Green method for efficiently enriching olive pomace polyphenol by coupling steam explosion pretreatment with subcritical water extraction

Abstract

The invention discloses a green method for efficiently enriching olive pomace polyphenol by utilizing a water vapor explosion pretreatment coupled subcritical water extraction technology, which comprises the following specific steps: adding water into dry olive pomace for wetting, feeding into a steam explosion machine for efficient wall breaking pretreatment, collecting pomace after explosion pretreatment, and performing subcritical water extraction to obtain the olive pomace polyphenol. And filtering and collecting an extracting solution rich in polyphenol while the solution is hot. The content of the polyphenol in the olive pomace can be maximally increased by 2.6 times in comparison with that in the combination of the non-steam-explosion pretreatment and the ultrasonic-assisted alcohol extraction and the combination of the steam-explosion pretreatment and the ultrasonic-assisted alcohol extraction in the combination of the steam-explosion pretreatment and the subcritical water extraction; and the combined processing time of coupling the water vapor explosion pretreatment with the subcritical water extraction is only 22 minutes in total. Therefore, the steam and the extraction solvent adopted by the method only need water, no organic solvent is needed, the wall breaking effect is remarkable, and the method has the characteristics of simplicity and convenience in operation, environment friendliness, high industrialization potential and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction and enrichment of effective components of natural products chemistry, and relates to a green method for double high-efficient enrichment of polyphenols from olive pomace by steam explosion pretreatment coupled with subcritical water extraction. Background Art

[0002] Olea europaea, also known as Olea europaea, is an oil crop of the genus Olea in the family Oleaceae and is a world-famous subtropical economic forest. In China, there are multiple varieties of Olea europaea, such as Frantoio, Picholine, Coratina, and Leccino. Olea europaea in China is mainly planted in Longnan, Gansu; Guangyuan, Sichuan; Hanzhong, Shaanxi; and Yongren, Yunnan. Olea europaea is an evergreen small tree that can grow up to 10 meters in height, and its bark is gray. This plant does not have high requirements for soil. As long as the pH value of the soil is between 6.5 and 8.0 and the soil is loose and breathable, the olive can grow healthily. Olive oil contains rich nutrients and functional active ingredients, so it has the reputation of "the queen of vegetable oils".

[0003] With the continuous expansion of the olive oil market, the planting area of olives and the fresh fruit yield increase year by year. Therefore, the yield of olive pomace is also increasing continuously. The fresh fruit yield of Olea europaea in Wudu District, Longnan City, the largest olive-producing area in China, reached 47,000 tons in 2022, and the oil production was 6,200 tons, with year-on-year increases of 12.50% and 6.90% respectively. The oil production has doubled compared with that in 2018. According to statistics, 35-40 kg of pomace will be produced after every 100 kg of fresh olive fruits are pressed for oil. Olive pomace is the main representative high-quality residual resource in the olive oil processing industry. Olive pomace is a kind of waste composed of pulp residues, broken olive kernel shells, and wastewater, with a water content as high as 65%. Olive pomace is rich in water-soluble phenolic substances and also contains active substances such as terpenoids, flavonoids, and phytosterols. In addition, olive pomace also contains water-soluble fats, proteins, water-soluble carbohydrates, and has a high potassium content and low phosphorus and trace element contents.

[0004] Olive pomace polyphenols are natural active substances extracted from the pomace, a by-product after olive oil extraction, and possess extremely high nutritional value and health benefits. Its core components such as hydroxytyrosol and oleuropein have super antioxidant capacity, can effectively scavenge free radicals, delay cell aging, and at the same time significantly inhibit inflammatory responses, playing an important role in preventing chronic diseases such as cardiovascular diseases and diabetes. Compared with other plant polyphenols, olive pomace polyphenols have higher bioavailability, stronger stability, and act more persistently in the body. In addition, it can regulate blood lipids, improve vascular function, promote metabolism, and has potential neuroprotective and intestinal health regulatory effects. From an environmental protection perspective, the extraction of olive pomace polyphenols realizes resource recycling, reduces waste pollution, and has both economic and ecological benefits. Currently, it has been widely applied in the fields of functional foods, dietary supplements, pharmaceuticals, and cosmetics, becoming a natural active ingredient with great development potential in the global health industry.

