CN118085125A - Method for extracting pumpkin pectin with antioxidant activity for seeds - Google Patents
Method for extracting pumpkin pectin with antioxidant activity for seeds Download PDFInfo
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- CN118085125A CN118085125A CN202410151026.XA CN202410151026A CN118085125A CN 118085125 A CN118085125 A CN 118085125A CN 202410151026 A CN202410151026 A CN 202410151026A CN 118085125 A CN118085125 A CN 118085125A
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- pectin
- seeds
- pumpkin
- ultrasonic
- extraction
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Landscapes
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Abstract
The invention relates to the field of biotechnology, in particular to a method for extracting pumpkin pectin for seeds with antioxidant activity, which adopts ultrasonic wave to assist a traditional acid method to extract the pumpkin pectin for seeds, designs a response curve model for ultrasonic parameters, has ultrasonic power of 49%, duty ratio of 50%, ultrasonic time of 15min and highest yield; the obtained pumpkin pectin for seeds belongs to low-ester pectin; has ABTS free radical scavenging ability, DPPH free radical scavenging ability and Fe 3+ reducing ability; can also inhibit the growth of diarrhea causing Escherichia coli, enterotoxigenic Escherichia coli, salmonella enterica subspecies of typhimurium serotype, hemorrhagic Escherichia coli, listeria monocytogenes, and Salmonella enteritidis serotype; promoting the proliferation of beneficial bacteria in the in-vitro fermentation process of intestinal samples of healthy people and constipation patients, reducing the relative abundance of harmful bacteria, and having the effects of promoting the proliferation of intestinal probiotics and improving the intestinal health.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for extracting pumpkin pectin with antioxidant activity for seeds.
Background
Pectin is a natural polysaccharide of the primary wall of the cell wall and the cell intermediate sheet of many natural plants, especially in fruits and vegetables, and contains a plurality of different monosaccharide residues, and is generally considered to be formed by connecting galacturonic acid through alpha-1, 4-glycosidic bonds, and the side chain is composed of neutral polysaccharides such as galactose, rhamnose and arabinose, and the like, the relative molecular mass of the pectin is 10-40 kDa, and pectic acid, fruit alcohol, arabinose, and the like are generated during pectin hydrolysis. Pectin is mainly present in natural plants, almost odorless, has a sticky and smooth taste, is milky and sticky in shape, is weak acid, has strong heat resistance, and is almost insoluble in organic solvents. Pectin is widely used as a thickener and gelator in the food industry of jams, jellies, jams, juice beverages, etc., due to its good thickening and setting properties. Meanwhile, they are used in pharmaceutical, cosmetic, agricultural and other industrial fields, such as pharmaceutical capsules, toothpaste, fertilizers, plant protection products, etc., because of their good stability and emulsifying properties.
Pectin extraction methods are numerous, but different extraction methods can produce pectins with different physicochemical properties. The pectin extraction method is usually acid extraction, ultrasonic extraction, microwave extraction, enzyme extraction, ultrahigh pressure extraction, ion exchange resin extraction, etc. But pectin extracted by different extraction methods has different physical and chemical properties such as molecular mass, monosaccharide composition, galacturonic acid content, esterification degree, surface morphology and the like, so that the physical and chemical properties and the functional properties of the pectin are different. The different structure and degree of esterification of pectin can affect the properties and function of pectin. The degree of methylation of pectin affects its water solubility and gelling properties; the molecular weight of pectin affects its thickening effect and gel properties; the branching structure of pectin also affects its function in food products. Pectin of different structures has different water solubility, gelling, stability and emulsifying properties and thus has different functions in the food industry. Compared with high-fat pectin, the low-fat pectin is more suitable for people pursuing low-calorie diet, has the similar benefits of promoting digestion health and controlling blood sugar as the high-fat pectin, and has wide food application value.
The inventor surprisingly found in the research process that the seed pumpkin by-product is taken as a raw material, the traditional acid method is adopted to assist ultrasonic waves, the extraction rate of the seed pumpkin pectin is obviously improved, and the extracted seed pumpkin pectin has the ABTS free radical scavenging capability, the DPPH free radical scavenging capability and the Fe 3+ reducing capability; can also inhibit the growth of diarrhea causing Escherichia coli (ESCHERICHIA COLI O127:63) CICC-10411, enterotoxigenic Escherichia coli (ESCHERICHIA COLI ETEC O78:80) CICC-10421, salmonella enterica subspecies of the typhimurium serotype (Salmonella enterica subsp. Enterica serovar typhimurium) CICC-10420, hemorrhagic Escherichia coli (ESCHERICHIA COLI EHEC O157:157:7) CICC-21530, listeria monocytogenes (Listeria monocytogenes) CGMCC1.9136-LS1, and Salmonella serovar enterica (Salmonella enterica subsp. Enterica) CGMCC1.10754-SM 1. The pumpkin pectin for seeds disclosed by the invention is found in the process of carrying out simulated fermentation on intestinal samples of healthy and constipation patients, promotes the heat proliferation of beneficial bacteria in the intestinal samples of healthy people and constipation patients, reduces the relative abundance of harmful bacteria, and has the effect of improving the intestinal health.
Disclosure of Invention
The primary aim of the invention is to provide a method for extracting pumpkin pectin for seeds with antioxidant activity, which comprises the following steps:
(1) Pretreatment: taking pumpkin, removing seeds from the pumpkin, cleaning, cutting into blocks, drying, crushing and sieving for later use;
(2) Pectin extraction: s1, accurately weighing the pumpkin powder sample obtained in the step (1), adding an HCL solution with a pH value of 1.0-3.0 into a reactor according to a feed-liquid ratio of 1:20-40, heating to 75-95 ℃, then heating in a constant-temperature water bath for 30-150min, and stirring the solution;
S2, carrying out ultrasonic auxiliary extraction on the solution obtained in the step S1, wherein the ultrasonic power is 30% -70%, the ultrasonic crushing time is 5-25min, and the ultrasonic duty ratio is 30% -70%;
S3, filtering, collecting filtrate, adding active carbon, carrying out constant-temperature water bath, filtering again, and collecting filtrate;
s4, evaporating and concentrating the filtrate collected in the step S3, concentrating the volume to one tenth of the original volume, adding the absolute ethyl alcohol solution with the volume which is one time of the original volume of the evaporating liquid again, uniformly mixing, standing and precipitating;
s5, centrifuging, and discarding the supernatant to obtain a pumpkin pectin crude extract for seeds; adding absolute ethyl alcohol, oscillating, cleaning and purifying the coarse extract of the pumpkin pectin for seeds, centrifuging, and discarding the supernatant to obtain the pumpkin pectin for seeds;
(3) And (3) drying: and (5) drying the pumpkin pectin for seeds obtained in the step (S5).
