CN116832079B - Preparation method of primary pulp for relieving purgation effect of phyllanthus emblica normal juice - Google Patents
Preparation method of primary pulp for relieving purgation effect of phyllanthus emblica normal juice Download PDFInfo
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- CN116832079B CN116832079B CN202310800579.9A CN202310800579A CN116832079B CN 116832079 B CN116832079 B CN 116832079B CN 202310800579 A CN202310800579 A CN 202310800579A CN 116832079 B CN116832079 B CN 116832079B
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- pectin
- phyllanthus emblica
- juice
- supernatant
- fresh
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Classifications
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Abstract
The invention provides a method for preparing primary pulp for relieving purgation of phyllanthus emblica juice, which comprises the following steps: a. taking fresh phyllanthus emblica fruits after de-enzyming, and squeezing to obtain pomace and fresh juice; b. decocting the fruit residue with water, cooling, centrifuging, and collecting supernatant; c. and d, mixing the fresh juice prepared in the step a with the supernatant prepared in the step b to obtain the phyllanthus emblica primary pulp. The invention also provides a method for detecting the quality in the process of preparing the primary pulp. The invention relieves diarrhea caused by taking fresh phyllanthus emblica juice by utilizing phyllanthus emblica pomace, fully utilizes the pomace resources after juicing to form relevant quality control indexes, and the prepared phyllanthus emblica pulp is safe to use, stable in quality and controllable.
Description
Technical Field
The invention relates to a preparation method of raw juice for relieving purgation effect of phyllanthus emblica juice, belonging to the field of food and medicine processing.
Background
Fructus Phyllanthi belongs to medicinal and edible varieties, and raw juice thereof is often used as a raw material of fruit juice beverage. Record and display phyllanthus emblica raw productHas strong purgation effect, which results in diarrhea and other symptoms of some people after taking the juice (Saini Rakshandha, shalma Nitin, oladeji Oluwole Solomon, sourirajan Anuradha, dev Kamal, zengin)ElShazly Mohamed,Kumar Vikas.Traditional uses,bioactive composition,pharmacology,and toxicology of Phyllanthus emblica fruits:A comprehensive review.[J].Journal of ethnopharmacology,2021,282.;Bhavesh C.Variya,Anita K.Bakrania,Snehal S.Patel.Emblica officinalis(Amla):A review for its phytochemistry,ethnomedicinal uses and medicinal potentials with respect to molecular mechanisms[J]Pharmacological Research,2016,111; xia Quan, shongbacon, wang Li, kong Jie national pharmaceutical research of traditional drug Phyllanthus emblica [ J]Journal of Chinese traditional medicine, 1997 (09): 3-6+13+62.). The traditional Chinese medicine holds that the phyllanthus emblica crude product is cold and cool, is easy to hurt stomach qi, and is easy to cause diarrhea when taken excessively (Li Saijin, gao Xiaoyu, li Guilan, luo Kailian, bai Zhongbin, hu Yifan, cheng Jun, tian Yang. The phyllanthus emblica water extract has the effect of improving the condition of the loperamide-induced constipation mice [ J]Food research and development 2022,43 (12): 16-21; liang Wenyi, chen Wenjing, wu Lingfang, li Shi, cui Yaping, flag, zhang Lanzhen based on the Phyllanthus emblica Tibetan medicine theory analysis of modern pharmacological research [ J ]]Modern world science and technology-traditional Chinese medicine-2016,18 (07): 1166-1170.). Therefore, when the phyllanthus emblica juice is applied as fruit juice or beverage, corresponding technological measures should be taken to avoid diarrhea in the process of taking the phyllanthus emblica fresh juice and processed products thereof. At present, common processing methods for relieving cold nature are milk processing, salt processing and the like, but the method is generally suitable for dry fruits of phyllanthus emblica. There is no corresponding processing method in the field of food application.
The existing processing method of the phyllanthus emblica juice comprises the following steps:
1. decocting (for medicinal materials): decocting with water for 3 times, the first time for 2h, and the second and third times for 1.5h. Milk dipping: soaking fructus Phyllanthi in milk for 12-24 hr, cleaning, and removing seed. Drying, grinding and sieving with a 40-mesh sieve.
2. Soaking in milk, decocting with water, soaking fructus Phyllanthi in milk for one night, taking out the fructus Phyllanthi the next day, cleaning with clear water, decocting in water until the water is dried, sieving fructus Phyllanthi, drying, grinding, and sieving with 40 mesh sieve. The unprocessed reference medicinal material is obtained by pulverizing the raw medicinal materials directly, and sieving.
3. Adding 200ml of salt water (containing 5g of edible salt) into about 100g of coarse phyllanthus emblica particles, uniformly stirring and absorbing, fully sealing for 12h, spreading on a porcelain plate, immediately placing into a 105 ℃ oven, taking out when the temperature is deviated to +0.5 ℃, cooling, crushing, sieving with a No. 4 sieve, and uniformly mixing for later use.
