaction between the two factors is not significant, and the elliptical contour line shows that the interaction between the two factors is significant.
As can be seen from FIGS. 7 to 12, the amount X of enzyme added3The response surface curve is steeper, the contour lines are denser, the influence is most obvious, and X is4The response surface curve of the enzymolysis time is gentle, the contour lines are sparse, and no obvious influence is caused. Among the four factors studied, the influence on the yield of the august peel SDF is as follows in sequence from big to small: enzyme addition amount X3>Enzymolysis temperature X2>Ratio of material to liquid X1>Time of enzymolysis X4. Observing the contour shape, the ratio of the feed to the liquid and the amount of enzyme added (X)1X3) Material-to-liquid ratio and enzymolysis time (X)1X4) And enzyme addition amount and enzymolysis time (X)3X4) The contour lines of (A) are elliptical, which shows that the interaction has significant influence on the SDF yield, and the ratio of the feed to the liquid and the enzymolysis temperature (X)1X2) Temperature of enzymolysis and enzyme addition amount (X)2X3) And the temperature and time of the enzymatic hydrolysis (X)2X4) The contour lines of (a) are circular, indicating that the interaction has no significant effect on the SDF yield. This is consistent with the results of the ANOVA analysis.
In order to further determine the optimal point of each factor, the optimal extraction process conditions of the August melon peel SDF are obtained after the obtained multivariate quadratic regression equation is calculated and analyzed by Design-Expert 10.0 software: the ratio of the feed to the liquid is 1:21.32(g/mL), the enzymolysis temperature is 48.34 ℃, the enzyme addition amount is 500.60(U/g), and the enzymolysis time is 95.78 min. The maximum value of Y in the regression equation, the theoretical value under the optimal extraction conditions, was 15.60%.
Example seven: response surface verification experiment
In order to verify the predictability of the model and the convenience and feasibility of actual operation, the extraction conditions are adjusted to be a feed-liquid ratio of 1:21(g/mL), an enzymolysis temperature of 48 ℃, an enzyme addition amount of 500(U/g) and an enzymolysis time of 95min, and a verification experiment is carried out on the result of response surface optimization under the conditions. The optimized extraction conditions were subjected to 3 repeatability validation experiments to obtain an average value of 15.47% of SDF yield actually measured. The difference between the SDF and the theoretical value is 0.13%, the SDF is closer to the theoretical value, the fitting degree is better, and the model is proved to optimize the SDF extraction process of the holboellia latifolia peel, so that the method is accurate and reliable and has better guiding significance. Therefore, the optimal extraction process conditions of the August melon and fruit peel SDF are that the ratio of material to liquid is 1:21(g/mL), the enzymolysis temperature is 48 ℃, the enzyme addition amount is 500(U/g) and the enzymolysis time is 95 min. The August melon and fruit peel SDF obtained under the condition is frozen and dried to obtain the SDF with the yield of 15.47 percent.
Experimental example eight: determination of physical and chemical properties of August melon and fruit peel SDF (soluble dietary fiber)
First, experiment method
1. Measurement of Water holding Capacity
Accurately weighing 0.100g of the August melon and fruit peel SDF sample, placing the sample in a 20mL centrifuge tube, adding 10mL of distilled water, uniformly stirring, placing the sample at normal temperature for 24h, centrifuging at 5000r/min for 15min, pouring out supernatant, draining the precipitated water, weighing, and calculating the water holding capacity.
Water holding capacity (g/g) — (sample wet weight-sample dry weight)/sample dry weight
2. Measurement of swelling Property
Weighing 0.100g of the Sphaete squash peel SDF sample, putting the sample into a test tube with scales, observing and recording the volume, adding 10mL of distilled water into the test tube, standing for 24h at normal temperature, reading the free expansion volume of the sample in the test tube, and calculating the expansion volume (mL/g) of each gram of fiber.
Swelling capacity (mL/g) — (after swelling SDF volume-sample volume)/dry weight of sample
3. Measurement of oil holding force
Weighing 0.100g of the Sphaeranthus Bayue peel SDF sample, placing the sample in a centrifuge tube, soaking the sample in edible soybean oil for 1h, stirring for 1 time every 5min, centrifuging for 15min at 5000r/min, removing redundant grease, weighing, and calculating the weight of the soybean oil carried by each gram of sample to be the oil holding capacity.
Oil holding capacity (g/g) — (sample wet weight-sample dry weight)/sample dry weight
4. Cholesterol adsorption assay
Two 1.00g SDF samples of the august peel were weighed into two 100mL conical flasks, respectively, and the samples were soaked with 50g egg yolk diluent (9 times of distilled water thoroughly whipping fresh egg yolk emulsion) and the solution was thoroughly stirred. Adjusting pH of the solution to 2.0 and 7.0 respectively to simulate gastric pH and intestinal pH, shaking in shaking table at 37 deg.C for 2 hr, centrifuging at 4000r/min for 20min, sucking 0.04mL supernatant, and measuring cholesterol content by o-phthalaldehyde method at 553 nm. Preparing a standard curve by using cholesterol as a standard substance, wherein y is 6.47x-0.0027, and R is2＝0.9998。
Second, experimental results
The results of the measurement of the physical and chemical properties of the august peel SDF are shown in Table 4, and the prepared august peel SDF has the water holding capacity of 8.31 +/-0.05 g/g, the swelling capacity of 5.53 +/-0.21 mL/g and the oil holding capacity of 6.42 +/-0.02 mg/g. Water retention and swelling are two important metrics of SDF. Because SDF absorbs toxic substances after water expansion, the peristalsis of intestines and stomach is enhanced, and the physiological function of defecation is promoted. The prepared august peel SDF has good water holding capacity and swelling property, and shows that the august peel SDF has certain hydrophilic application value.
