Preparation method of coffee pericarp source pectin
By combining ultrasonic treatment and enzymatic hydrolysis with alcohol precipitation and dialysis, high-RG-I type pectin was prepared, which solved the problem of the easily damaged structure of RG-I type pectin and achieved high-efficiency pectin extraction and improved economic benefits.
Patent Information
- Application Number
- CN202511190243.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-25
- Publication Date
- 2025-11-14
AI Technical Summary
In the preparation of pectin using existing technologies, the structure of RG-I type pectin is easily damaged, resulting in insufficient utilization of its biological activity. Furthermore, the overall utilization rate of coffee pericarps is low, and the added value is not effectively improved.
High RG-I type pectin was prepared by using coffee pericarps as raw material, which were then treated with ultrasound and mixed with vitamin C. The pH value was adjusted and cellulase, xylanase and mannanase were added for enzymatic hydrolysis. Combined with alcohol precipitation and dialysis, high RG-I type pectin was prepared.
It improves the yield of pectin and the proportion of RG-I type pectin, achieving efficient preparation and economic benefits of pectin, facilitating industrial production, and enhancing the utilization value of coffee pericarps.
Abstract
Description
Technical Field
[0001] This invention relates to the field of pectin extraction technology, and more particularly to a method for preparing coffee pericarp-derived pectin. Background Technology
[0002] Pectin is a complex acidic heteropolysaccharide, primarily comprising three domains: homogalacturon (HG), rhamnogalacturonan-I (RG-I), and rhamnogalacturonan-II (RG-II). In commercial production, to obtain pectin of consistent quality, producers typically treat raw materials under high-temperature, high-acid extraction conditions. The resulting commercial pectin is usually dominated by the HG domain, while the RG-I domain is disrupted under these conditions. However, recent studies have shown that RG-I pectin possesses superior in vitro probiotic, immunomodulatory, anti-cancer metastasis, and chronic metabolic disease regulation bioactivities.
[0003] Cascara, meaning "fruit skin" in Spanish, refers to the outer skin of the coffee cherry that wraps around coffee beans. It is a byproduct of coffee bean processing, rich in pectin, accounting for 5-20% of its dry weight, but its comprehensive development and utilization still have significant limitations. Using Cascara as a raw material to prepare pectin polysaccharides has low production costs, facilitates the full utilization of coffee fruit skin, increases its added value, and enables green and efficient production of pectin polysaccharides, making it easy to industrialize and improve economic benefits. Therefore, it is necessary to propose a method for preparing pectin with high RG-I content using Cascara as a raw material. Summary of the Invention
[0004] In view of this, the present invention provides a method for preparing coffee pericarp-derived pectin polysaccharide, thereby solving the above-mentioned problems.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] This invention provides a method for preparing coffee pericarp pectin, comprising the following steps:
[0007] S1. Mix coffee husk powder, vitamin C and water, and sonicate to obtain solution 1; the mass-to-volume ratio of coffee husk powder to water is 1g:18-22mL, and the concentration of vitamin C in solution 1 is 10-16mmol / L;
[0008] S2. Adjust the pH of solution 1 to 4.8-5.0, then add cellulase, xylanase and mannanase, and enzymatically hydrolyze at 43-47℃ for 85-95 min to inactivate the enzymes and obtain solution 2;
[0009] S3. Centrifuge solution 2 and collect the supernatant;
[0010] S4. The supernatant is concentrated by rotary evaporation to 30-36% of its original volume, ethanol is added for alcohol precipitation, and after standing, it is centrifuged to obtain crude pectin.
[0011] S5. Remove the protein from the crude pectin, dialyze, and freeze-dry to obtain coffee pericarp pectin.
[0012] Preferably, during enzymatic hydrolysis, the amount of cellulase used is 70-80 U / g coffee fruit peel powder, the amount of xylanase used is 40-50 U / g coffee fruit peel powder, and the amount of mannanase used is 55-65 U / g coffee fruit peel powder.
[0013] Preferably, the enzymatic hydrolysis is accompanied by stirring, and the stirring speed is 140-160 rpm.
[0014] Preferably, the power of the ultrasound is 160-200W and the duration of the ultrasound is 14-16 minutes.
[0015] Preferably, the rotary evaporation concentration temperature is 60-70°C, and the rotary evaporation concentration speed is 50-70 rpm.
[0016] Preferably, the volume ratio of the concentrated filtrate to ethanol is 1:1 to 1.2.
