A method for extracting feijoa pectin rich in RG-I high ester

Abstract

The invention discloses a method for extracting RG-I high-ester feijoa pectin, comprising the following steps: pre-treating feijoa pulp into small pieces and inactivating pectinase, extracting pectin in a microwave oven using a 50mM sodium citric acid salt solution, cooling, enzymatic hydrolysis, centrifugation, purifying with ethanol, and drying to obtain RG-I high-ester pectin. The extraction method disclosed by the invention can fully extract pectin from feijoa pulp by microwave-assisted enzymatic hydrolysis, shortening the enzyme treatment time, greatly improving the extraction efficiency and product yield; and the pectin prepared by the invention is high-esterification pectin, with a more complete structure, an RG-Ι content of 41.38%, and a high viscosity.

Description

Technical Field

[0001] The invention belongs to the technical field of pectin extraction, and particularly relates to a method for extracting feijoa pectin rich in RG-I high ester. Background Art

[0002] Feijoa, also known as Brazilian guava, is a perennial subtropical evergreen shrub of the genus Myrtaceae, and is a new type of fruit in my country in recent years. Feijoa is rich in nutrients and functional active ingredients, such as protein, vitamins, minerals and polyphenols. It is reported that the pectin content in feijoa fruit reaches 20%, so feijoa is a treasure trove of pectin resources. However, the large-scale planting of feijoa in my country has yet to be promoted, and its processed products have yet to be developed. There are few reports and patents on related processed products, especially pectin extraction.

[0003] Pectin is an acidic heteropolysaccharide widely found in the middle layer of the cell wall of dicotyledonous plants. Pectin is widely used in food, medicine, cosmetics and other industries due to its good emulsification, gelling, stability and thickening properties. At the same time, pectin is also a natural water-soluble dietary fiber that can reduce the risk of cancer, diabetes, obesity, regulate the human intestinal microenvironment, and reduce blood lipids. High-ester pectin refers to pectin with an esterification degree (the ratio of methyl-esterified galacturonic acid units to total galacturonic acid units in pectin) ≥ 50%. Because its morphological structure is closest to natural pectin molecules, it has good application prospects. The degree of esterification will affect the structure, functional properties and ability of pectin to form complexes with other substances. It is of great significance to retain the complete structure of pectin as much as possible and improve the performance of pectin for better application in food and other industries.

[0004] The main structural domains of pectin include homogalacturonan (HG), rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and a small amount of substituted galacturonan. Commercial pectin is mainly composed of the HG region and is often used as a thickener and gelling agent in food-related fields. Most of the RG-I region can be removed by high temperature and strong acid to improve the stability and gel properties of pectin. More and more studies have shown that pectin rich in RG-I has better anti-cancer effects, regulates chronic metabolic diseases, and has immune activity and prebiotic properties. Therefore, how to scientifically extract pectin with complete RG-I has attracted the attention of more and more researchers and consumers.

[0005] Existing pectin extraction methods mainly include ion exchange, acid / base extraction, supercritical extraction and bio-enzyme extraction, but these extraction methods are not ideal. In addition, strong acids or strong bases are not only corrosive to equipment, but the wastewater after extraction will also cause environmental pollution. Therefore, how to prepare high-ester pectin with a complete structure (RG-I), good performance and high yield is one of the problems that need to be solved at present. Summary of the invention

[0006] The purpose of the present invention is to provide a method for extracting feijoa pectin rich in RG-I high ester, which can obtain feijoa high ester pectin rich in RG-I structure, good in quality and high in yield.

[0007] To achieve the above object, the present invention provides a method for extracting RG-I high-ester feijoa pectin, comprising the following steps:

[0008] (1) boiling feijoa pulp and drying it until the water content is less than 6%, crushing it and mixing the powder with a buffer solution;

[0009] (2) The mixed solution is subjected to microwave heating, followed by enzymatic hydrolysis, centrifugation, and purification, and the pectin is dried;

[0010] The power of microwave heating treatment is 350-400W, and the time is 5-15min.