[0005] In recent years, scholars at home and abroad have carried out extensive research on the high-value utilization of olive pomace. In terms of extraction processes, currently, various extraction methods such as heating reflux, solid-liquid extraction, enzymatic hydrolysis, ultrasonic-microwave assisted extraction, and supercritical CO2 extraction are adopted at home and abroad. Chinese Patent 202310238325.2 discloses a method for improving the total phenolic content of olive fruits and the extraction rate of polyphenolic compounds. Using a natural deep eutectic solvent, under ultrasonic conditions, olive fruits are extracted to obtain an olive fruit extract rich in total phenols. Although this method uses a natural deep eutectic solvent and ultrasonic extraction to improve the total phenol yield, there are still deficiencies such as difficult solvent recovery, sensitivity to process parameters, large-scale production difficulties, and potential solvent residue risks, and the extraction process needs to be further optimized. Chinese Patent 202310166044.0 discloses a method for improving the extraction rate of polyphenols from olive pomace by combining ultrasonic waves and medium-intensity electric fields. The olive pomace is dried to a constant weight and ground to obtain olive pomace powder; an ethanol solution is added to the sieved olive pomace powder, and polyphenols in the olive pomace powder are extracted by combining ultrasonic waves and medium-intensity electric fields. This patent uses the ultrasonic-electric field combination technology to improve the polyphenol extraction rate, but there are still deficiencies such as high energy consumption, complex process control, limited solvent selectivity, and strict pretreatment requirements, and the energy efficiency and process stability need to be further optimized. Chinese Patent 201910243826.3 discloses a method for extracting olive polyphenols assisted by composite enzymes. In this invention, cellulase, ligninase, and pectinase are used, the solvent is an ethanol aqueous solution of 50-70%, the auxiliary gas is nitrogen, the nitrogen flow rate is 0.6-1.0 L / min, the solid-liquid ratio of olive pomace to ethanol aqueous solution is 1:6-1:10, and the mass ratio of olive pomace, cellulase, ligninase, and pectinase is 100:0.5:0.5:0.3, and the reaction temperature is 45-60 , with a pH of 6.0 - 7.5 and a time of 30 - 60 min. This method has a relatively high cost of enzyme preparation and is prone to inactivation. Nitrogen protection increases the process complexity, and the use of organic solvents brings pressure on recovery and environmental protection. The multi - enzyme ratio and reaction condition control are required to be strict, and the physical damage effect on the cell wall is limited, affecting the final extraction efficiency.

[0006] Nie Longying et al. optimized the extraction process of total polyphenols from olive pomace by ultrasonic - assisted enzymatic hydrolysis. The research shows that under the optimal extraction conditions, the extraction yield of total polyphenols reaches 1.81 ± 0.32%. Compared with the traditional organic solvent extraction method, which has problems such as low extraction efficiency and organic solvent residues, this method significantly improves the dissolution rate of polyphenols. However, although the enzymatic hydrolysis method has a relatively high extraction efficiency, its cost is relatively high, which limits its industrial application. In addition, Difonzo et al. used supercritical CO2 extraction technology to extract polyphenols from olive pomace, but this technology has poor selectivity for polar polyphenols, affecting its extraction effect. And currently, the commonly used extraction technologies generally face bottleneck problems such as incomplete cell wall destruction, high production cost, high organic solvent usage rate, and low polyphenol yield. Therefore, developing an efficient, low - cost, and environmentally friendly extraction technology for olive pomace polyphenols is still the key research direction at present.