[0017] Preferably, the volume fraction of ethanol is 90-95%, the temperature of alcohol precipitation is 3-5°C, and the standing time is 8-10 hours.
[0018] Preferably, the centrifugation speed in S3 is 3000-4000 rpm and the centrifugation time is 8-12 min; the centrifugation speed in S4 is 4500-5500 rpm and the centrifugation time is 13-17 min.
[0019] Preferably, the molecular weight cutoff of the dialysis bag is 8–14 kDa.
[0020] The present invention also provides coffee pericarp pectin obtained according to the preparation method described above.
[0021] By adopting the above technical solution, the present invention has the following beneficial effects:
[0022] 1. This invention adds vitamin C during enzymatic hydrolysis, which promotes pectin dissolution while preventing the degradation of RG-I type pectin. The synergistic effect of cellulase, xylanase, and mannanase increases the pectin yield and the proportion of RG-I type pectin. Using the method of this invention, the pectin yield is high, reaching 12.5%, and the proportion of RG-I type pectin exceeds 60%.
[0023] 2. The production cost of pectin polysaccharide prepared from coffee pericarp (Cascara) is low, which is conducive to making full use of coffee pericarp, increasing its added value, realizing green production and efficient preparation of pectin polysaccharide, and making it easy to realize industrial production and improve economic benefits. Detailed Implementation
[0024] This invention provides a method for preparing coffee pericarp pectin, comprising the following steps:
[0025] S1. Mix coffee husk powder, vitamin C and water, and sonicate to obtain solution 1; the mass-to-volume ratio of coffee husk powder to water is 1g:18-22mL, and the concentration of vitamin C in solution 1 is 10-16mmol / L;
[0026] S2. Adjust the pH of solution 1 to 4.8-5.0, then add cellulase, xylanase and mannanase, and enzymatically hydrolyze at 43-47℃ for 85-95 min to inactivate the enzymes and obtain solution 2;
[0027] S3. Centrifuge solution 2 and collect the supernatant;
[0028] S4. The supernatant is concentrated by rotary evaporation to 30-36% of its original volume, ethanol is added for alcohol precipitation, and after standing, it is centrifuged to obtain crude pectin.
[0029] S5. Remove the protein from the crude pectin, dialyze, and freeze-dry to obtain coffee pericarp pectin.
[0030] This invention involves mixing coffee fruit peel powder, vitamin C, and water, followed by ultrasonication to obtain solution 1. The mass-to-volume ratio of coffee fruit peel powder to water is 1g:18-22mL, preferably 1g:19-21mL, and more preferably 1g:20mL. The concentration of vitamin C in solution 1 is 10-16mmol / L, preferably 12-14mmol / L, and more preferably 13mmol / L. The ultrasonic power is 160-200W, preferably 170-190W, and more preferably 180W. The ultrasonic time is 14-16min, preferably 15min. The addition of vitamin C can promote the dissolution of pectin polysaccharides while preventing the degradation of RG-I type polysaccharides. The purpose of ultrasonication is to disrupt the structure of plant cell walls, making it easier for enzymes to contact pectin and improving enzymatic hydrolysis efficiency.
[0031] Then, adjust the pH of solution 1 to 4.8–5.0, more preferably 4.9; add cellulase, xylanase, and mannanase, adjust the pH, and perform enzymatic hydrolysis, then inactivate the enzymes to obtain solution 2; the amount of cellulase used is 70–80 U / g coffee fruit peel powder, preferably 73–78 U / g coffee fruit peel powder, more preferably 75 U / g coffee fruit peel powder; the amount of xylanase used is 40–50 U / g coffee fruit peel powder, preferably 42–48 U / g coffee fruit peel powder, more preferably 45 U / g coffee fruit peel powder; the amount of mannanase used is 55–65 U / g coffee fruit peel powder, preferably 57–62 U / g coffee fruit peel powder, more preferably 60 U / g Coffee fruit peel powder; the enzymatic hydrolysis temperature is 43-47℃, preferably 44-46℃, more preferably 45℃; the enzymatic hydrolysis time is 85-95 min, preferably 87-92 min, more preferably 90 min; the enzymatic hydrolysis is accompanied by stirring at a speed of 140-160 rpm, preferably 145-155 rpm, more preferably 150 rpm; cellulase, xylanase, and mannanase have a synergistic effect, which can effectively destroy the structure of plant cell walls, making the cell walls loose, thereby reducing the resistance from the cell walls and intercellular matrix during solvent extraction, accelerating the rate of pectin dissolution from cells, and improving the pectin extraction rate. Xylanase and mannanase can avoid destroying the RG-I domain of pectin, and this specific enzyme action helps to preserve the structural integrity of pectin during extraction. After enzymatic hydrolysis, the enzyme is inactivated by boiling for 13-17 min, preferably 14-16 min, more preferably 15 min, to obtain solution 2.