[0011] Furthermore, the boiling temperature in step (1) is 85-100° C., and the boiling time is 3-8 min.

[0012] Furthermore, the drying temperature in step (1) is 45-55°C.

[0013] Furthermore, the powder after crushing in step (1) is passed through a 60-mesh sieve.

[0014] Furthermore, in step (1), the ratio of the powder to the buffer is 10 g: 120-180 mL, and the buffer is 50 mM sodium citrate buffer with a pH of 6.8.

[0015] Furthermore, the enzymatic hydrolysis process is specifically as follows: after β-glucosidase and feijoa powder are uniformly mixed in an amount of 250 U / g, enzymatic hydrolysis is performed for 90-100 minutes, the enzymatic hydrolysis temperature is 45-55° C., and the enzymatic hydrolysis speed is 120-180 rpm.

[0016] Furthermore, after the enzymatic hydrolysis in step (2), the extracted solution is heated in a water bath at 90-100° C. for 3-5 minutes.

[0017] Furthermore, the centrifugal speed in step (2) is 4500-5500 rpm, and the centrifugal time is 8-12 min.

[0018] Furthermore, the purification specifically includes the following steps: collecting the supernatant after centrifugation, adding twice the volume of anhydrous ethanol to the supernatant, mixing and standing for precipitation for 2 hours, centrifuging at 4500-5500 rpm after the pectin is precipitated, and washing the pectin in the test tube with anhydrous ethanol 2-3 times.

[0019] Furthermore, the pectin is dried at a temperature of 45-60°C.

[0020] In summary, the present invention has the following advantages:

[0021] 1. The extraction method disclosed in the present invention can fully extract pectin from feijoa pulp by microwave-assisted enzymolysis, shorten the enzyme treatment time, and greatly improve the extraction efficiency and product yield; and the pectin prepared by the present invention is high-esterification degree (esterification degree 68.43%) pectin, with a more complete structure, an RGI content of 41.38%, and a higher viscosity.

[0022] 2. The extraction method disclosed in the present invention has low energy consumption and little pollution, which improves the utilization value of feijoa and provides theoretical guidance for the further development and utilization of feijoa.

[0023] 3. The method of the present invention retains more of the RG-I regional structure of pectin and extracts high-ester pectin rich in RG-I. The pectin can be separated from the raw material by microwave treatment of feijoa pulp and then using biological enzymes, and more original pectin is converted into soluble pectin for extraction. Therefore, the extraction method disclosed in the present invention can obtain high-yield pectin. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The stable shear flow curves of pectins obtained by microwave-assisted enzymatic method (MEHE), enzymatic method (EHE) and conventional hot acid water method (CHE). DETAILED DESCRIPTION

[0025] The principles and features of the present invention are described below in conjunction with the examples, which are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0026] The microwave heating method used in the present invention is a microwave oven heating method, and the microwave oven model used is MM823ESJ-PA Midea microwave oven, and the gear during heating is medium and low fire. Therefore, the present invention also attaches a detection method for the frequency of the medium and low fire gear to avoid the problem of unclear heating parameters. The detection method is: the microwave power is detected by the IEC705 method. First, prepare two 500mL plastic beakers and fill them with 500mL of water at a temperature of about 10°C, place the beakers in the center of the cooking area, set the microwave oven to full power and turn on the power, let the microwave oven run accurately for 60s, and the magnetic tube reaches the working temperature for 2s. Then take out the beaker from the microwave oven, stir each beaker with a magnetic stirrer immediately before measuring the temperature of each beaker with a thermometer and quickly measure the temperature, and repeat each group twice to take the average value.

[0027] Example 1

[0028] This embodiment provides a method for extracting feijoa pectin rich in RG-I high ester, comprising the following steps:

[0029] (1) After washing and drying the harvested feijoa, peel and cut them into pieces, and then put them into deionized water at 90°C and boil them for 5 minutes to inactivate the pectinase. After controlling the water content appropriately, dry them in a 50°C forced air drying oven until the moisture content is less than 6%. Grind them with a grinder and pass them through a 60-mesh sieve to make feijoa pulp powder (pulp and pulp), seal them in a ziplock bag, and store them in a desiccator for later use.