[0007] In order to overcome the limitations of traditional extraction methods for olive pomace polyphenols, the present invention designs a new green and low - cost extraction and enrichment process coupling steam explosion pretreatment and sub - critical water extraction. This process efficiently destroys the cell structure of raw materials through steam explosion pretreatment and combines the high extraction efficiency of sub - critical water extraction under high temperature and high pressure. It can not only significantly improve the dissolution rate of polyphenols but also better retain the biological activity of active ingredients, and at the same time has excellent environmental protection characteristics. Summary of the Invention

[0008] The present invention provides a method for efficient and green enrichment of olive pomace polyphenols, which solves problems such as high energy consumption, low efficiency, complex process control, and the risk of organic solvent residues in traditional extraction methods. It is an integrated innovative production process applied to the enrichment of olive pomace.

[0009] 1. A green method for double - high enrichment of olive pomace polyphenols by coupling steam explosion pretreatment and sub - critical water extraction, characterized in that the steps are as follows: The first step, steam explosion pretreatment: Weigh the soaked and wetted olive pomace and use a steam explosion device to perform steam explosion pretreatment with saturated steam water at a certain pressure. The pretreatment is carried out at a temperature range of 152 - 212 °C under water - saturated steam pressure, and the pressure holding time range is 60 s - 240 s; The second step, drying and pulverizing: Dry the steam - exploded olive pomace at a constant temperature of 50 °C, pulverize it through a high - speed pulverizer and then sieve it to obtain a uniform powder, and place it in a dryer at room temperature for standby; Step 3, subcritical water extraction: Use a subcritical water extraction device (HAWG type reactor) to perform subcritical water extraction on the steam-exploded (under the treatment conditions of a saturated vapor pressure of 1.5 MPa and a holding time of 120 s) olive pomace. Conduct subcritical water extraction experiments on the treated samples under certain pressure, extraction temperature, and time conditions. After heating the extraction kettle to the target temperature and maintaining the set time, immediately cool it to room temperature, filter and collect the extract, repeat the extraction 2 times, combine the filtrates and make up the volume, and store them at 4 °C for further analysis; Step 4, liquid-phase analysis and detection of polyphenol content: Analyze and detect the changes in the content of olive polyphenols under different conditions in a specific mobile phase by gradient elution at a certain wavelength, temperature, flow rate, time, and injection volume.

[0010] 2. The process method for enriching polyphenols from olive pomace according to claim 1, wherein the saturated water vapor pressure for steam explosion pretreatment in the first step is 0.5, 1.0, 1.5, or 2.0 MPa; 3. The moisture content range of the dried olive pomace in the second step according to claim 1 is 3 - 5%; 4. In the third step of subcritical water extraction according to claim 1, the fixed extraction pressure is 3.0 MPa; the extraction temperature range is 120 - 140 °C; the extraction time range is 10 - 30 min; the solid-liquid ratio range is 1:20 - 1:40, with the unit of g / mL; [[ID=eleven]]5. The specific liquid-phase detection and analysis conditions in the fourth step according to claim 1 are: gradient elution 0 - 6 min: 90 - 70% B, 6 - 13 min: 70 - 60% B, 13 - 17 min: 60 - 55% B, 17 - 19 min: 55 - 51.7% B, 19 - 27 min: 51.7 - 48.5% B, 27 - 28 min: 48.5 - 45% B, 28 - 35 min: 45 - 23% B, 35 - 40 min: 23 - 95% B, 40 - 45 min: 95 - 90% B, 45 - 60 min: 90 - 90% B, the wavelength is 280 nm, the injection volume is 20 μL, the elution time is 60.01 min, and the flow rate is 1.0 mL / min. The column used is Agilent ZORBAX SB-Aq (250×4.6 mm, 5 μm).

[0011] Beneficial effects: 1. The present invention uses steam explosion (saturated vapor pressure 1.5 MPa / holding time 120 s) to effectively destroy the lignocellulose structure, enabling the highest increase in the polyphenol content of up to 260% in subsequent subcritical water extraction (extraction temperature 130 °C / extraction time 20 min).

[0012] 2. The whole aqueous phase system of the present invention avoids the use and residue of organic solvents, reduces costs and increases safety at the same time.

[0013] 3. The production process of the present invention is energy-saving compared with the traditional hot extraction method (usually more than 6 hours). The treatment time is short, only 22 minutes in total, with high efficiency. And the process parameters (saturated vapor pressure 1.5 MPa, solid-liquid ratio 1:30, etc.) are optimized to have the feasibility of large-scale production.