[0032] Solution 2 is centrifuged, and the supernatant is collected. The supernatant is then concentrated by rotary evaporation at a temperature of 60–70°C, preferably 62–68°C, more preferably 65°C; the rotation speed is 50–70 rpm, preferably 55–65 rpm, more preferably 60 rpm; the concentration is reduced to 30–36% of the original volume, preferably 32–34%, more preferably 33%. The filtrate after rotary evaporation is mixed with ethanol for alcohol precipitation, with a volume ratio of filtrate to ethanol of 1:1–1.2, more preferably 1:1.1. The volume fraction is 90-95%, preferably 91-94%, more preferably 93%; the alcohol precipitation temperature is 3-5℃, preferably 3.5-4.5℃, more preferably 4℃; after alcohol precipitation, the mixture is allowed to stand and then centrifuged; the standing time is 8-10h, preferably 8.5-9.5h, more preferably 9h; the centrifugation speed is 4500-5500rpm, preferably 4800-5200rpm, more preferably 5000rpm; the centrifugation time is 13-17min, preferably 14-16min, more preferably 15min; after centrifugation, the precipitate is collected to obtain crude pectin.
[0033] This invention removes protein from crude pectin using the sevage method. The protein-removed pectin solution is then diluted and placed in a dialysis bag for dialysis. The concentration of the diluted pectin solution is 0.5–2% (w / v), preferably 1–1.5% (w / v), and more preferably 1.2% (w / v). The molecular weight cutoff of the dialysis bag is 10 kDa.
[0034] The present invention also provides coffee pericarp pectin prepared according to the preparation method described above.
[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0036] The coffee husk powder used in this embodiment of the invention was purchased from Bolivia, but the scope of application of the method of the present invention is not limited to the coffee husk powder used in the embodiment, but is also applicable to all other coffee husk powders.
[0037] The cellulase, xylanase, mannanase, and acidic protease used in the embodiments of the present invention and the comparative example were purchased from Angel Enzyme Preparations Co., Ltd.
[0038] Example 1
[0039] (1) Accurately weigh 120g of dry coffee fruit peel powder and add it to 2400mL of ultrapure water. Add vitamin C to make the final concentration 13mmol / L. Sonicate at 180W for 15min to obtain solution 1.
[0040] (2) Adjust the pH of solution 1 to 4.9, then add cellulase, xylanase and mannanase, and enzymatically hydrolyze at 45℃ for 85-95 min, and inactivate the enzyme at 120℃ for 30 s to obtain solution 2; the amount of cellulase used is 75 U / g coffee fruit peel powder, the amount of xylanase used is 45 U / g coffee fruit peel powder, and the amount of mannanase used is 60 U / g coffee fruit peel powder;
[0041] (3) Centrifuge the obtained solution 2 at 3500 rpm for 10 min and collect the supernatant;
[0042] (4) Evaporate the supernatant at 65℃ and 60rpm to 33% of the original volume, add an equal volume of 95% (volume fraction) ethanol, let stand at 4℃ for 10h, centrifuge at 5000rpm for 15min, collect the precipitate, wash 3 times to obtain crude pectin.
[0043] (5) Sevage method for protein removal: Prepare a 5% solution of crude pectin. Prepare a chloroform:n-butanol mixture of 4:1 (v / v) in a fume hood. Add the mixture to centrifuge tubes at a pectin:mixed solvent (w / v) ratio of 3:1. Vortex for 20 min, centrifuge at 3000 rpm for 15 min. The solution separates into three layers: upper layer of pectin, middle layer of denatured protein and lipids, and lower layer of chloroform-n-butanol.
[0044] (6) Take out the upper solution, separate the pectin layer, mix it with an appropriate amount of water, put it into a dialysis bag with a molecular weight cutoff of 3kDa, immerse the dialysis bag in dialysis solution at 4℃, and dialyze with ultrapure water for 48h. Change the dialysis solution every 4h for 3 times, and then change it every 8h in the later stage.
[0045] (7) The liquid in the dialysis bag is rotary evaporated to 1 / 5 of its original volume and then freeze-dried at -55°C to obtain coffee fruit peel powder pectin. The rotary evaporation conditions are the same as in step (4).