[0030] (2) Take 10 g of the feijoa powder prepared in step (1) and place it in a 250 mL conical flask. Add 150 mL of 50 mM sodium citrate buffer (solid-liquid ratio 1:15), adjust the pH to 6.8 with citric acid, and mix well.

[0031] (3) Place the mixture from (2) in a microwave oven (MM823ESJ-PA Midea microwave oven) and set the heat to medium-low (380W IEC) for 9 minutes. Remove from the oven and allow to cool until it is not too hot.

[0032] (4) Weigh β-glucosidase (250 U / g, ratio of enzyme activity to substrate) into a conical flask, mix well, and perform enzymatic hydrolysis in a constant temperature shaking incubator for 99 min. The enzymatic hydrolysis temperature is set at 50° C. and the shaking speed is 150 rpm.

[0033] (5) After extraction, heat in a 95°C water bath for 5 minutes to inactivate the enzyme.

[0034] (6) The extracted solution was centrifuged at 5000 rpm for 10 min, the supernatant was collected, and the residue was washed twice with distilled water. The supernatants were combined and the volume was recorded.

[0035] (7) Add twice the volume of anhydrous ethanol to the supernatant, mix well and let stand for two hours to settle. After the pectin is precipitated, centrifuge at 5000 rpm and wash the pectin in the test tube twice with anhydrous ethanol.

[0036] (8) Dry the cleaned pectin in an electric blast drying oven at 50°C, grind it with a mortar, and pass it through a 100-mesh sieve for later use.

[0037] Example 2

[0038] (1) After washing and drying the harvested feijoa, peel and cut them into pieces, and then put them into deionized water at 95°C and boil them for 5 minutes to inactivate the pectinase. After controlling the water content appropriately, dry them in a 55°C forced air drying oven until the moisture content is less than 6%. Grind them with a grinder and pass them through a 60-mesh sieve to make feijoa pulp powder (pulp and pulp), seal them in a ziplock bag, and store them in a desiccator for later use.

[0039] (2) Take 10 g of the feijoa powder prepared in step (1) and place it in a 250 mL conical flask. Add 160 mL of 50 mM sodium citrate buffer (solid-liquid ratio 1:16), adjust the pH to 6.8 with citric acid, and mix well.

[0040] (3) Place the mixture from (2) in a microwave oven (MM823ESJ-PA Midea microwave oven) and set the heat to medium-low (350W IEC) for 10 minutes. Remove from the oven and allow to cool until it is not too hot.

[0041] (4) Weigh β-glucosidase (250 U / g, ratio of enzyme activity to substrate) into a conical flask, mix well, and perform enzymatic hydrolysis in a constant temperature shaking incubator for 90 min. The enzymatic hydrolysis temperature is set at 50° C. and the shaking speed is 150 rpm.

[0042] (5) After extraction, heat in a 95°C water bath for 5 minutes to inactivate the enzyme.

[0043] (6) The extracted solution was centrifuged at 5500 rpm for 8 min, the supernatant was collected, and the residue was washed twice with distilled water. The supernatants were combined and the volume was recorded.

[0044] (7) Add two volumes of anhydrous ethanol to the supernatant, mix well and let stand for two hours to settle. After the pectin is precipitated, centrifuge at 5000 rpm and wash the pectin in the test tube three times with anhydrous ethanol.

[0045] (8) Dry the cleaned pectin in an electric blast drying oven at 50°C, grind it with a mortar, and pass it through a 100-mesh sieve for later use.

[0046] Example 3

[0047] (1) After washing and drying the harvested feijoa, peel and cut them into pieces, and then put them into deionized water at 90°C and boil them for 5 minutes to inactivate the pectinase. After controlling the water content appropriately, dry them in a 50°C forced air drying oven until the moisture content is less than 6%. Grind them with a grinder and pass them through a 60-mesh sieve to make feijoa pulp powder (pulp and pulp), seal them in a ziplock bag, and store them in a desiccator for later use.