[0014] 4. The present invention uses raw materials that are widely available and inexpensive, and the process equipment is relatively mature, which is more economical and environmentally friendly compared with other methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a flow chart provided by the present invention.

[0016] Figure 2 is a process flow chart of steam explosion pretreatment.

[0017] Figure 3 is a diagram of subcritical water extraction equipment and schematic diagram.

[0018] Figure 4 is a liquid chromatogram of five olive pomace polyphenols in liquid phase detection.

[0019] Figure 5 SEM images of olive pomace under different treatment conditions; A, untreated olive pomace; B, olive pomace pretreated by steam explosion; C, olive pomace after subcritical water extraction; D, olive pomace after steam explosion pretreatment and subcritical water extraction. DETAILED DESCRIPTION OF THE INVENTION (1) Steam explosion pretreatment: Weigh the soaked olive pomace and use steam explosion equipment to carry out steam explosion treatment with saturated steam water under a certain pressure. The treatment temperature is 152 - 212 °C, and the pressure holding time is 60 - 240 s for steam explosion pretreatment.

[0020] (2) Drying and pulverizing: Vacuum-dry the olive pomace obtained in step (1) at 60 for 24 hours, pulverize it with a high-speed pulverizer and sieve it to obtain uniform powder, and place it in a dryer for standby.

[0021] (3)Subcritical water extraction: The olive pomace obtained in step (2) was subjected to subcritical water extraction using a subcritical water extraction device (HAWG type reactor). Each time, a certain amount of the treated sample was accurately weighed, the extraction pressure was fixed at 3.0 MPa, the extraction temperatures were 120, 130 or 140 °C respectively, the extraction times were 10, 20 or 30 min respectively, and the solid-liquid ratio was 1:20, 1:30 or 1:40 g / mL. Under these conditions, subcritical water extraction treatment was carried out. After the treatment was completed, it was immediately cooled to room temperature, the extract was filtered and collected, the extraction was repeated 2 times, the filtrates were combined, and the volume was made up.

[0022] (4)Liquid phase analysis and detection of polyphenol content: The extract obtained in (3) was analyzed by liquid phase. Under the conditions of a wavelength of 280 nm, a column temperature of 30 °C, a flow rate of 1.0 mL / min, and an injection volume of 20 μL, the content changes of different olive polyphenols were analyzed and detected by gradient elution in a specific mobile phase. Through polyphenol content determination and liquid phase detection, the polyphenol content increased by 100 - 260% compared with the traditional method.

[0023] In step (1), the water content of the soaked olive pomace is about 30 - 40%.

[0024] In step (2), it was sieved through a 80-mesh sieve after crushing.

[0025] In step (3), the collected filtrate should be stored in an environment of 4 - 8 °C in time to facilitate the stability of the measurement results.

[0026] In step (3), the collected filtrate was made up to 400 mL.

[0027] In step (4), the liquid phase used was reverse phase high performance liquid chromatography (RP-HPLC) for detection and analysis. The gradient elution conditions were 0 - 6 min: 90 - 70% B, 6 - 13 min: 70 - 60% B, 13 - 17 min: 60 - 55% B, 17 - 19 min: 55 - 51.7% B, 19 - 27 min: 51.7 - 48.5% B, 27 - 28 min: 48.5 - 45% B, 28 - 35 min: 45 - 23% B, 35 - 40 min: 23 - 95% B, 40 - 45 min: 95 - 90% B, 45 - 60 min: 90 - 90% B. The column used was Agilent ZORBAX SB-Aq (250×4.6 mm, 5 μm). Example 1

[0028] (1)Steam explosion pretreatment: Accurately weigh 550 ± 20 g of water-soaked olive pomace and treat it with a steam explosion device. Under a saturated steam pressure of 1.5 MPa, the treatment temperature is 200 °C, and the pressure retention time is 120 s for steam explosion pretreatment.