[0046] Example 2
[0047] (1) Accurately weigh 120g of dry coffee fruit peel powder and add it to 2160mL of ultrapure water. Add vitamin C to make the final concentration 10mmol / L. Sonicate at 160W for 16min to obtain solution 1.
[0048] (2) Adjust the pH of solution 1 to 4.8, then add cellulase, xylanase and mannanase, and enzymatically hydrolyze at 43℃ for 95 min, and inactivate the enzyme at 120℃ for 30 s to obtain solution 2; the amount of cellulase used is 70 U / g coffee fruit peel powder, the amount of xylanase used is 50 U / g coffee fruit peel powder, and the amount of mannanase used is 55 U / g coffee fruit peel powder;
[0049] (3) Centrifuge the obtained solution 2 at 3000 rpm for 12 min and collect the supernatant;
[0050] (4) Evaporate the supernatant at 60℃ and 50rpm to 36% of the original volume, add an equal volume of 90% (volume fraction) ethanol, let stand at 3℃ for 8h, centrifuge at 4500rpm for 17min, collect the precipitate, wash 3 times to obtain crude pectin.
[0051] Steps (5), (6), and (7) are the same as in Example 1.
[0052] Example 3
[0053] (1) Accurately weigh 120g of dry coffee fruit peel powder and add it to 2640mL of ultrapure water. Add vitamin C to make the final concentration 16mmol / L. Sonicate at 200W for 14min to obtain solution 1.
[0054] (2) Adjust the pH of solution 1 to 5.0, then add cellulase, xylanase and mannanase, and enzymatically hydrolyze at 47℃ for 85 min, and inactivate the enzyme at 120℃ for 30 s to obtain solution 2; the amount of cellulase is 80 U / g coffee fruit peel powder, the amount of xylanase is 40 U / g coffee fruit peel powder, and the amount of mannanase is 65 U / g coffee fruit peel powder;
[0055] (3) Centrifuge the obtained solution 2 at 4000 rpm for 8 min and collect the supernatant;
[0056] (4) Evaporate the supernatant at 70℃ and 70rpm to 30% of the original volume, add an equal volume of 93% (volume fraction) ethanol, let stand at 5℃ for 9h, centrifuge at 5500rpm for 13min, collect the precipitate, wash 3 times to obtain crude pectin.
[0057] Steps (5), (6), and (7) are the same as in Example 1.
[0058] Comparative Example 1. Acid Extraction Method
[0059] Add 120g of dry coffee fruit peel powder to 2400mL of ultrapure water, adjust the pH to 2 with citric acid, sonicate at 200W for 15min, extract by continuous stirring at 80℃ for 90min, filter, centrifuge at 3500g for 10min, collect the supernatant, adjust the pH to 6, centrifuge again, collect the supernatant, add 3 times the volume of alcohol, and store overnight at 4℃. Centrifuge at 4000g for 10min, collect the precipitate, remove protein using the sevage method, dialyze under running water at 4℃ for 48h, the molecular weight cutoff of the dialysis bag is 3kDa, rotary evaporate the liquid in the dialysis bag to 1 / 5 of the original volume, and freeze-dry at -55℃ to obtain refined pectin.
[0060] Comparative Example 2. Alkali Extraction Method
[0061] Add 120g of dry coffee fruit peel powder to 2400mL of distilled water, adjust the pH to 10.0 using 6M NaOH solution, sonicate for 15min at 200W, extract by continuous stirring at 80℃ for 90min, filter, centrifuge at 3500g for 10min, collect the supernatant, adjust the pH to 6, centrifuge again, collect the supernatant, add 3 times the volume of alcohol, and store overnight at 4℃. Centrifuge at 4000g for 10min to collect the precipitate, remove protein using the sevage method, dialyze under running water at 4℃ for 48h, the molecular weight cutoff of the dialysis bag is 3kDa, rotary evaporate the liquid in the dialysis bag to 1 / 5 of the original volume, and freeze-dry at -55℃ to obtain refined pectin.
[0062] Comparative Example 3
[0063] Unlike Example 1, enzymatic hydrolysis was performed using only cellulase.
[0064] Comparative Example 4
[0065] Unlike Example 1, enzymatic hydrolysis was performed using only xylanase.
[0066] Comparative Example 5
[0067] Unlike Example 1, enzymatic hydrolysis was performed using only mannanase.
[0068] Comparative Example 6
[0069] Unlike Example 1, mannanase was replaced with acidic protease.