4. Salting: the salt amount is 3% of the raw material of the phyllanthus emblica, the water adding amount is 1.2 times of the phyllanthus emblica, and the phyllanthus emblica is soaked for 10d. Or the salt is 5% of the raw material of the emblic leafflower fruit, the water adding amount is 2 times of the emblic leafflower fruit, and the processing temperature is 105 ℃.
5. Fresh phyllanthus emblica fruit juice is prepared, cleaning, removing cores, crushing, inactivating enzyme (90 ℃ for 5 min), rough filtering, clarifying (0.02% pectase is treated for 1h at 45 ℃), filtering, centrifuging and obtaining phyllanthus emblica juice.
6. Fructus phyllanthi clear juice: selecting fruits, washing fruits, scalding, removing cores, pulping, performing enzymolysis, leaching, juicing, straining, clarifying, blending, fine filtering, filling, sterilizing and obtaining the phyllanthus emblica juice beverage.
7. Fresh phyllanthus emblica, sorting, cleaning, crushing and stoning, squeezing, rough filtering, centrifugal separation and low-temperature preservation of clear juice
8. The process for composite enzymolysis of phyllanthus emblica juice comprises the following steps: the optimal technological parameters of the pectase-cellulase complex enzyme enzymolysis of the phyllanthus emblica juice are that the mass ratio of the pectase to the cellulase is 1.1:1, the addition amount of the compound enzyme is 0.01%, the enzymolysis time is 2.9h, and the enzymolysis temperature is 55 ℃.
9. Mild phyllanthus emblica treatment: pretreatment, wherein one group of samples is placed in a climatic chamber with the temperature of 38 ℃ and the humidity of 85 percent to 100 percent for heat treatment for 2 days. The cold shock treatment is to immerse the fruits completely in ice water at 0deg.C for 30min, drain and air dry.
Two groups of cold shock, heat shock and normal untreated samples are respectively weighed by 200g, one group of the samples containing the cold shock, the heat shock and the untreated samples are sealed into a test group A by a vacuum packaging machine, the other group of the samples are placed in a plastic basket without being packaged into a test group B, the test group A, B is repeatedly weighed, and the samples are respectively placed in a refrigerator at 4 ℃ and a constant-temperature incubator at 20 ℃ with the humidity of 77%.
10. Composite fermented phyllanthus emblica drink: pulverizing fructus Phyllanthi, extracting in water bath, filtering to obtain fructus Phyllanthi water extract, mixing with cherry tomato juice, homogenizing, sterilizing, and fermenting.
11. High-temperature steam treatment: fresh phyllanthus emblica (with the diameter of about 10-15 mm) is selected and cleaned, then is placed into high-temperature steam with the steam pressure of 0.3MPa for treatment for 130s, taken out, immediately cooled, enucleated, squeezed into juice in a spiral way and filtered centrifugally for standby (marked as D-shaped). Control sample: the fresh phyllanthus emblica fruits are selected and cleaned and then directly spirally taken.
12. And (3) clarifying: fresh fruits are selected for pretreatment of phyllanthus emblica juice, the fresh fruits are cleaned, the seeds are removed, the fresh fruits are soaked in 1% sodium chloride solution at 50 ℃ for 1h, the soaked fruits are cleaned by clear water, the pulp is liquefied by complex enzyme, the juice is obtained through squeezing, and the pre-squeezed juice of phyllanthus emblica is obtained through rough filtration. 1.2.2 clarification method (1) Cold treatment method: cooling changes the colloid properties to form a precipitate. And (3) placing the primary juice at 0-4 ℃ for cooling, precipitating, centrifuging and leaving supernatant. (2) Complex enzymatic method: adding complex enzyme for enzymolysis, centrifuging, and collecting supernatant. (3) gelatin method: adding gelatin, clarifying, centrifuging, and collecting supernatant.
The fructus Phyllanthi pulp or extract prepared by the above process still has diarrhea problem after administration.
Regarding the utilization of phyllanthus emblica pomace, there are also reports in the literature such as: song Guobin comprehensive utilization of fructus Phyllanthi fruit residue, china forestry science institute, discloses extracting dietary fiber from fructus Phyllanthi fruit residue, or preparing into fermentation product. Tan Qingchu, etc., and the preparation process research of the antibacterial leave-on gel of the phyllanthus emblica fruit residue, the world traditional Chinese medicine, the volume 17 and the phase 19 of 10 months in 2022, discloses that the phyllanthus emblica fruit residue is prepared into the antibacterial leave-on gel. At present, no report of the effect of relieving the purgation of the natural juice of the phyllanthus emblica by utilizing the phyllanthus emblica fruit residues exists.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for preparing raw juice for relieving the purgation effect of the natural juice of phyllanthus emblica.
The invention provides a method for preparing primary pulp for relieving purgation of phyllanthus emblica juice, which comprises the following steps:
a. taking fresh phyllanthus emblica fruits after de-enzyming, and squeezing to obtain pomace and fresh juice;
b. decocting the fruit residue with water, cooling, centrifuging, and collecting supernatant;
c. and d, mixing the fresh juice prepared in the step a with the supernatant prepared in the step b to obtain the phyllanthus emblica primary pulp.