Preferably, the ratio of feed to liquid as described in step S1 is 1:20-40.
Preferably, the pH value in the step S1 is 1.0-1.5.
Preferably, the temperature described in step S1 is 80-90 ℃.
Preferably, the time described in step S1 is 60-120min.
Preferably, the ultrasonic power in the step S2 is 40% -60%.
Preferably, the crushing time in the step S2 is 10-20min.
Preferably, the ultrasonic duty cycle in step S2 is 40% -60%.
Preferably, the drying mode in the step (3) is normal pressure drying and vacuum freeze drying.
Preferably, the normal pressure drying in the step (3) is performed in an oven, the temperature of the oven is set to 45 ℃, the drying time is about 10 hours, and the drying is performed until the weight is constant; vacuum freeze drying is carried out in a vacuum freeze dryer, the extracted wet seeds are frozen for 12 hours at the temperature of minus 80 ℃ and then enter the vacuum freeze dryer, the pre-cooling stage temperature is 3 hours at the temperature of minus 50 ℃, the vacuumizing stage temperature is minus 40 ℃, and the temperature is set as follows after the vacuum degree is 0: -30 ℃, -20 ℃, -10 ℃ for 1h, then sequentially setting the temperature to be: the vacuum freeze drying process of the pumpkin pectin for seeds is completed at the temperature of 0 ℃, 5 ℃, 10 ℃, 15 ℃, 20 ℃ and 25 ℃ for 3 hours.
The second object of the invention is to provide pumpkin pectin for seeds extracted by the extraction method.
The third object of the invention is to provide the application of the pumpkin pectin for seeds in preparing antibacterial products.
The fourth object of the invention is to provide the application of the pumpkin pectin for seeds in antioxidation or preparation of antioxidation products.
The fifth object of the invention is to provide the application of the pumpkin pectin for seeds in preparing medicines, food additives or health products for improving intestinal health.
The beneficial effects of the invention are as follows: (1) The invention provides a method for extracting seed pumpkin pectin, which adopts an ultrasonic-assisted traditional acid method to extract the seed pumpkin pectin, designs a response surface model for ultrasonic parameters, and shows that the ultrasonic power is 49%, the actual duty ratio is 50%, the actual ultrasonic time is 15min, and the yield of the seed pumpkin pectin is highest.
(2) The invention also provides pumpkin pectin for seeds, which is prepared by the extraction method, and belongs to low-ester pectin, wherein the average molecular weight of NCP is 920.019kDa, and the average molecular weight of NUP is 743.615kDa;
(3) The pumpkin pectin for seeds has the ability of removing ABTS free radicals, DPPH free radicals and Fe 3+ reducing ability; also useful are the growth of Escherichia coli (ESCHERICHIA COLI O127:63) CICC-10411, enterotoxigenic Escherichia coli (ESCHERICHIA COLI ETEC O78:80) CICC-10421, salmonella enterica subspecies of the typhimurium serotype (Salmonella enterica subsp. Enterica serovar typhimurium) CICC-10420, escherichia coli hemorrhagic (ESCHERICHIA COLI EHEC O157:7) CICC-21530, listeria monocytogenes (Listeria monocytogenes) CGMCC1.9136-LS1, and Salmonella enterica of the serotype (Salmonella enterica subsp. Enterica) CGMCC1.10754-SM 1.
(4) The pumpkin pectin for seeds disclosed by the invention is found in the process of fermenting the feces of healthy and constipation people, has the effects of promoting the proliferation of beneficial intestinal bacteria of healthy people and constipation patients, reducing the abundance of harmful intestinal bacteria and improving the health of the intestinal tracts.
Drawings
FIG. 1 Single factor diagram of pectin extraction by conventional acid method
FIG. 2 is a single factor chart of ultrasonic assisted acid extraction of pectin
FIG. 3 is a scanning electron microscope image of pumpkin pectin for seed according to the different extraction methods
FIG. 4 Infrared Spectrum of pumpkin pectin for seeds according to different extraction methods
FIG. 5 pectin improvement in intestinal tract health fungus change patterns of different populations
Detailed Description
The scope of the invention is described in detail below in connection with specific examples.
In the following examples, conventional acid-heated extraction of pumpkin pectin from seeds is abbreviated as NCP; ultrasonic assisted acid heating extraction method seed pumpkin pectin is simply called NUP; commercially available pectin is abbreviated CP. Each experiment was repeated three times.
Example one extraction of pumpkin pectin for seed
1. Pretreatment of raw materials
Seeds collected from the Altai area of Xinjiang are removed by pumpkin, cleaned, cut into blocks with the size of 3 cm-5 cm, and put into an oven to be dried for 18-24 hours at the temperature of 70 ℃. The dried pumpkin slices for seeds are crushed by a high-speed crusher and then are sieved by a 80-mesh sieve, and pumpkin powder for seeds is collected and stored in a drying place for standby.
2. Calculation of pectin yield of pumpkin for seeds
The yield of the pumpkin pectin for seeds in the experiment is that the mass of the pumpkin pectin for seeds obtained by extraction accounts for the proportion of the pumpkin powder for seeds. The calculation formula is as follows:
wherein: y is the extraction yield (%) of pumpkin pectin for seeds; a is the quality (g) of pumpkin pectin for seed extraction; b is the mass (g) of the weighed pumpkin powder for seeds.