TABLE 4 Spanish melon peel SDF physicochemical Properties
The cholesterol adsorption amount of the August melon and fruit peel SDF is 5.28 +/-0.07 mg/g under the condition of simulating a gastric juice environment (pH2.0) of a human body, and the cholesterol adsorption amount of the August melon and fruit peel SDF is 9.75 +/-0.11 mg/g under the condition of simulating a small intestine environment (pH7.0) of the human body. These two data indicate that the octopus peel SDF has a higher capacity for cholesterol adsorption under neutral conditions (mimicking the pH of the small intestine) than under acidic conditions (mimicking the pH of the stomach). Therefore, the prepared august peel SDF can adsorb cholesterol, and can be used for preparing functional food beneficial to weight reduction or used as a beverage additive for supplementing dietary fibers to human bodies.
Example nine: determination of SDF (glucose-lowering activity) of holboellia latifolia peel
The alpha-glucosidase can hydrolyze 4-nitrophenyl-D-glucopyranoside (PNPG) to generate p-nitrophenol (pNP), PNP has a maximum absorption value at 405nm under alkaline conditions, and the concentration of the pNP in a reaction solution is measured by an enzyme-labeled analyzer, so that the activity of a sample for inhibiting the alpha-glucosidase can be detected.
1. Experimental methods
Alpha-glucosidase activity assay: alpha-glucosidase solution (3.92U/mL) and p-nitrophenyl-alpha-D-glucopyranoside solution (PNPG: 6mM) were prepared by 100mM phosphate buffer (pH 6.9). Compounds were dissolved in 3% DMSO to prepare the various concentrations required, and each concentration of test compound was mixed with 10 μ L of α -glucosidase and reacted at 37 ℃ for 10 minutes, followed by addition of 200 μ L of LPNPG and reaction for another 30 minutes. 1mLNa was added2CO3And further diluted with 4mL of distilled water. Absorbance was measured at 405 nm. A solution without sample (replaced with 3% DMSO) was used as a control, and acarbose was used as a positive control. The percent inhibition of alpha-glucosidase was calculated according to the following formula:
alpha-glucosidase inhibition (%) - [1- (A)Sample (I)-ABackground/ABlank space-ABackground)]×100
In the formula, ABlank spaceAbsorbance of control solution without sample (substituted with 3% DMSO), ABackgroundAbsorbance of a background solution (replaced with NaCl solution) containing no alpha-glucosidase, ASample (I)Represents the absorbance of the test sample.
2. Results of the experiment
The results are shown in fig. 13 and indicate that: the invention provides the holboellia latifolia peel SDF which has obvious activity of inhibiting alpha-glucosidase. Compared with positive acarbose, when the concentration of the sample is more than 4mg/mL, the capacity of the Brucea triphylla peel SDF for inhibiting the alpha-glucosidase is obviously higher than the capacity of the acarbose for inhibiting the alpha-glucosidase. When the sample concentration reached the maximum, the august peel SDF had an alpha-glucosidase inhibition rate 1.27 times that of acarbose.
The alpha-glucosidase inhibitor is a medicament with a better effect suitable for the diabetics at present, the natural alpha-glucosidase inhibitor mainly comprises polysaccharide, glycoside, flavonoid, alkaloid, phenolic acid, terpene and the like, a large amount of flavonoid compounds are glucose-dependent enzyme inhibitors, and the inhibition effect can cause the glucose absorption of intestinal tracts to be blocked, reduce the blood sugar level, slow down the development of diabetes and alleviate the diabetic complications.
From the above results, it can be inferred that the august peel SDF contains α -glucosidase inhibitors such as flavonoids, but since the experiment employs an in vitro screening model (enzyme-inhibitor model) in which the α -glucosidase inhibitory activity of the august peel SDF is measured using PNPG as a substrate, the model cannot directly evaluate the in vivo pharmacological effects of the screened hypoglycemic substance. Therefore, whether the august peel SDF has the effect of reducing blood sugar needs to be further verified by animal experiments.
Experimental example ten: influence of different dietary fiber enzymolysis conditions on physicochemical properties of soluble dietary fibers of holboellia latifolia
The experiment is designed for researching the influence of different cellulase extraction conditions on the physicochemical properties of the soluble dietary fibers of the holboellia latifolia.