The water-removing method in the step a adopts microwave water-removing, and the microwave water-removing method comprises the following steps: heating fresh fructus Phyllanthi at 4kw and 48deg.C for 15min.
And b, adding water into the pomace obtained in the step b for reflux decoction, wherein the decoction temperature is 100 ℃, the decoction time is 20-40min, and the centrifugation speed is 3000-5000 r/min after cooling.
Further preferably, the pomace obtained in the step b is added with water for reflux decoction, the decoction temperature is 100 ℃, the pH value is 1.7, the decoction time is 80 minutes, and the extraction liquid-to-liquid ratio is 1:25, centrifuging after cooling, wherein the centrifuging speed is 4500r/min.
And c, mixing the fresh juice and the supernatant fluid, and then heating and decocting by microwaves, and carrying out microwave vacuum reflux heating at 50 ℃ and minus 0.076MPa to obtain the phyllanthus emblica primary pulp.
And c, mixing the fresh juice obtained in the step with the supernatant, and carrying out reflux decoction at 100 ℃ for 15min to obtain the phyllanthus emblica primary pulp.
And c, mixing the fresh juice obtained in the step with the supernatant, sterilizing, fermenting, inoculating 0.5% (1×107 cfu/mL) of activated lactobacillus into the sterilized combined juice, and fermenting at 40 ℃ for 48 hours to obtain phyllanthus emblica primary pulp.
The invention also provides a method for detecting the quality of the primary pulp in the preparation process, which is to control the quality of the primary pulp by detecting the content of pectin extracted from the pomace in the step b.
Wherein the pectin content in the supernatant is not less than 10% w/w.
The monosaccharide in the pectin comprises arabinose, galactose, glucose, xylose and galacturonic acid; the weight average molecular weight (Mw) was 539238, the number average molecular weight (Mn) was 269797, and the peak molecular weight (Mp) was 384163; the ion chromatogram of pectin is shown in figure 9.
The beneficial effects of the invention are as follows: the problem of diarrhea caused by taking fresh phyllanthus emblica juice is relieved by utilizing phyllanthus emblica pomace, the pomace resources after juicing are fully utilized, relevant quality control indexes are formed, and the prepared phyllanthus emblica pulp is safe to use, stable in quality and controllable.
Drawings
FIG. 1 effect of feed to pectin extraction
FIG. 2 influence of extraction time on pectin extraction
FIG. 3 influence of ethanol consumption on pectin extraction
FIG. 4 influence of pH dose on pectin extraction
FIG. 5 influence of extraction temperature on pectin extraction
FIG. 6 response surface graph ((A) heating time and feed liquid ratio, (B) heating time and ethanol amount, (C) influence of feed liquid ratio and ethanol amount on synergistic extraction rate of phyllanthus emblica pectin and polyphenol)
FIG. 7 molecular weight map of Emblica officinalis
FIG. 8 is a mixed-label ion chromatogram
FIG. 9 ion chromatography of Phyllanthus emblica pectin
FIG. 10 pectin infrared spectrum
FIG. 11 pectin SEM image
Detailed Description
EXAMPLE 1 preparation of the phyllanthus emblica puree of the invention (microwave method)
Step 1: selecting and cleaning phyllanthus emblica: selecting mature high-quality phyllanthus emblica fruits, and cleaning;
step 2: microwave de-enzyming: heating fresh fructus Phyllanthi at 4kw and 48 deg.C for 15min.
Step 3: squeezing fructus Phyllanthi, recovering fruit residue, adding 12 times of water, decocting under reflux (100deg.C) for 30min, cooling, centrifuging (4000 r/min), collecting supernatant, and mixing with fresh juice.
Step 4: microwave heating and decocting: and (5) carrying out microwave vacuum reflux heating at 50-0.076 MPa.
Quality control index: the pectin content is not less than 10%.
EXAMPLE 2 preparation of the phyllanthus emblica puree of the invention (cooking method)
Step 1: selecting and cleaning phyllanthus emblica: selecting mature high-quality phyllanthus emblica fruits, and cleaning;
step 2: microwave de-enzyming: heating fresh fructus Phyllanthi at 4kw and 48 deg.C for 15min.
Step 3: squeezing fructus Phyllanthi, recovering fruit residue, decocting with 12 times of water under reflux (100deg.C) for 30min, cooling, centrifuging (4000 r/min), collecting supernatant, and mixing with the juice.
Step 4: heating the mixed juice of the phyllanthus emblica obtained in the step 4 (reflux-decocting at 100 ℃ for 15 min) to obtain phyllanthus emblica primary pulp.
EXAMPLE 3 preparation of the phyllanthus emblica puree of the invention (fermentation method)
Step 1: selecting and cleaning phyllanthus emblica: selecting mature high-quality phyllanthus emblica fruits, and cleaning;
step 2: microwave de-enzyming: heating fresh fructus Phyllanthi at 4kw and 48 deg.C for 15min.