3. Pumpkin pectin for seed extraction by traditional acid method
(1) Influence of feed liquid on extraction yield of pumpkin pectin for seeds
Accurately weighing 5g of pretreated pumpkin powder samples, respectively adding HCL solution with pH of 2.0 into a reactor according to the feed liquid ratio of 1:20, 1:25, 1:30, 1:35 and 1:40, keeping the temperature of a water bath kettle at 80 ℃, keeping the temperature of the water bath for 1h, and stirring the solution at intervals to prevent caking. After the water bath heating is completed, the filter liquor is immediately filtered by a buchner funnel, 0.5 percent of active carbon is added into the filter liquor, the temperature is kept for 30 minutes in a constant-temperature water bath kettle at 50 ℃, and the filter liquor is collected again. Evaporating and concentrating the filtrate by using a rotary evaporator, stopping after the volume is concentrated to one tenth of the original volume, adding an absolute ethyl alcohol solution with the volume being 1 time of the original volume of the evaporating liquid into an evaporating bottle, uniformly mixing, and standing at 4 ℃ for 2 hours to precipitate the seeds by using pumpkin pectin. Centrifuging the precipitated seed pectin suspension at 8000rpm for 20min, and removing supernatant to obtain seed pectin crude extract. Adding appropriate amount of absolute ethyl alcohol into the crude extract of the pumpkin pectin for seeds, sufficiently oscillating, sufficiently cleaning and purifying the crude extract of the pumpkin pectin for seeds, continuously centrifuging at 8000rpm for 20min, discarding the supernatant to obtain the pumpkin pectin for seeds, repeating the cleaning process twice to obtain almost transparent gelatinous substance, and drying in an oven at 45 ℃ until the weight is constant. The weight of pumpkin pectin for seed was weighed with an electronic balance. The influence of the feed liquid on the pectin yield in the pumpkin powder for seeds is examined.
As can be seen from FIG. 1-A, when the seed pumpkin pectin is extracted by the conventional acid heating extraction method, the seed pumpkin pectin yield tends to increase and then decrease along with the reduction of the feed-liquid ratio, and the pectin yield is maximum when the feed-liquid ratio is 1:30.
(2) Influence of pH on extraction yield of seed pumpkin pectin
Other steps are the same as (1), the feed-liquid ratio is 1:30, and the pH is respectively set as: five levels of HCL solutions, 1.0, 1.5, 2.0, 2.5, 3.0, were examined for the effect of pH on pectin yield in the seed pumpkin powder.
As can be seen from FIG. 1-B, when the conventional acid heating extraction method is used for extracting the pectin from the pumpkin, the yield of the pectin from the pumpkin is increased and then decreased along with the continuous increase of the pH value, and the yield of the pectin from the pumpkin is maximum when the pH value is 1.5.
(3) Influence of the extraction temperature on the extraction yield of seed pumpkin pectin
Other steps are the same as those in the step (1), the feed-liquid ratio is 1:30, and the leaching temperatures of the water bath are respectively set to be: five levels of 75 ℃, 80 ℃, 85 ℃, 90 ℃ and 95 ℃ were examined for the effect of the leaching temperature on the pectin yield in the pumpkin powder for seeds.
As can be seen from FIG. 1-C, when the conventional acid heating extraction method is used for extracting the seed pumpkin pectin, the yield of the seed pumpkin pectin tends to increase and decrease with the increase of the heating extraction temperature, and the yield of the seed pumpkin pectin is maximum when the extraction temperature is 80 ℃.
(4) Influence of the extraction time on the extraction yield of seed pumpkin pectin
Other steps are the same as those of the step (1), the ratio of the feed liquid to the liquid is 1:30, and the leaching time is respectively set as follows: five levels of 30min, 60min, 90min, 120min and 150min were examined for the effect of the leaching time on the pectin yield in the seed pumpkin powder.
As can be seen from FIG. 1-D, when the conventional acid heating extraction method is used for extracting the seed pumpkin pectin, the yield of the seed pumpkin pectin tends to increase and then decrease with the increase of the heating extraction time, and the yield of the seed pumpkin pectin is maximum when the extraction time is 40 min.
4. Ultrasonic-assisted acid method for extracting pumpkin pectin for seeds
(1) Influence of ultrasonic power on extraction yield of pumpkin pectin for seeds
The water bath leaching is the same as in step 2. After the water bath leaching is completed, ultrasonic auxiliary extraction is carried out, leaching liquor is put into an ultrasonic breaker for ultrasonic breaking, and ultrasonic power is respectively set as follows: five levels of 30%, 40%, 50%, 60%, 70%, ultrasonic crushing time of 15min, ultrasonic duty cycle of 50%, suction filtration using buchner funnel immediately after ultrasonic completion and collection of filtrate. The filtrate is treated in the same manner as in step 2.
As can be seen from fig. 2-a, when the ultrasonic-assisted acid heating extraction method is used for extracting the seed pumpkin pectin, the yield of the seed pumpkin pectin tends to increase and decrease with the increase of the ultrasonic extraction power, and when the ultrasonic extraction power is 50%, the yield of the seed pumpkin pectin is the maximum. The optimal extracted ultrasonic power was determined to be 50%.
(2) Influence of ultrasonic extraction time on extraction yield of pumpkin pectin for seeds
Other steps are the same as (1), the ultrasonic power is set to 50%, and the ultrasonic extraction time is set to: five levels of 5min, 10min, 15min, 20min, 25 min.
As can be seen from FIG. 2-B, when the ultrasonic-assisted acid heating extraction method is used for extracting the seed pumpkin pectin, the yield of the seed pumpkin pectin tends to increase and decrease with the increase of the ultrasonic extraction time, and the yield of the seed pumpkin pectin is maximum when the ultrasonic extraction time is 15 min.
(3) Influence of ultrasonic duty ratio on extraction rate of seed pumpkin pectin
Other steps are the same as (1), the ultrasonic power is set to 50%, and the ultrasonic duty ratio is set to: five levels of 30%, 40%, 50%, 60%, 70% were examined for the effect of the ultrasonic duty cycle on pectin yield in the seed pumpkin powder.
As can be seen from fig. 2-C, when the ultrasonic-assisted acid heating extraction method is used for extracting the seed pumpkin pectin, the yield of the seed pumpkin pectin tends to increase and decrease with the increase of the ultrasonic extraction duty ratio, and when the ultrasonic extraction duty ratio is 50%, the yield of the seed pumpkin pectin is the largest.