[0029] (2)Drying and pulverizing: The olive pomace obtained in step (1) is dried in vacuo at 50 °C, pulverized by a high-speed pulverizer, and passed through an 80-mesh sieve to obtain a uniform powder, which is placed in a dryer for standby.

[0030] (3)Subcritical water extraction: The olive pomace obtained in step (2) is subjected to subcritical water extraction using a subcritical water extraction device. Accurately weigh 5.00 ± 0.10 g of the sample after steam explosion pretreatment, fix the extraction pressure at 3.0 MPa, the extraction temperatures are 130 °C respectively, the extraction times are 20 min respectively, and under the condition of a solid-liquid ratio of 1:30 g / mL, carry out subcritical water extraction treatment. After the treatment is completed, it is immediately cooled to room temperature, the extraction liquid is filtered and collected, extracted 2 times repeatedly, the filtrates are combined, and the volume is fixed to 400 mL.

[0031] (4)Liquid-phase analysis and detection of polyphenol content: The extraction liquid obtained in (3) is analyzed by liquid phase. Under the conditions of a wavelength of 280 nm, a column temperature of 30 °C, a flow rate of 1.0 mL / min, and an injection volume of 20 μL, the content changes of different olive polyphenols are analyzed and detected in a specific mobile phase by gradient elution. After polyphenol content determination and liquid-phase detection, the polyphenol content is increased by 260% compared with the traditional method. Example 2

[0032] (1)Steam explosion pretreatment: Accurately weigh 550 ± 20 g of water-soaked olive pomace and treat it with a steam explosion device. Under a saturated steam pressure of 1.5 MPa, the treatment temperature is 200 °C, and the pressure retention time is 120 s for steam explosion pretreatment.

[0033] (2)Drying and pulverizing: The olive pomace obtained in step (1) is dried in vacuo at 50 °C, pulverized by a high-speed pulverizer, and passed through an 80-mesh sieve to obtain a uniform powder, which is placed in a dryer for standby.

[0034] (3)Subcritical water extraction: The olive pomace obtained in step (2) is subjected to subcritical water extraction using a subcritical water extraction device. Accurately weigh 5.00 ± 0.10 g of the sample after steam explosion pretreatment, fix the extraction pressure at 3.0 MPa, the extraction temperatures are 120 °C respectively, the extraction times are 30 min respectively, and under the condition of a solid-liquid ratio of 1:30 g / mL, carry out subcritical water extraction treatment. After the treatment is completed, it is immediately cooled to room temperature, the extraction liquid is filtered and collected, extracted 2 times repeatedly, the filtrates are combined, and the volume is fixed to 400 mL.

[0035] (4)Liquid analysis and detection of polyphenol content: The extract obtained in (3) was analyzed by liquid chromatography. Under the conditions of a wavelength of 280 nm, a column temperature of 30 °C, a flow rate of 1.0 mL / min, and an injection volume of 20 μL, the content changes of different olive polyphenols were analyzed and detected in a specific mobile phase by gradient elution. After polyphenol content determination and liquid chromatography detection, the polyphenol content increased by 98.14% compared with the traditional method. Example 3

[0036] (1)Steam explosion pretreatment: Accurately weigh 550 ± 20 g of soaked olive pomace and treat it with a steam explosion device. Under a saturated steam pressure of 1.5 MPa, the treatment temperature is 200 °C, and the pressure retention time is 120 s for steam explosion pretreatment.

[0037] (2)Drying and pulverizing: The olive pomace obtained in step (1) was vacuum-dried at 50 °C, pulverized by a high-speed pulverizer, and passed through an 80-mesh sieve to obtain a uniform powder, which was placed in a desiccator for later use.

[0038] (3)Subcritical water extraction: The olive pomace obtained in step (2) was subjected to subcritical water extraction using a subcritical water extraction device. Accurately weigh 5.00 ± 0.10 g of the sample after steam explosion pretreatment, fix the extraction pressure at 3.0 MPa, the extraction temperature at 130 °C, the extraction time at 30 min, and the solid-liquid ratio at 1:20 g / mL for subcritical water extraction treatment. After the treatment, it was immediately cooled to room temperature, filtered to collect the extract, extracted 2 times repeatedly, the filtrates were combined, and the volume was fixed to 400 mL.