Step 3: squeezing fructus Phyllanthi, recovering fruit residue, decocting with 12 times of water under reflux (100deg.C) for 30min, cooling, centrifuging (4000 r/min), collecting supernatant, mixing with fresh juice, sterilizing, and fermenting.
Step 4: inoculating activated lactobacillus 0.5% (1×107 cfu/mL) into sterilized mixed juice, fermenting at 40deg.C for 48 hr to obtain fructus Phyllanthi stock.
Example 4 preparation Process screening test of Emblica officinalis puree of the present invention and method for detecting and controlling quality in the preparation Process of the puree
1 instrument and reagents
1.1 monomer samples
Residue of fructus Phyllanthi (purchased from the lotus pool medicinal material market) residue after squeezing fresh fruit is dried, crushed and sieved with a No. three sieve.
1.2 instruments
TABLE 1 Main instruments
1.3 reagents
TABLE 2 Main reagents
2 Experimental methods
2.1 pectin extraction and content determination
Extracting pectin by adopting a heating reflux extraction-alcohol precipitation method. The technical route is as follows: precisely weighing 2g of phyllanthus emblica residue powder, adding a certain volume of solvent (RO water), regulating pH to 1.7 by using hydrochloric acid, weighing, reflux-extracting for a certain time on an electrothermal sleeve, naturally cooling, and then supplementing the weight to the previous weight. Centrifuging at 4500r for 15min, and separating fructus Phyllanthi residue powder and pectin-containing supernatant.
Pectin component separation and calculation: ethanol (95%) was added at a certain ratio v/v to precipitate a liquid phase, which was centrifuged at 4500r for 15min at high speed, and the lower precipitate was collected. And volatilizing residual ethanol from the precipitate in a vacuum drying oven, and drying overnight in a freeze drying oven to obtain the dry pectin.
And weighing pectin, dividing the pectin by the weight of the residue, and calculating the pectin extraction rate.
2.2 Single factor experiment
The pre-experiment shows that the polyphenol content in the phyllanthus emblica pomace is far lower than the pectin content, the pectin extraction rate is five times or more than that of the polyphenol, and the weight is large, so that the single factor experiment only uses pectin as a response to ensure that the extraction rate is large.
According to a single factor experiment method, setting the influence factors as extraction time (30, 50, 70, 90, 120 min), feed-liquid ratio (30, 50, 70, 90, 110 g/ml), ethanol amount (1:1, 1:2, 1:3, 1:4, 1:5 ml/ml), pH value (1.5, 2, 2.5, 3, 3.5), and extraction temperature (60, 70, 80, 90, 100 ℃) to respectively examine the influence of the five factors on the pectin extraction rate in the phyllanthus emblica fruit residues, determining the optimal range of each factor, adopting a response surface analysis method to design subsequent experiments and establish a model, and obtaining the optimal extraction condition.
2.3 response surface optimization test scheme
Based on the single factor experimental result, the fixed pH is 1.7, the reflux heating temperature is 100 ℃, the Box-Behnken experimental design is adopted, and the A value represents the reflux heating extraction time, the B value represents the feed liquid ratio and the C value represents the ethanol consumption as response to the influence of pectin extraction rate. The response value is the comprehensive score of the pectin extraction rate, and the calculation is carried out by adopting a weight method, and the formula (2) is as follows:
in the formula (2), R 1 For pectin extraction rate, R 2 The extraction rate of the polyphenol is obtained.
The test factors and horizontal codes are shown in table 3. Both calculation and graphics were performed by design experert 8.0 software.
Table 3 response surface experimental factor level coding
2.4 physicochemical Properties of Phyllanthus emblica pectin
2.4.1 molecular weight determination of Phyllanthus emblica pectin
And (3) preparation of a reagent: precisely preparing 0.05M NaCl solution, filtering with 0.45 μm filter membrane, ultrasonic treating for 10min, and preserving under RT condition
Sample and standard solution preparation: the sample and standard were precisely weighed, the sample was prepared as a 5mg/ml solution, centrifuged at 12000rpm for 10min, the supernatant was filtered through a 0.22 μm microporous filter membrane, and the sample was transferred to a 1.8ml sample-in vial.
The chromatographic method comprises the following steps: chromatographic column: BRT105-104-102 series gel column (8X 300 mm); mobile phase: 0.05M NaCl solution; flow rate: 0.6ml/min, column temperature: 40 ℃; sample injection amount: 20 μl; a detector: differential detector RID-10A.
2.4.2 Phyllanthus emblica pectin monosaccharide composition
And (3) preparation of a reagent: 2.4g of 50% NaOH solution is dissolved in 2L of water, 15mM NaOH solution is prepared precisely, and the mixture is preserved under RT conditions; 1.2g of 50% NaOH solution, 8.2mg of NaOAC was dissolved in 1L of water, and 15mMNaOH&100mM NaOAC solution was prepared precisely and stored at RT.
Sample preparation: 10mg of the sample was precisely weighed into an ampoule, 10ml of 3M TFA was added and hydrolyzed at 120℃for 3 hours. Accurately sucking the acid hydrolysis solution, transferring to a pipe, blowing with nitrogen, drying, adding 5ml of water, mixing, sucking 100uL, adding 900uL of deionized water, and centrifuging at 12000rpm for 5min. The supernatant was analyzed by IC.