[0048] (2) Take 10 g of the feijoa powder prepared in step (1) and place it in a 250 mL conical flask. Add 140 mL of 50 mM sodium citrate buffer (solid-liquid ratio 1:14), adjust the pH to 6.8 with citric acid, and mix well.

[0049] (3) Place the mixture from (2) in a microwave oven (MM823ESJ-PA Midea microwave oven) and set the heat to medium-low (400W IEC) for 9 minutes. Remove from the oven and allow to cool until it is not too hot.

[0050] (4) Weigh β-glucosidase (250 U / g, ratio of enzyme activity to substrate) into a conical flask, mix well, and perform enzymatic hydrolysis in a constant temperature shaking incubator for 100 min. The enzymatic hydrolysis temperature is set to 45° C. and the shaking speed is 150 rpm.

[0051] (5) After extraction, heat in a 90°C water bath for 5 minutes to inactivate the enzyme.

[0052] (6) The extracted solution was centrifuged at 5000 rpm for 10 min, the supernatant was collected, and the residue was washed twice with distilled water. The supernatants were combined and the volume was recorded.

[0053] (7) Add two volumes of anhydrous ethanol to the supernatant, mix well and let stand for two hours to settle. After the pectin is precipitated, centrifuge at 5500 rpm and wash the pectin in the test tube three times with anhydrous ethanol.

[0054] (8) Dry the cleaned pectin in an electric blast drying oven at 50°C, grind it with a mortar, and pass it through a 100-mesh sieve for later use.

[0055] Comparative Example 1

[0056] This comparative example provides a method for extracting feijoa pectin by a bioenzymatic method, comprising the following steps:

[0057] 10 g of feijoa pulp powder was accurately weighed into a conical flask, 150 mL (1:15) of 50 mM sodium citrate solution with a pH of 6.8 was added, and β-glucosidase (250 U / g) was added for enzymatic hydrolysis for 99 min, the enzymatic hydrolysis temperature was set at 50° C., and the shaking speed was 150 rpm. Other treatments were the same as in Example 1.

[0058] Comparative Example 2

[0059] This comparative example provides a method for extracting feijoa pectin using a conventional hot acid water method, comprising the following steps:

[0060] (1) Weigh 10 g of feijoa pulp powder into a beaker, add 150 mL of hydrochloric acid solution with a pH of 1, and extract in a heat-collecting magnetic stirrer set at 95°C for 130 min.

[0061] (2) After the extraction is completed, the pulp residue is separated from the soluble compounds using a centrifuge, and the separated pulp residue is washed with distilled water.

[0062] (3) The filtrate was collected and centrifuged at 5000 rpm for 10 min to remove fine powder, and 2 volumes of anhydrous ethanol were added to precipitate pectin for 2 h.

[0063] (4) The precipitated pectin was centrifuged at 5000 rpm for 10 min, washed with anhydrous ethanol three times and centrifuged again. The final pectin was dried in an electric heated air drying oven at 50°C.

[0064] Test Example 1-Monosaccharide Determination

[0065] The determination method of monosaccharides adopts the trichloroacetic acid (TFA) method. 10 mg of polysaccharide sample is accurately weighed, and 1 mL of TFA (4M) solution is added to hydrolyze at 110°C for 4 hours. Subsequently, the hydrolyzate is co-distilled with ethanol under reduced pressure several times to completely remove TFA. The hydrolyzed sample and standard monosaccharides are acetylated by adding a mixture of hydroxylamine hydrochloride, pyridine and acetic anhydride. The analysis was performed using a Shimadzu GC / MS gas chromatography (QP 2010Plus) system. The initial temperature was set to 150°C and maintained for 17 minutes. The temperature program was increased from 150°C to 165°C at a rate of 1°C / min, then increased to 200°C at a rate of 10°C / min, and then increased to 380°C at a rate of 50°C / mi. The sample injection volume was 1 μL, the split ratio was 1:20, the carrier gas was helium, and the flow rate was 1 mL / min; the ion source temperature was 230°C, the electron impact voltage was 70 eV, the mass scan range was m / z20-650, the frequency was 0.2 s / scan, and the solvent delay time was set to 5.5 min. β-phenyl-glucoside (0.05% w / v) was used as the internal standard for standard quantification, and the RG-I content was calculated according to the following formula:

[0066] RG-I(%)=2Rha(%)+Ara(%)+Gal(%)

[0067] Test Example 2 - Determination of Degree of Esterification (DE)

[0068] The method for determining the degree of esterification is carried out according to the titration method in the national food safety standard GB25533-2010.