Example two, ultrasonic-assisted acid extraction orthogonal analysis
1. Ultrasonic-assisted pumpkin pectin response curved surface design for seed extraction
In a single-factor experiment of an ultrasonic assisted acid extraction method, ultrasonic power, ultrasonic time and duty ratio are used as influencing factors, the pectin yield of the pumpkin for seeds is used as a response value, a three-factor and three-level Box-Behnken Design (BBD) response surface Design experiment (Response Surface Methodology, RSM) is adopted, and the optimal pectin yield of the pumpkin for seeds and each factor condition under the optimal response value are researched. The experimental factor levels and numbers are shown in table 1. The whole experimental design is 17 groups, and the center point experiment is repeated for 5 groups.
The experimental design and data analysis used Design Expert software (Version 10.0). The response value can be expressed as a quadratic polynomial as follows:
Where Y is the response value, βo, β i、βii, and β ij are regression coefficients of intercept term, linear term, square term, and interaction term, respectively, and x i and x j represent number independent variables, which are numbered according to the following equation:
xi＝(Xi-Xo)/ΔX (3)
Where X i is the numbered value of the variable Xi, xo is the center point of its factor and ΔX is the step size. Table 1 shows the complete BBD response surface experimental design, the data and number values of the arguments used in the table.
TABLE 1Box-Behnken ultrasound-assisted extraction response surface Experimental design
2. Orthogonal analysis
(1) Ultrasonic-assisted seed extraction pumpkin pectin response surface model fitting and statistical analysis
There are significant differences between the results of RSM experiments under different experimental combinations, and multiple regression analysis was performed on experimental data by Design Expert software (Version 10.0), and the predictive regression model of seed pumpkin pectin yield was as follows:
Y＝16.78+2.5A-0.56B+0.22C-3.61A2-3.11B2-3.67C2 (4)
Wherein Y (%) is the pectin yield of the pumpkin for seeds, A, B, C is the ultrasonic power of the pumpkin for independent seeds, the ultrasonic time of the pumpkin for seeds and the number value of the duty ratio of the pumpkin for seeds. The significance of the regression model was checked by analysis of variance (AVOVA) on the F statistic, response surface quadratic polynomial model.
The results are shown in table 2, with larger F statistics and smaller p values and less than 0.05 indicating more significant model and corresponding variables. Having a higher F statistic (15.49) and a very low p value (< 0.0001) in the model means that the model is very significant. The value of the determination coefficient (R 2) calculated by the quadratic regression model is 0.8229, and most of the changes can be fit to the designed model. The adjusted determination coefficientIs close to R 2, there is a high correlation between actual experimental data and predicted experimental data values. From the above analysis, the designed model can be used to predict pectin release rates over a range of individual factor levels and in every two combinations. The significance of each regression coefficient is determined by the F value and the p value in the table, and researches show that the primary term coefficient (A, B, C) and the secondary term coefficient (A 2、B2、C2) obviously influence the pectin release rate (p < 0.05), and the order of the influence factors is that the duty ratio is higher than the ultrasonic extraction power and the ultrasonic extraction time in sequence.
TABLE 2 analysis of variance (ANOVA) and parameter significance test of multiple regression model of pumpkin for seeds
Note that: AR 2＝0.8229、R2 Adj = 0.7697, c.v. =8.94
(2) Extraction parameter optimization and model verification
As shown in table 3, the optimal extraction conditions for obtaining the yield of pumpkin pectin for seeds by taking the derivative of the quadratic polynomial model are as follows: the ultrasonic power of the pumpkin for seeds is 54.51%, the ultrasonic duty ratio is 52.75%, the ultrasonic extraction treatment time is 14.52min, and the maximum yield of the pumpkin pectin for seeds predicted under the conditions is 17.13%. Considering the feasibility of the experiment, the optimal extraction conditions were slightly modified as follows: the actual ultrasonic power of the seed pumpkin is 55%, the actual ultrasonic duty ratio is 53%, the actual ultrasonic extraction treatment time is 15min, and the yield of the seed pumpkin pectin obtained through experiments under actual conditions is 17.18%, which is close to a predicted value and far superior to the traditional acid heating extraction method. The actual result again verifies that the model is suitable for predicting the research of ultrasonic-assisted extraction on the yield of pumpkin pectin for seeds.
TABLE 3 comparison of pectin yield response surface predicted optimal value and actual optimal value for ultrasonic-assisted acid extraction of seed pumpkin pectin
Example III physical and chemical Property analysis of pumpkin pectin for seed
1. Scanning electron microscope image analysis
And uniformly adhering a small amount of powdery pectin sample to a sample stage by using a dried pumpkin pectin sample for seeds and commodity pectin by using a scanning electron microscope, spraying gold for 90s by using an ion sputtering method after air drying, and then, coating a conductive film, and observing the surface morphology of pectin samples with different sources and extraction methods by using a machine.
From fig. 3, it can be seen that the ultrasonic-assisted extraction and the conventional acid extraction have an effect on the surface morphology of the pumpkin pectin for seeds. Compared with the traditional acid extraction method, the surface net structure of the pumpkin pectin for seeds, which is crushed by ultrasonic waves, is crushed more fully, so that the surface of the pumpkin pectin for seeds is rougher. The coarse pectin surface network structure may enhance its adsorption capacity, improve its stability and facilitate nutrient release.
2. Physical and chemical index
(1) Determination of total sugar
An anthrone colorimetric method is adopted. The absorbance values measured at wavelength A620 are plotted on the abscissa (0 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 700 μg, 800 μg) for each milliliter of glucose standard. The standard curve is plotted as follows: the regression equation is y=0.0065x+0.0042, and the fitting degree is R 2 =0.9995. Taking 250 mu L of pectin sample and 625 mu L of anthrone solution, quickly mixing, standing at room temperature for 10-15 min, and measuring the absorbance value under A 620. Substituting the measured absorbance into a regression equation to calculate the total sugar content in the sample.
(2) Determination of galacturonic acid content
The measurement of galacturonic acid content adopts m-hydroxybiphenyl method. Preparation of galacturonic acid standard solution: accurately weighing 10mg of galacturonic acid standard substance, dissolving in 100mL of distilled water, and preparing into 0.1mg/mL of galacturonic acid standard solution; preparation of sodium tetraborate-sulfuric acid solution: accurately weighing 0.954g of sodium tetraborate, adding 100mL of concentrated sulfuric acid solution, carrying out ultrasonic treatment to dissolve the sodium tetraborate, and then adding the concentrated sulfuric acid solution to 200 mL; preparation of m-hydroxydiphenyl reagent: accurately weighing 0.15g of m-hydroxybiphenyl reagent, and adding 100mL of 0.5% NaOH solution.