The preparation and calculation method of the standard substance solution comprises the following steps: 16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid, mannuronic acid) were prepared to about 10mg/ml standard solution. Standard solutions of monosaccharides were precisely prepared to obtain 0.01, 0.1, 0.5, 1, 5, 10 and 20mg/L gradient concentration standards as standards 1 to 7. The mass of the different monosaccharides was determined according to the absolute quantitative method and the molar ratio was calculated on the basis of the molar mass of the monosaccharides, the results being given in Table 4.
The chromatographic method comprises the following steps: chromatographic column: dionexCarbopacTMPA20 (3×150); mobile phase: AH 2 O;B:250mMNaOHC:50mMNaOH&500mM NaOAC; flow rate: 0.3ml/min; sample injection amount: 5. Mu.L; column temperature: 30 ℃; a detector: an electrochemical detector.
Linear relationship:
TABLE 4 Standard Linear relationship
2.4.3 analysis of the functional group Structure of Phyllanthus emblica pectin
Precisely weighing 2mg of sample and 200mg of potassium bromide, pressing into tablets, and tabletting the blank control by adopting potassium bromide powder. Respectively placing the images on a Fourier transform infrared spectrometer FT-IR650 for scanning and recording.
2.4.4 tissue Structure analysis of Phyllanthus emblica pectin
And sticking a proper amount of phyllanthus emblica pectin sample on a sample platform. And observing morphological characteristics and tissue structures of the sample under an acceleration voltage of 5KV under an electron microscope, and photographing.
3 results and discussion
3.1 Single factor experimental analysis
3.1.1 Effect of feed to pectin extraction Rate
According to the description in 2.1, the heating reflux time is fixed for 70min, the ethanol consumption is 1:2.5, the pH is 2, the heating temperature is 100 ℃, and the influence of different feed liquid ratios on the pectin extraction rate is studied. As can be seen from fig. 1, the pectin extraction rate initially increases with increasing feed to liquid ratio, when the feed to liquid ratio is 1: the highest concentration is reached at 50g/ml, because the ratio of feed to liquid is increased, which is favorable for hydrolyzing pectin into pectin and increasing the pectin concentration in the solution. The pectin extraction rate is reduced along with the increase of the feed-liquid ratio, and the extraction rate of pectin is obviously reduced due to the fact that the solvent volume is too large, the dissolution of non-pectin substances is promoted, and the subsequent steps of alcohol precipitation, centrifugation and the like are affected by the increase of the whole volume. Thus 25, 30, 35 are chosen for optimization.
3.1.2 Effect of extraction time on pectin extraction Rate
According to the description in 2.1, the fixed feed-to-liquid ratio is 1:2.5, the ethanol consumption is 1:2.5, the pH is 2, the heating temperature is 100 ℃, and the influence of different extraction times on the pectin extraction rate is studied. As can be seen from fig. 2, when the extraction time was increased from 30 to 50 minutes, the pectin extraction rate of phyllanthus emblica was low and did not increase significantly, when the extraction time was increased from 50 to 90 minutes, the pectin extraction rate was increased significantly and reached the maximum, and when the extraction time was over 90 minutes, the pectin extraction rate was decreased. Therefore, when the extraction time is increased within a certain range, the solubility and the diffusion coefficient of pectin can be increased, and the extraction rate of the phyllanthus emblica pectin can be improved. However, when the extraction time is too long, or when the pectin molecules are damaged or degraded by long-time heating, the quality of the pectin is lowered, and the extraction rate tends to be lowered. Thus 60, 70, 80min optimization was chosen.
3.1.3 Effect of ethanol amount on pectin extraction yield
According to the method described in 2.1, the heating time is fixed for 70min, the feed-liquid ratio is 1:2.5, the pH is 2, the heating temperature is 100 ℃, and the influence of different ethanol consumption on pectin extraction rate is studied. As can be seen from fig. 3, the pectin extraction rate reaches the highest at a volume of ethanol of 1:2, which is lower than 1:2, pectin precipitation is incomplete, and the extraction rate is obviously reduced. When the volume of ethanol is higher than 1:2, other components except pectin are precipitated, which can interfere with precipitation separation of pectin to reduce pectin extraction.
3.1.4 influence of pH on pectin extraction
According to the description in 2.1, the heating time is fixed for 70min, the ethanol consumption is 1:2.5, the feed-liquid ratio is 1:2.5, the heating temperature is 100 ℃, and the influence of different pH values on the pectin extraction rate is studied. As can be seen from the analysis in FIG. 4, the pH reached the highest at 1.5-2, and above 2, the pH dropped sharply and the extraction rate was extremely low, so the fixed pH was 1.7.
3.1.5 Effect of extraction temperature on pectin extraction Rate
According to the description in 2.1, the heating time is fixed for 70min, the ethanol consumption is 1:2.5, the feed-liquid ratio is 1:2.5, the pH is 2, and the influence of different extraction temperatures on the pectin extraction rate is studied. As can be seen from fig. 5, the extraction temperature is increased, and the pectin extraction rate is significantly increased, so that the extraction temperature is fixed at 100 ℃.