1. Experimental methods
Taking 2g of holboellia latifolia peel, adjusting the material-liquid ratio, the enzymolysis temperature, the enzyme addition amount and the influence of the enzymolysis time on the water retention property, the swelling property, the oil retention capacity and the cholesterol adsorption of the soluble dietary fibers of the holboellia latifolia, wherein the conditions are the same as in example 1 except for the adjustment of the indexes, the determination method of each index is the same as above, and the specific experimental design and results are shown in the following table.
TABLE 5 influence of different dietary fiber enzymolysis conditions on physicochemical properties of the soluble dietary fiber of Bacopa monnieri (Benth.) Kuntze
As can be seen from the data in Table 5, in the technical scheme, the enzymolysis condition of the cellulase can improve the yield of the dietary fiber, and meanwhile, the cellulase also has an influence on the physicochemical properties of the obtained dietary fiber. By optimizing the feed-liquid ratio, the enzymolysis temperature, the enzyme addition amount and the enzymolysis time during the enzymolysis of the cellulase, the water holding capacity, the swelling property and the oil holding capacity of the obtained dietary fiber can be improved, even the biological activity of the dietary fiber is influenced, and the cholesterol adsorption effect and the alpha-glucosidase inhibition rate of the dietary fiber can be improved.
The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (10)
1. An extraction process of a soluble dietary fiber of holboellia latifolia peel with a blood sugar reducing function is characterized by comprising the following steps: the method comprises the steps of taking holboellia latifolia peels as raw materials, sequentially carrying out enzymolysis treatment on the holboellia latifolia peels through papain, alpha-amylase and cellulase, concentrating, carrying out alcohol precipitation and drying to obtain the holboellia latifolia peel extract, and carrying out high-temperature enzyme deactivation treatment after single enzymolysis.
2. The extraction process of the soluble dietary fiber of the holboellia latifolia peel with the blood sugar reducing function according to claim 1, which is characterized in that: the holboellia latifolia peel is pretreated before enzymolysis, and the pretreatment operation comprises cleaning holboellia latifolia peel, drying at 50-70 deg.C for 24-48h, pulverizing, sieving, and soaking in phosphate buffer solution for extraction.
3. The extraction process of the soluble dietary fiber of the holboellia latifolia peel with the blood sugar reducing function according to claim 2, which is characterized in that: the material-liquid ratio of the August melon and fruit peel powder to the phosphate buffer solution is 1: 10-30.
4. The extraction process of the soluble dietary fiber of the holboellia latifolia peel with the blood sugar reducing function according to claim 3, wherein the extraction process comprises the following steps: the enzymolysis conditions of the papain are that the enzymolysis pH is 6-7, the enzymolysis temperature is 60-70 ℃, the enzymolysis time is 30-60min, and the enzyme addition amount is 500-.
5. The extraction process of the soluble dietary fiber of the holboellia latifolia peel with the blood sugar reducing function according to claim 4, wherein the extraction process comprises the following steps: the enzymolysis conditions of the alpha-amylase are that the enzymolysis pH is 6.5-7, the enzymolysis temperature is 85-90 ℃, the enzymolysis time is 30-60min, and the enzyme addition amount is 20-40U/g.
6. The extraction process of the soluble dietary fiber of the holboellia latifolia peel with the blood sugar reducing function according to claim 5, wherein the extraction process comprises the following steps: the enzymolysis conditions of the cellulase are that the enzymolysis pH is 4-8, the enzymolysis temperature is 40-60 ℃, the enzymolysis time is 40-120min, and the enzyme addition amount is 80-720U/g.
7. The extraction process of the soluble dietary fiber of the holboellia latifolia peel with the blood sugar reducing function according to claim 6, wherein the extraction process comprises the following steps: the enzymolysis conditions of the cellulase are that the ratio of feed to liquid is 1:21, the enzymolysis pH is 5, the enzymolysis temperature is 48 ℃, the enzymolysis time is 95min, and the enzyme addition amount is 500U/g.
8. The extraction process of the soluble dietary fiber of the holboellia latifolia peel with the blood sugar reducing function according to claim 7, wherein the extraction process comprises the following steps: centrifuging and filtering the holboellia latifolia peel enzymolysis liquid in the concentration process, performing rotary evaporation concentration, extracting with hot water of 70 ℃, repeatedly centrifuging twice, and collecting and combining supernate; the rotary evaporation temperature is 55-65 ℃, and the volume ratio of the solution before and after rotary evaporation is 4:1-5: 1.
9. The extraction process of the soluble dietary fiber of the holboellia latifolia peel with the blood sugar reducing function according to claim 8, wherein the extraction process comprises the following steps: the alcohol precipitation process conditions are that the concentration of ethanol is 95%, the adding amount of the ethanol is 4 times of the volume of the solution after rotary evaporation, the alcohol precipitation temperature is 4 ℃, the alcohol precipitation time is 12-36h, the precipitate is filtered and collected after alcohol precipitation, and then the precipitate is frozen and dried to obtain the holly fruit peel soluble dietary fiber.
10. An application of August melon and fruit peel soluble dietary fiber in preparing hypoglycemic drugs or hypoglycemic functional foods.
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