Drawing a standard curve: respectively sucking 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL and 0.6mL of galacturonic acid standard solution into a test tube with a plug, and adding distilled water to 1mL. Precooling in ice water bath, adding prepared sodium tetraborate-sulfuric acid solution to 6mL, mixing with vortex instrument, heating in boiling water bath for 12min, taking out, cooling to room temperature, adding 80 μl of 0.15% m-hydroxybiphenyl solution, shaking to room temperature for 40min, and measuring absorbance at 525 nm. And drawing a standard curve regression equation with y=0.0083x+0.0054 and fitting degree R 2 =0.9975 by taking the standard galacturonic acid amount as an abscissa and the light absorption value at 525nm as an ordinate.
Determination of seed pumpkin pectin samples: the seeds extracted by different methods are prepared into pectin solution of 0.1mg/mL by pumpkin pectin, 1mL is added into a test tube with a plug, precooled in ice bath, then sodium tetraborate-sulfuric acid solution which is prepared is added to 6mL, vortex mixing is carried out by a vortex machine, heating is carried out for 12min in boiling water bath, the ice bath is taken out to room temperature, then 80 mu L of 0.15% m-hydroxybiphenyl solution is added, shaking is carried out to room temperature for 40min, and the absorbance value is measured at 525 nm. Substituting the measured absorbance into a regression equation to calculate the galacturonic acid content in the sample.
(3) Determination of the degree of esterification
20Mg of a seed pumpkin pectin sample was weighed, dissolved in 4ml of distilled water, added with 100. Mu.L of ethanol, and stirred at a speed of 250r/min for 30min. After the pectin is completely dissolved, 3-5 drops of phenolphthalein are added. The mixture was titrated with 0.1mol/L NaOH solution until the solution turned pink, and the reading recorded the volume fraction (V1) of NaOH consumed. Then, 2ml of a 0.1mol/L NaOH solution was added, and after stirring manually for 2 minutes, a 0.1mol/L HCL solution was added to the solution until the color of the solution became dark. The solution was then stirred for 15min, titrated again with 0.1mol/L NaOH solution until a pink color again developed, and the record was read of the volume of NaOH consumed (V2).
The degree of esterification of the seed pumpkin pectin was calculated according to the following formula.
Wherein: DE is the degree of esterification%
V1 is the volume of NaOH solution consumed for the first time, mL
V2 is the volume of the NaOH solution consumed for the second time, mL
(4) Determination of protein content
The absorbance values measured at wavelength a595 are plotted on the abscissa (50 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg) with the concentration of bovine serum albumin per ml and the standard curve y=0.0023x+0.0038, fitting degree R 2 =0.9982. Taking 20 mu L of sample and 20 mu LBradord of working solution, quickly and uniformly mixing, reacting for 5min at the room temperature of 25-30 ℃, and measuring the absorbance value under A595. And (3) calculating the protein concentration of the diluted sample on a standard curve, selecting a sample with proper dilution to calculate the final sample protein concentration, and calculating the protein concentration of the pumpkin pectin sample for the primordium according to the dilution multiple.
(5) Results
Specific data of pumpkin pectin (NCP) for seed by conventional acid heating extraction method, pumpkin pectin (NUP) for seed by ultrasonic assisted acid heating extraction method, and commercially available pectin (CP) are shown in Table 4. The pectin esterification degree obtained by different methods is less than 50% by measuring the pectin esterification degree of the pumpkin for seeds, and the pectin of the pumpkin for seeds is low-fat pectin.
TABLE 4 physicochemical index and content of pumpkin pectin for seed
3. Determination of molecular weight and distribution thereof
Molecular weight and purity analysis were performed on seed pumpkin pectin samples using High Performance Gel Permeation Chromatography (HPGPC). Weighing pumpkin pectin for seeds, dissolving with deionized water, and preparing into solution with concentration of 10.0 mg/ml.
Detection conditions: TSK-GEL G4000SWxl column (300X 7.8 mm); deionized water is used for the mobile phase, and the flow rate is 1.0mL/min; column temperature is 30 ℃; the sample injection amount is 20 mu L; maximum holding pressure 25bar; and (5) RID detection.
Drawing a standard curve: a series of standards such as Dextran et al were added to deionized water to make a standard solution of 5 mg/mL. The sample loading was 20 μl and the standard curve was plotted by GPC analysis software against retention time (Rt) as log weight average molecular weight (log Mw) of the Dextran standard. The retention time of the seed pumpkin pectin sample is substituted into a standard curve regression equation to calculate the molecular weight.
As shown in Table 5, the results of the measurement of the average molecular weight of the pumpkin pectin for seeds and the distribution thereof were analyzed, and it was found that the molecular weights of the pumpkin pectin for seeds were different in the different extraction methods, and that the average molecular weight of NCP was 920.019kDa and the average molecular weight of NUP was 743.615kDa.
Since the pumpkin pectin for seeds obtained by extraction is coarse pectin, different peaks appear in the measurement of the molecular weight of the pumpkin pectin for seeds, and the molecular weight of the pumpkin pectin for seeds is finally represented by the average molecular weight. The molecular weight also has an effect on the function and properties of the seed pumpkin pectin. The molecular weight of the pumpkin pectin for seeds can be influenced by different extraction methods of the pumpkin pectin for seeds, and the detection result shows that compared with the traditional acid heating method, the ultrasonic auxiliary acid extraction method can reduce the average molecular weight of the pumpkin pectin for seeds.