3.2 response surface collaborative optimization and statistical analysis
The research adopts Box-Behnken experimental design, and optimizes the extraction conditions of pectin and polyphenol in phyllanthus emblica by taking the extraction rate of pectin and the synergistic extraction rate of polyphenol as output under three input conditions of feed-liquid ratio, extraction time and ethanol consumption. The design and experimental results are shown in table 5. The extraction rates of pectin and polyphenol are respectively 11.45-14.03% and 1.62-2.53%, and the synergistic extraction rate of pectin and polyphenol is 11.44557% -14.03149% according to 2.3. Regression analysis was performed on the 15 experimental point impact values using the Design-Expert 8.0.6 program, with pectin extraction and polyphenol extraction as impact values, and the statistical model of the suggested pectin and polyphenol extraction was as follows:
synergistic extraction rate
=+13.36+0.3197A-0.0895B-0.1334C-0.4103AB+0.2516AC+0.1292BC-0.0332A2-0.0693B2-1.04C2
Analysis of variance was performed on the regression equation, and the results are shown in Table 6. Table 6 shows that the primary term factor (A, C) has significant influence on the results by the interactive terms (AB, AC) and the secondary term (C2) (P is less than or equal to 0.05). While the primary term factor (B) interacts with the term (BC) and the secondary term (A2, C2) has significantly less effect on the result (P > 0.05).
TABLE 5 response surface analysis protocol and experimental results
TABLE 6 regression model analysis of variance
The experiment takes the synergistic extraction rate of pectin and polyphenol as an influence value, designs an influence analysis experiment of 15 experimental points of three factors and three levels, and repeats the zero point experiment for 3 times to judge the experimental error.
Fixed parameters: precisely weighing 2g of powder, extracting at pH1.7, heating at 100deg.C as fixed parameter, and keeping the rest in table.
The optimal technological parameters obtained by combining the mathematical analysis of the regression model are that the feed-liquid ratio is 1:25, the extraction time is 80min, and the ethanol consumption is 1:1.2. The theoretical value of the synergistic extraction under this optimal process condition is 14.0735%. In order to further test the reliability of the experimental method, the optimal extraction condition is adopted for extraction, and the actual measured synergistic extraction rate of phyllanthus emblica fruits is 14.42%, and the relative error is about 1.62%. Therefore, the extraction condition parameters obtained by optimization of the response surface analysis method can be considered to be reliable, and the method has reference value.
3.3 analysis of any two factor interactions
The relationship between the synergistic extraction rate of phyllanthus emblica pectin and polyphenol and the response values can be better understood by using FIGS. 6 (A-C). Fig. 6A, B, C respectively fixes the level of zero for 1 variable, and the response varies with the other two variables. The contour line density represents the obvious change of the response value, the ellipse represents the optimal proportion of two factors with obvious effect on the dependent variable, and the dot with red middle is simulated. Fig. 6 and A, B, C all conform to the analysis of variance results of table 6, and for the extraction yield of pectin and polyphenol, the heating time is significant with the feed-liquid ratio and the ethanol amount, respectively, and the interaction between the feed-liquid ratio and the ethanol amount is insignificant.
3.4 pectin molecular weight
By using the retention time of dextran series of known molecular weight on BRT105-104-102 tandem gel column, a standard curve was made to give lgMp-RT (peak molecular weight), lgMw-RT (weight average molecular weight), lgMn-RT (number average molecular weight) calibration curve equation:
lgMp-RT correction curve equation: y= -0.195x+12.375r2= 0.9913
lgMw-RT correction curve equation: y= -0.2078x+12.968r2=0.993
lgMn-RT correction curve equation: y= -0.181x+11.734r2= 0.9972
The spectrum of the phyllanthus emblica pectin passing through the gel column is shown in fig. 7, and the peak of the mobile phase is about 47.1 min. Rt= 34.823min, mp= 384163, mw= 539238, mn= 269797 are calculated according to the calculation formula.
3.5 analysis of monosaccharide composition results
The ion chromatogram obtained by mixing the standard substances is shown in figure 8. (solvent peak: 2.0min is the peak of sodium hydroxide, 40min is the peak of sodium acetate). The ion chromatogram of phyllanthus emblica pectin is shown in figure 9.
The analysis and calculation are carried out according to the method of 2.4.2, and the composition of the phyllanthus emblica pectin molecular monosaccharide is shown in table 7.