[0069] Test Example 3 - Determination of rheological properties

[0070] The pectin sample was dissolved in deionized water to prepare a concentration of 1.0% (w / v). The rotational rheometer was Haake RS6000 from Thermo Corporation of the United States. The probe used a flat circular steel plate (60 mm in diameter) with a gap setting of 1.0 mm to measure the apparent viscosity of the sample. The viscosity of the sample was measured at 25°C with a shear rate of 0.1 s -1 Gradually increase to 100s -1 The apparent viscosity curve below.

[0071] The experimental results are shown in Table 1 and Figure 1 shown.

[0072] Table 1 Physicochemical parameters of pectin

[0073]

[0074] From Table 1 and Figure 1 It can be seen that the extraction method disclosed in the present invention can greatly improve the yield of pectin, and maintain a high level of esterification degree and RG-I content. The extraction method disclosed in the present invention can retain more RG-I regional structure of pectin and extract high-ester pectin rich in RG-I. At the same time, the esterification degree of pectin extracted by the present invention is 68.43%, which belongs to high-ester pectin, and the viscosity is equivalent to that of pectin extracted by enzyme method, which is better than that of pectin extracted by conventional hot acid water method.

[0075] Although the specific implementation of the present invention has been described in detail, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A method for extracting feijoa pectin rich in RG-I high ester, characterized in that: The following steps are involved: (1) boiling feijoa pulp and drying it until the water content is less than 6%, crushing it, and then mixing the powder with a buffer solution; the ratio of the powder to the buffer solution in step (1) is 10 g: 120-180 mL, and the buffer solution is a 50 mM sodium citrate buffer solution with a pH of 6.8; (2) The mixed solution is subjected to microwave heating, followed by enzymolysis, centrifugation, purification, and drying of the pectin; the enzymolysis process is specifically as follows: β-glucosidase and feijoa powder are mixed evenly in an amount of 250 U / g, and enzymolysis is performed for 90-100 minutes at a temperature of 45-55° C. and a rotation speed of 120-180 rpm; The power of the microwave heating treatment is 350-400W, and the time is 5-15min.

2. The method for extracting RG-I high ester feijoa pectin according to claim 1, characterized in that: The boiling temperature in step (1) is 85-100° C., and the boiling time is 3-8 minutes.

3. The method for extracting RG-I high ester feijoa pectin as claimed in claim 1, characterized in that: The drying temperature in step (1) is 45-55°C.

4. The method for extracting RG-I high ester feijoa pectin as claimed in claim 1, characterized in that: The powder after pulverization in step (1) is sieved through a 60-mesh sieve.

5. The method for extracting RG-I high ester feijoa pectin as claimed in claim 1, characterized in that: After the enzymatic hydrolysis in step (2), the extracted solution is heated in a water bath at 90-100° C. for 3-5 minutes.

6. The method for extracting RG-I high ester feijoa pectin as claimed in claim 1, characterized in that: The centrifugal speed in step (2) is 4500-5500 rpm, and the centrifugal time is 8-12 min.

7. The method for extracting RG-I high ester feijoa pectin as claimed in claim 1, characterized in that: The purification specifically includes the following steps: collecting the supernatant after centrifugation, adding twice the volume of anhydrous ethanol to the supernatant, mixing and then standing for precipitation for 2 hours, centrifuging at 4500-5500 rpm after the pectin is precipitated, and washing the pectin in the test tube with anhydrous ethanol 2-3 times.

8. The method for extracting feijoa pectin rich in RG-I high ester according to claim 1, characterized in that: The pectin is dried at a temperature of 45-60°C.

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