TABLE 5 average molecular weight of NCP, NUP
4. Determination of monosaccharide composition
And determining the monosaccharide composition and the molar ratio in the pumpkin pectin sample for seeds by adopting PMP pre-column derivatization high performance liquid chromatography. Pre-column derivatization step: mu.L of the mixed control solution was pipetted into a 5mLEP tube, 250. Mu.L of 0.6mol/LNaOH solution, 500. Mu.L of 0.4 mol/LPMP-methanol was added and reacted at 70℃for 1 hour. Cooled in cold water for 10min, neutralized by adding 500. Mu.L of 0.3mol/LHCL solution, vortexed with 1mL of chloroform for 1min, centrifuged at 3000r/min for 10min, extracted 3 times, and the supernatant was used for HPLC. Preparing a standard solution: mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, N-acetyl-glucosamine, glucose, N-acetyl-galactosamine, galactose, xylose, arabinose, fucose mixed standard solution with a concentration of 50 μg/mL was prepared. Seed pumpkin pectin hydrolysis sample preparation: 3.0mg of the seed pumpkin pectin sample is weighed into an ampere bottle, and 3.0ml of 2 mol/LTFA solution is added for acidolysis at 120 ℃ for 4 hours. 1.0ml of hydrolysis solution was measured, pH was adjusted to 7.0 with NaOH solution, and the volume was set to 2.0ml. The standard solution and the pre-column derivative of the pumpkin pectin for seeds are prepared by the method and are detected by liquid chromatography. Chromatographic conditions: mobile phase 0.05M potassium dihydrogen phosphate solution (PH adjusted to 6.70 with NaOH solution): acetonitrile=83:17 (v/v), column temperature: 30 ℃, flow rate: 1.0ml/min, detection wavelength: 250nm, sample injection amount: 20 μl, column: xtimate C184.6.6 x 200mm,5 μm. And calculating the monosaccharide composition in the pumpkin pectin sample for seeds according to the peak-out time and the peak area.
Results: table 6 shows the pumpkin pectin (NUP) monosaccharide composition for the ultrasound-assisted acid heating process seeds. Since the main components of pectin are galacturonic acid (GalA), rhamnose (Rha), galactose (Gal) and the like, the content of GalA is large in the monosaccharide composition of CUP and NUP, but the high content of glucose (Ghl) can be seen in the table, probably because of the presence of some non-pectin components or the lack of purification of the resulting pectin, so that some soluble sugar is not removed, it can be deduced that the detected NUP is pumpkin pectin for coarse seeds. If the test sample is purified, a significant amount of non-pectic soluble sugars can be removed. Xylose (Xyl), mannose (Man), fucose (Fur) and the like were also found by examination, and the presence of these monosaccharides may be linked on the side chain of pectin, and thus the content was also small.
TABLE 6 monosaccharide composition of NCP, NUP
5. Fourier transform infrared spectroscopy
And detecting main functional groups of different components of the pumpkin pectin sample for seeds and the commercial pectin by adopting Fourier infrared spectroscopy. Mixing 1mg of seed pumpkin pectin powder sample with a proper amount of KBr, grinding uniformly in an agate mortar, putting the uniformly ground mixture into a sample preparation device to prepare a sheet, measuring and analyzing by an upper machine, scanning the wave number range to 400-4000cm -1, and analyzing and processing the experimental result by OMNIC software, wherein the resolution is 4cm -1.
As can be seen from fig. 4: the Fourier infrared spectra of NCP and NUP are basically consistent with that of CP, and the pectin is extracted by the traditional acid heating extraction method and the ultrasonic assisted acid heating method. The two stronger temperature absorption peaks at 1600-1750cm -1 are respectively due to the existence of free carboxyl and esterified carboxyl, and the areas of the two absorption peaks can confirm that the results of the pectin with low esterification degree are identical. Pectin derived from the same raw material may also have different properties and structures if the extraction methods are different.
Example IV functional Property measurement of pumpkin pectin for seed
1. Antioxidant capacity
(1) ABTS radical scavenging ability
Preparing an ABTS test solution: ABTS stock (7.4 mmol/L,0.4 mL): ABTS 0.0045g, distilled water 1.1025mL (mw=548.7); k 2S2O8 stock (2.6 mmol/L,1.43 mL): k 2S2O8 0.0025.0025 g was taken, 3.575mL of distilled water (mw= 270.32) was added, the two were mixed, and after 12 hours of standing at room temperature in the dark, they were diluted 50-fold with PBS. A0 value detection: the absorbance was measured at 734nm after 1.6mL of the ABTS solution was thoroughly mixed with 0.6mL of PBS. And (3) detecting an A value: the absorbance at 734nm was measured after 1.6mL of LABSS test solution was mixed with 0.6mL of gradient sample, and the experiment was repeated three times independently.
ABTS clearance = (A0-a)/A0 x 100%
Wherein the parameters represent: a0: 2mLPBS+2mLABSS; a:2m L samples+2 mLABSS;
The results are shown in Table 7, where the ABTS radical scavenging ability of pectin increases significantly with increasing pectin concentration. The ABTS free radical scavenging ability of the pumpkin pectin for seeds is far more than that of the pectin sold in the market, and the ABTS free radical scavenging ability of the pumpkin pectin for seeds is more than 3 times of that of the pectin sold in the market under the same concentration gradient. Whereas the ABTS radical scavenging ability of NUP in seed pumpkin pectin was greater than that of NCP at the same concentration gradient.
Table 7NCP, NUP, CP ABTS radical scavenging Capacity
(2) DPPH free radical scavenging ability
The DPPH radical scavenging ability of pectin from different sources was assessed in comparison. Pectin (1 mg/mL,2mg/mL,3mg/mL,4mg/mL,5 mg/mL) was reacted with DPPH ethanol solution (0.2 mmol/L) at different concentration gradients, and absorbance was measured at 517nm after 20min of dark reaction.
DPPH radical clearance (%) = (1- (A1-A2)/A0) ×100
Wherein the parameters represent: a0:2mL of methanol+2 mLDPPH; a1:2m L samples +2mLDPPH; a2:2mL of sample solution+2 mL of methanol
From table 8 it can be seen that the DPPH radical scavenging capacity of pectin increases significantly with increasing pectin concentration. The DPPH free radical scavenging ability of the pumpkin pectin for seeds is far more than that of the commercial pectin, and the DPPH free radical scavenging ability of the pumpkin pectin for seeds is more than 1.5 times of that of the commercial pectin under the same concentration gradient. Whereas the DPPH radical scavenging capacity of NUP in seed pumpkin pectin was greater than that of NCP at the same concentration gradient.