TABLE 1 molecular composition of Emblica officinalis pectin
3.6 analysis of Infrared Spectroscopy results
The infrared results of pectin samples of polysaccharide fraction are shown in figure 10. The absorption band is a telescopic vibration absorption peak of-OH at 3600-3200cm-1, and the absorption peak in this region is a characteristic peak of saccharides. The method comprises the following steps: 3417cm-1 is the stretching vibration absorption peak of O-H, and is a characteristic peak of saccharide. There was an absorption peak at 2927cm-1, which was attributed to C-H stretching vibration. There was an absorption peak at 1739cm-1, ascribed to C=O stretching vibration. The absorption peak at 1627cm-1 may be attributed to water of crystallization. Absorption peaks at 1440cm-1, 1147cm-1 and 1101cm-1 are attributed to C-O stretching vibration. Absorption peaks at 1367cm-1 and 1328cm-1 are attributed to C=O symmetrical telescopic vibration. Absorption peaks at 1236cm-1 and 1049cm-1 and 1018cm-1 were attributed to O-H angular vibration. There was an absorption peak at 971cm-1, which was attributed to the rolling vibration of the terminal methine group of the pyran ring. There was an absorption peak at 917cm-1, and the asymmetric ring was ascribed to stretching vibration of the pyran ring.
3.7 pectin electron microscope tissue structure
The pectin electron microscope image is shown in figure 11, and the phyllanthus emblica pectin has rough surface and more crystalline particles. No obvious cracks and pore structures, and shows that the pectin tissue structure is relatively complete.
The extraction rate of pectin in waste phyllanthus emblica pomace extracted by an acid extraction and alcohol precipitation method is improved through response surface optimization, the optimized condition is that the feed-liquid ratio is 1:25, the extraction time is 80min, the ethanol consumption is 1:1.2, and the extraction is carried out according to the optimal parameters, so that the extraction rate is 14.42%. The obtained phyllanthus emblica pectin is light yellow and transparent. The infrared spectrum of the sample showed a characteristic absorption peak with saccharides, indicating that it is indeed a pectic component. And the molecular weight of the phyllanthus emblica pectin is not high, which is favorable for dissolution and absorption, so that the phyllanthus emblica pectin has greater activity potential. The pectin contains arabinose, galactose, glucose, xylose and galacturonic acid as main monosaccharide components. And further observing the pectin structure of the phyllanthus emblica under an electron microscope, and finding that the tissue structure of the phyllanthus emblica is relatively good. In summary, since the waste phyllanthus emblica pomace accounts for about 65% of the total weight of fresh fruits, further research on pectin, polyphenol and other substances in the pomace is one of important preconditions for recycling the waste phyllanthus emblica pomace. As one of the main components in the waste pomace, a more clear and effective direction is provided for recycling the waste pomace of the emblic leafflower fruit.
The beneficial effects of the invention are demonstrated by efficacy tests below.
Test example 1 evaluation of diarrhea Effect of Emblica officinalis puree of the present invention
(1) Experimental method
1.1 formulation of loperamide
400mg of loperamide is accurately weighed and dissolved in 50mL of 0.9% physiological saline to make the final concentration of the loperamide 8mg/mL for later use.
1.2 preparation of Cannabis pellet suspension
The hemp seed pill (6 g/pill) is dissolved in 0.9 percent physiological saline to a final concentration of 60mg/mL for standby.
1.3. Grouping and administration of animals
The loperamide-induced constipation model is mainly developed by inhibiting intestinal motility, affecting intestinal water metabolism and the enteric nervous system. The experiment set a blank control group, a model control group and a positive control group (n=12), while setting a 600 mg/(kg·d) phyllanthus emblica juice dose group (n=12). Gastric lavage of the blank group is performed with 0.9% physiological saline; the model control group irrigates 8 mg/(kg.d) of loperamide; gastric lavage 900 mg/(kg.d) of the fructus cannabis pill suspension of the positive control group; the administration group was respectively perfused with 8 mg/(kg.d) of the test substance and loperamide at the corresponding concentrations, and all samples were prepared to the desired concentrations using 0.9% physiological saline. The stomach was orally irrigated 1 time a day for 7 days, and the amount of the stomach was 0.125mL/10g bw.
1.4 stool test
And (3) carrying out a mouse defecation experiment according to the health food function evaluation guiding principle. At night on day 6 of subject intervention, the mice of each group were not deprived of water. After 12h, each dose group was perfused with gastric saline and the corresponding test subjects, and after 1h, each group of mice was perfused with ink and timing was started. Placing mice after stomach filling in independent cages, providing sufficient water source, recording black stool discharge time of each mouse, collecting feces discharged from each mouse within 6 hr, and counting the number of grains and total mass of feces
(2) Experimental results
By comparing the 6-hour defecation amount of different treatment groups with that of a control group, compared with a blank group, the defecation amount of mice can be obviously increased by microwave processing of the emblic juice, cooking of the emblic juice and fermentation of the emblic juice. However, the processed phyllanthus emblica juice group has significantly lower defecation (P < 0.01) than the phyllanthus emblica fresh juice. Wherein, the defecation amount of the fermentation processing phyllanthus emblica juice has the most obvious effect, the average defecation amount is 412mg, which is obviously higher than the average defecation amount (P < 0.05) of a blank group; the defecation amount of the microwave processed emblic juice and the cooking processed emblic juice is lower than that of the control group (P < 0.01). Meanwhile, the defecation amount of the hemp seed pill group is also obviously higher than that of the control group, and the difference is extremely obvious (P < 0.01), which is a positive control group. Overall, the defecation of the processed phyllanthus emblica juice set is significantly lower. Therefore, the conclusion is that the purgation effect of the processed phyllanthus emblica juice is relieved compared with that of the raw juice, and the steaming processing effect is better.