[0070] Comparative Example 7
[0071] Unlike Example 1, the amount of xylanase used was 70 U / g coffee fruit peel powder.
[0072] Comparative Example 8
[0073] Unlike Example 1, no vitamin C was added during the polysaccharide hydrolysis process.
[0074] The yield of pectin and the proportion of RG-I type pectin in Examples 1-3 and Comparative Examples 1-8 were determined respectively, and the results are shown in Table 1.
[0075] Pectin yield = (Mass of extracted pectin / Mass of dry coffee fruit powder) × 100%
[0076] Table 1. Pectin yield and RG-I type pectin percentage
[0077] Group Pectin yield (%) Percentage of RG-I type pectin (%) Example 1 12.5 64.8 Example 2 11.9 63.5 Example 3 12.3 63.1 Comparative Example 1 7.6 43.7 Comparative Example 2 5.2 52.6 Comparative Example 3 2.6 30.2 Comparative Example 4 2.2 39.8 Comparative Example 5 3.8 55.4 Comparative Example 6 8.3 48.3 Comparative Example 7 9.1 58.9 Comparative Example 8 10.2 58.0
[0078] As shown in Table 1, the pectin extraction method of this invention yields a high pectin yield, and the extracted pectin contains a high proportion of RG-I type pectin, exceeding 60%. The pectin yield and the proportion of RG-I type pectin of this invention are both higher than those of acid extraction and alkali extraction methods. Comparative Examples 3-4 show that the pectin yield extracted by this invention is higher than the sum of the pectin yields obtained by extracting pectin using cellulase, xylanase, and mannanase individually, indicating a synergistic effect among these three enzymes. Comparative Examples 6 and 7 show that changing the type and amount of enzyme decreases the pectin yield and the proportion of RG-I type pectin. Comparative Example 8 shows that adding vitamin C during enzymatic hydrolysis can improve the pectin yield and prevent the degradation of RG-I type pectin.
[0079] As can be seen from the above embodiments, the present invention provides a method for preparing coffee pericarp pectin, which yields a high pectin yield and a high proportion of RG-I.
[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing coffee fruit pericarp pectin, characterized in that, Includes the following steps: S1. Mix coffee husk powder, vitamin C and water, and sonicate to obtain solution 1; the mass-to-volume ratio of coffee husk powder to water is 1g:18-22mL, and the concentration of vitamin C in solution 1 is 10-16mmol / L; S2. Adjust the pH of solution 1 to 4.8-5.0, then add cellulase, xylanase and mannanase, and enzymatically hydrolyze at 43-47℃ for 85-95 min to inactivate the enzymes and obtain solution 2; S3. Centrifuge solution 2 and collect the supernatant; S4. The supernatant is concentrated by rotary evaporation to 30-36% of its original volume, ethanol is added for alcohol precipitation, and after standing, it is centrifuged to obtain crude pectin. S5. Remove the protein from the crude pectin, dialyze, and freeze-dry to obtain coffee pericarp pectin.
2. The preparation method according to claim 1, characterized in that, During enzymatic hydrolysis, the dosage of cellulase is 70-80 U / g coffee fruit pulp, the dosage of xylanase is 40-50 U / g coffee fruit pulp, and the dosage of mannanase is 55-65 U / g coffee fruit pulp.
3. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis is accompanied by stirring at a speed of 140–160 rpm.
4. The preparation method according to claim 1, characterized in that, The ultrasonic power is 160-200W, and the ultrasonic time is 14-16 minutes.
5. The preparation method according to claim 1, characterized in that, The rotary evaporation concentration temperature is 60–70°C, and the rotary evaporation concentration speed is 50–70 rpm.
6. The preparation method according to claim 1, characterized in that, The volume ratio of the concentrated filtrate to ethanol is 1:1 to 1.
2.
7. The preparation method according to claim 7, characterized in that, The volume fraction of ethanol is 90-95%, the alcohol precipitation temperature is 3-5°C, and the standing time is 8-10 hours.
8. The preparation method according to claim 7, characterized in that, The centrifugation speed in S3 is 3000-4000 rpm, and the centrifugation time is 8-12 min; the centrifugation speed in S4 is 4500-5500 rpm, and the centrifugation time is 13-17 min.
9. The preparation method according to claim 1, characterized in that, The molecular weight cutoff of the dialysis bag is 3 kDa.
10. Coffee pericarp pectin obtained by the preparation method according to any one of claims 1 to 9.