Table 8NCP, NUP, CP DPPH radical scavenging ability
(3) Fe 3+ reducing ability
The reducing power of pectin Fe 3+ from different sources was evaluated in comparison. Sample solutions of different mass concentration gradients (1 mg/mL,2mg/mL,3mg/mL,4mg/mL,5 mg/mL) were accurately removed, vitamin C solution was used as a control, and 2.5mL of 0.2mol/L phosphate buffer (PBS, pH 6.6) and 2.5mL of 1% potassium ferricyanide solution were added. After being evenly mixed, the mixed solution is placed in a constant-temperature water bath kettle with the temperature of 50 ℃ for water bath for 20min. Then adding 2.5mL of 10% trichloroacetic acid (TCA), centrifuging (4000 r/min,10 min), sucking 2.5mL of supernatant, adding 2.5m L distilled water, finally adding 0.5mL of 0.1% ferric trichloride solution, shaking, mixing uniformly, and standing for 10min. Absorbance was measured at a wavelength of 700 nm.
Total reducing power = A1-A0
Wherein the parameters represent: a1: absorbance of the sample solution; a0: absorbance of blank group
From table 9 it can be seen that the Fe 3+ reducing power of pectin increases significantly with increasing pectin concentration. The Fe 3+ reducing ability of the pumpkin pectin for seeds is far more than that of the commercial pectin, and the Fe 3+ reducing ability of the pumpkin pectin for seeds is more than 2 times of that of the commercial pectin under the same concentration gradient. The Fe 3+ reducing power of NUP in the pumpkin pectin for seeds is larger than the Fe 3+ reducing power of NCP under the same concentration gradient.
Table 9NCP, NUP, CP shows Fe 3+ reducing power
2. Pectin bacteriostatic ability
(1) Cultivation of 6 pathogenic bacteria
The sources of the 6 pathogenic bacteria and the culture methods are shown in Table 10.
Table 10 pathogenic bacteria name and culture method
(2) Evaluation of bacteriostasis ability of pectin
Pectin is added into a liquid culture medium of 6 pathogenic bacteria according to different proportions (0%, 1% and 2%), sterilization is carried out for 20min at 115 ℃ for standby, the activated 6 pathogenic bacteria are inoculated into the culture medium with different pectin concentrations for culture according to 2% (v/v), dilution coating counting is carried out after 24 hours, and whether the pectin can inhibit the growth of pathogenic bacteria is judged according to the dilution coating result.
As shown in Table 11, the bacterial colony count of the pathogenic bacteria culture medium after the pectin is added is obviously less than that of the control group without the apple pectin after the pathogenic bacteria culture medium is diluted and coated to culture 6 pathogenic bacteria, even no pathogenic bacteria grow, which indicates that the pectin can obviously inhibit the growth of pathogenic bacteria and has good antibacterial effect.
TABLE 11 pathogenic bacteria inhibition results by different concentration pectin additions
Note that: wherein-means sterile colony growth; + represents the colony number less than or equal to 10; ++ means colony numbers between 10 and 100; ++ means that the colony count is not less than 200.
Example five, strain Change during in vitro fermentation of seed pumpkin pectin
1. Sample collection
Volunteers not taking prebiotics or probiotic products within approximately 3 months of collection, nor receiving antibiotic treatment, were sampled for their intestinal tract for study. Stool samples from healthy adult human were collected from student volunteers at the university of stone river, intestinal fluid samples from constipation patients were collected from the digestive system department of the first affiliated hospital at the university of stone river, and intestinal fluid was extracted from the intestinal tract of the patient at the time of enteroscopy after the patient had agreed. The intestinal samples of healthy volunteers were collected in total, and the intestinal fluid samples of constipation volunteers were collected in total, in 4. Constipation volunteer samples were divided into A, B groups. Group A (3 females, 1 male, age group 50-60) and group B (4 females, age group 70-90). Fresh samples were mixed with sterile physiological saline (0.9%, w/v) to prepare a 10% (w/v) suspension.
2. In vitro simulated fermentation and data analysis
The 1L culture medium comprises the following components: 2.0g peptone, 2.0g yeast extract ,0.1g NaCl,0.04g K2HPO4,0.04g KH2PO4,0.01gMgSO4·7H2O、0.01g CaCl2·2H2O,2.0g NaHCO3,0.5g g cysteine HCL salt, 0.5g bile salt, 2mL Tween 80,0.01mL vitamin K1,1mL heme solution. The medium contained 100mg of a carbon source (final concentration of 1%) per 9mL of the medium, and the pH was adjusted to 7.4.+ -. 0.02. The experimental group uses pumpkin pectin for seeds as a carbon source, and the positive control uses commercial apple pectin (CP) as a carbon source.
1.0ML of the sample suspension was poured into penicillin bottles containing 9.0mL of intestinal microbial medium for in vitro simulated fermentation. All experimental groups were subjected to in vitro simulated fermentation in an anaerobic tank in triplicate. Collecting fermentation liquid of 0h and 48h, and collecting thalli after centrifugation. According to the instructions, total DNA of each group was extracted using a fecal genomic DNA extraction kit (Tiangen), and the purity of the total DNA was measured using 1% agarose gel electrophoresis. And preparing a PCR reaction system by the qualified intestinal microbial genome DNA sample and universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3'), setting PCR reaction conditions for amplification, and carrying out 16S rRNA high-throughput sequencing on the PCR product on a Illumina Miseq double-end sequencing platform. Subsequently, all chimeric sequences were excluded by denoising algorithm using vsearch tools. The remaining sequences were clustered into OTUs with 97% similarity cut and unique representative OTU sequences were identified. The SILVAribosomal RNAdatabase (Sliva) database was selected for species alignment of OUT.
3. Results
From fig. 5, it can be seen that the apple pectin has a lower probiotic effect than the seed pumpkin pectin, which is more abundant than the apple pectin in the variety of promoting the proliferation of probiotics in the fermentation process of the feces of healthy people. Apple pectin only promotes a relatively rich increase in lactobacillus mucilaginosus (Limosilactobaillus) and a decrease in the relative abundance of the conditionally pathogenic bacteria Prevotella (Prevotella) when fermented to 48 hours. Whereas pumpkin pectin for seeds increases the relative abundance of Bifidobacterium (bifidobacteria), lactobacillus (Lactobacillus), lactobacillus mucilaginosus (Limosilactobaillus) by 8.28%, 37.03% and 24.79%, respectively; at the same time, the relative abundance of the conditional pathogenic bacteria Prevotella and Klebsiella is reduced by 9.41% and 0.39%, respectively.