TABLE 8 defecation conditions of mice of each group after feeding the drugs
Note that: p <0.05 compared to control group; * P <0.01
The experimental results show that the weight of the mice in the three plasmogen groups is more normal than that of the mice in the raw group, the feeding amount is larger, and the defecation condition is more normal. The method shows that the raw juice phyllanthus emblica prepared by three processing methods of steaming, microwave and fermentation can obviously improve diarrhea symptoms of mice, wherein the effect of the phyllanthus emblica processed by the steaming method is optimal.
Claims (3)
1. A method for preparing raw pulp for relieving purgation effect of phyllanthus emblica raw juice is characterized by comprising the following steps: it comprises the following steps:
a. taking fresh phyllanthus emblica fruits after de-enzyming, and squeezing to obtain pomace and fresh juice;
b. decocting the fruit residue with water, cooling, centrifuging, and collecting supernatant;
c. mixing the fresh juice prepared in the step a with the supernatant prepared in the step b to obtain phyllanthus emblica primary pulp;
the water-removing method in the step a adopts microwave water-removing, and the microwave water-removing method comprises the following steps: heating fresh fructus Phyllanthi at 4kw and 48deg.C for 15min;
b, adding water into the pomace for reflux decoction, wherein the decoction temperature is 60-100 ℃, the decoction time is 60-80min, cooling and centrifuging, and the centrifuging speed is 3000-5000 r/min;
c, mixing the fresh juice obtained in the step C with the supernatant, and performing microwave heating decoction, and performing microwave vacuum reflux heating at 50 ℃ and minus 0.076MPa to obtain phyllanthus emblica primary pulp; or (b)
c, mixing the fresh juice obtained in the step c with the supernatant, and carrying out reflux decoction at 100 ℃ for 15min to obtain phyllanthus emblica primary pulp; or (b)
c, mixing the fresh juice obtained in the step C with the supernatant, sterilizing, fermenting, inoculating activated 0.5% lactobacillus with concentration of 1×107cfu/mL into the sterilized combined juice, and fermenting at 40 ℃ for 48 hours to obtain phyllanthus emblica primary pulp.
2. The method of manufacturing according to claim 1, characterized in that: b, adding water into the pomace for reflux decoction, wherein the decoction temperature is 100 ℃, the pH value is 1.7, the decoction time is 80min, and the extraction liquid-to-liquid ratio is 1:25, centrifuging after cooling, wherein the centrifuging speed is 4500r/min.
3. A method for determining the pectin content of a pulp as claimed in claim 1 or 2, characterized in that: measuring the pectin content in the pomace of the step b; the method for measuring the pectin content comprises the following steps:
a. extracting pectin by adopting a heating reflux extraction-alcohol precipitation method: precisely weighing 2g of phyllanthus emblica residue powder, adding a certain volume of solvent RO water, adjusting pH to 1.7 by using hydrochloric acid, carrying out reflux extraction on an electric heating sleeve after weighing, and supplementing the weight to the previous weight after natural cooling; centrifuging at 4500r for 15min, and separating fructus Phyllanthi residue powder and pectin-containing supernatant;
b. pectin component separation and calculation: adding 95% ethanol to precipitate liquid phase, centrifuging at 4500r for 15min, and collecting lower precipitate; volatilizing residual ethanol from the precipitate in a vacuum drying oven, and drying overnight in a freeze drying oven to obtain dry pectin;
c. weighing pectin, dividing by the weight of the residue, and calculating to obtain pectin extraction rate; the pectin content in the supernatant is not lower than 10% w/w; the monosaccharide in the pectin comprises arabinose, galactose, glucose, xylose and galacturonic acid; the weight average molecular weight Mw was 539238, the number average molecular weight Mn was 269797, and the peak molecular weight Mp was 384163.
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CN101617844A (en) * | 2008-06-30 | 2010-01-06 | 昆明格林斯通生物工程有限公司 | Fructus phyllanthi fruit juice beverage and preparation method thereof |
CN104592416A (en) * | 2015-01-04 | 2015-05-06 | 砀山海升果胶有限责任公司 | High-quality natural pectin extraction technology |
CN110214878A (en) * | 2019-07-05 | 2019-09-10 | 深圳市永沣香料有限公司 | Without astringent sense emblic composite beverage preparation method |
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CN101617844A (en) * | 2008-06-30 | 2010-01-06 | 昆明格林斯通生物工程有限公司 | Fructus phyllanthi fruit juice beverage and preparation method thereof |
CN104592416A (en) * | 2015-01-04 | 2015-05-06 | 砀山海升果胶有限责任公司 | High-quality natural pectin extraction technology |
CN110214878A (en) * | 2019-07-05 | 2019-09-10 | 深圳市永沣香料有限公司 | Without astringent sense emblic composite beverage preparation method |
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