[0039] (4)Liquid analysis and detection of polyphenol content: The extract obtained in (3) was analyzed by liquid chromatography. Under the conditions of a wavelength of 280 nm, a column temperature of 30 °C, a flow rate of 1.0 mL / min, and an injection volume of 20 μL, the content changes of different olive polyphenols were analyzed and detected in a specific mobile phase by gradient elution. After polyphenol content determination and liquid chromatography detection, the polyphenol content increased by 113.02% compared with the traditional method. Implementation case comparative experiment: As shown in Table 1 and Table 2, the enrichment of polyphenols from olive pomace by steam explosion pretreatment coupled with subcritical water extraction has a significant improvement in polyphenol yield, operation time, and solvent usage cost compared with other combinations such as ultrasonic-assisted ethanol extraction. At the same time, it can also promote a higher enrichment of polyphenols released from bound phenols such as flavonoids.

[0040]

[0041]

Claims

1. A green method for efficiently enriching polyphenols from olive pomace by coupling steam explosion pretreatment with subcritical water extraction, characterized in that, The steps are as follows: The first step, steam explosion pretreatment: Weigh the soaked and wetted olive pomace and use steam explosion equipment to conduct steam explosion pretreatment with saturated steam water under a certain pressure. The pretreatment is carried out at a temperature range of 152 - 212 °C under water-saturated steam pressure, and the pressure retention time range is 60 s - 240 s; The second step, drying and pulverizing: Dry the olive pomace after steam explosion at a constant temperature of 50 °C, pulverize it with a high-speed pulverizer and then sieve it to obtain a uniform powder, and place it in a dryer at room temperature for standby; The third step, subcritical water extraction: Use a subcritical water extraction device (HAWG type reactor) to conduct subcritical water extraction on the olive pomace pretreated by steam explosion (under the treatment conditions of a saturated vapor pressure of 1.5 MPa and a pressure retention time of 120 s). Conduct subcritical water extraction experiments on the treated samples under certain pressure, extraction temperature and time conditions; After heating the extraction kettle to the target temperature and maintaining the set time, immediately cool it to room temperature, filter and collect the extract, repeat the extraction 2 times, combine the filtrates and make up the volume, and store it at 4 °C for testing; The fourth step, liquid-phase analysis and detection of polyphenol content: Analyze and detect the content change of olive polyphenols under different conditions in a specific mobile phase by gradient elution at a certain wavelength, temperature, flow rate, time and injection volume.

2. The process for enriching polyphenols from olive pomace according to claim 1, wherein, The water-saturated pressure of the steam explosion pretreatment in the first step is 0.5, 1.0, 1.5 or 2.0 MPa.

3. The moisture content range of the dried olive pomace in the second step as described in claim 1 is 3 - 5%.

4. In the third step of subcritical water extraction as described in claim 1, the fixed extraction pressure is 3.0 MPa; the extraction temperature range is 120 - 140 °C; the extraction time range is 10 - 30 min; the solid-liquid ratio range is 1:20 - 1:40, with the unit of g / mL.

5. The specific liquid-phase detection and analysis conditions in the fourth step as described in claim 1 are: Gradient elution 0 - 6 min: 90 - 70% B, 6 - 13 min: 70 - 60% B, 13 - 17 min: 60 - 55% B, 17 - 19 min: 55 - 51.7% B, 19 - 27 min: 51.7 - 48.5% B, 27 - 28 min: 48.5 - 45% B, 28 - 35 min: 45 - 23% B, 35 - 40 min: 23 - 95% B, 40 - 45 min: 95 - 90% B, 45 - 60 min: 90 - 90% B, the wavelength is 280 nm, the injection volume is 20 μL, the elution time is 60.01 min, and the flow rate is 1.0 mL / min. The column used is Agilent ZORBAX SB-Aq (250×4.6 mm, 5 μm).

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