In the process of in vitro fermentation of constipation patient intestinal juice by using seed pumpkin pectin as carbon source, seed pumpkin pectin increases relative abundance of Bacteroides (bacilli), bifidobacterium (Bifidobacterium), coliform (Collinsella) and Pediococcus (Pediococcus) by 16.29%, 0.85%, 3.44% and 0.41%, respectively; at the same time, the relative abundance of the conditional pathogenic bacteria Escherichia coli (Escherichia-Shigella) in the intestinal microorganisms of the patients is obviously reduced by 35.56 percent.
Most bacteroides have polysaccharide utilization sites, and can decompose complex polysaccharide; bifidobacteria are used as intestinal probiotics, and have various important physiological functions of improving gastrointestinal functions, resisting aging and the like; the genus coliform can effectively prevent infection and severe disease of new coronavirus. The increase of the beneficial bacteria can keep balance of intestinal microbiota, inhibit growth of harmful bacteria, generate beneficial metabolites, and help to improve and maintain intestinal health. In general, the pumpkin pectin for seeds can remarkably promote the proliferation of intestinal probiotics of healthy people and constipation patients, reduce the relative abundance of intestinal harmful bacteria, and can promote the proliferation of the intestinal probiotics in the process of fermenting the feces of healthy people, but the pumpkin pectin for seeds and the commercial apple pectin have more advantages in promoting the proliferation of the probiotics.
In summary, the invention provides a method for extracting seed pumpkin pectin, which adopts an ultrasonic-assisted traditional acid method to extract seed pumpkin pectin, and designs a response curved surface model for ultrasonic parameters, and results show that the ultrasonic power is 49%, the actual duty ratio is 50%, the actual ultrasonic time is 15min, and the yield of seed pumpkin pectin is highest. The invention also provides pumpkin pectin for seeds, which is prepared by the extraction method, and belongs to low-ester pectin. The average molecular weight of NCP is 920.019kDa and the average molecular weight of NUP is 743.615kDa; the pumpkin pectin for seeds has the ability of removing ABTS free radicals, DPPH free radicals and Fe 3+ reducing ability; can also inhibit the growth of diarrhea causing Escherichia coli (ESCHERICHIA COLI O127:63) CICC-10411, enterotoxigenic Escherichia coli (ESCHERICHIA COLI ETEC O78:80) CICC-10421, salmonella enterica subspecies of the serotype typhimurium (Salmonella enterica subsp. Enterica serovar typhimurium) CICC-10420, hemorrhagic Escherichia coli (ESCHERICHIA COLI EHEC O157:157:H 7) CICC-21530, listeria monocytogenes (Listeria monocytogenes) CGMCC1.9136-LS1, and Salmonella serovar enterica (Salmonella enterica subsp. Enterica) CGMCC1.10754-SM 1; can promote the proliferation of beneficial bacteria in the in vitro fermentation process of intestinal samples of healthy people and constipation patients, reduce the relative abundance of harmful bacteria, and has the effects of promoting the proliferation of intestinal probiotics and improving the intestinal health.
Claims (9)
1. A method for extracting pumpkin pectin with antioxidant activity for seeds is characterized by comprising the following steps:
(1) Pretreatment: taking pumpkin, removing seeds from the pumpkin, cleaning, cutting into blocks, drying, crushing and sieving for later use;
(2) Pectin extraction: s1, accurately weighing the pumpkin powder sample obtained in the step (1), adding an HCL solution with a pH value of 1.0-3.0 into a reactor according to a feed-liquid ratio of 1:20-40, heating to 75-95 ℃, then heating in a constant-temperature water bath for 30-150min, and stirring the solution;
S2, carrying out ultrasonic auxiliary extraction on the solution obtained in the step S1, wherein the ultrasonic power is 30% -70%, the ultrasonic crushing time is 5-25min, and the ultrasonic duty ratio is 30% -70%;
S3, filtering, collecting filtrate, adding active carbon, carrying out constant-temperature water bath, filtering again, and collecting filtrate;
s4, evaporating and concentrating the filtrate collected in the step S3, concentrating the volume to one tenth of the original volume, adding the absolute ethyl alcohol solution with the volume which is one time of the original volume of the evaporating liquid again, uniformly mixing, standing and precipitating;
s5, centrifuging, and discarding the supernatant to obtain a pumpkin pectin crude extract for seeds; adding absolute ethyl alcohol, oscillating, cleaning and purifying the coarse extract of the pumpkin pectin for seeds, centrifuging, and discarding the supernatant to obtain the pumpkin pectin for seeds;
(3) And (3) drying: and (5) drying the pumpkin pectin for seeds obtained in the step (S5).
2. The extraction method according to claim 1, wherein the feed liquid ratio in step S1 is 1:25-35, pH value of 1.0-1.5, temperature of 80-90 deg.C, and time of 60-120min.
3. The extraction method according to claim 1, wherein the ultrasonic power in step S2 is 40% -60%, the crushing time is 10-20min, and the ultrasonic duty cycle is 40% -60%.
4. The method according to claim 1, wherein the drying in the step (3) is performed by normal pressure drying and vacuum freeze drying.
5. The extraction method according to claim 4, wherein the normal pressure drying in the step (3) is performed in an oven, the temperature of the oven is set to 45 ℃, the drying time is about 10 hours, and the drying is performed until the weight is constant; vacuum freeze drying is carried out in a vacuum freeze dryer, the extracted wet seeds are frozen for 12 hours at the temperature of minus 80 ℃ and then enter the vacuum freeze dryer, the pre-cooling stage temperature is 3 hours at the temperature of minus 50 ℃, the vacuumizing stage temperature is minus 40 ℃, and the temperature is set as follows after the vacuum degree is 0: -30 ℃, -20 ℃, -10 ℃ for 1h, the temperature being set in sequence: the vacuum freeze drying process of the pumpkin pectin for seeds is completed at the temperature of 0 ℃,5 ℃,10 ℃, 15 ℃, 20 ℃ and 25 ℃ for 3 hours.
6. The pumpkin pectin for seeds extracted by the extraction method according to any one of claims 1 to 5.
7. Use of the pumpkin pectin for seeds according to claim 6 for preparing bacteriostatic products.
8. Use of the pumpkin pectin for seeds according to claim 6 for antioxidation or for preparing antioxidation products.
9. Use of the pumpkin pectin for seeds according to claim 6 for preparing a medicament, food additive or health product for improving intestinal health.
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