Aspergillus foetidus slp7-15 and application thereof in extracting polyphenols from pomegranate peel

Abstract

This invention discloses a *Aspergillus spp.* SLP7-15 and its application in the extraction of polyphenols from pomegranate peel. The fermentation broth obtained from *Aspergillus spp.* SLP7-15 culture is filtered, and the filtrate is mixed with pomegranate peel powder. Enzymatic hydrolysis is performed at 35–40℃ for 4–6 hours, followed by the addition of deionized water and anhydrous ethanol. The mixture is then subjected to ultrasonic extraction, filtered, evaporated under reduced pressure, dissolved in anhydrous ethanol, and vacuum dried to obtain the polyphenol extract. Before the ultrasonic extraction of pomegranate peel polyphenols with ethanol, the addition of crude enzyme solution prepared from *Aspergillus spp.* SLP7-15 fermentation facilitates the dissolution of polyphenols, significantly improving the polyphenol extraction yield. The microbial enzymatic hydrolysis-assisted extraction method for pomegranate peel polyphenols provided by this invention can increase the polyphenol extraction yield by 26.6% compared to the direct ultrasonic extraction method with ethanol.

Description

(I) Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a strain of Aspergillus oryzae SLP7-15 and its application in the extraction of polyphenols from pomegranate peel. (II) Background Technology

[0002] Pomegranate (Punica granatum), also known as Anshiliu, is a deciduous shrub or small tree belonging to the genus Punica in the family Lythraceae. The fruit of the pomegranate is what people commonly refer to as "pomegranate." The fresh fruit can be eaten directly or used to produce pomegranate juice, pomegranate wine, and other beverages. The peel of the pomegranate contains various bioactive components, including polyphenols, organic acids, pectin, alkaloids, and polysaccharides. Its extracts exhibit good antibacterial, antiviral, antitumor, antioxidant, immunomodulatory, and digestive-improving effects. Pomegranate peel is a commonly used traditional Chinese medicine, included in all editions of the Chinese Pharmacopoeia (medicinal name: Pericarpium Granati). It is acidic and astringent in nature, warm in taste, and enters the large intestine meridian; it has astringent, antidiarrheal, hemostatic, and anthelmintic functions, and is mainly used to treat chronic diarrhea, chronic dysentery, hematochezia, rectal prolapse, metrorrhagia, leukorrhea, and abdominal pain due to intestinal parasites.

[0003] According to incomplete statistics, my country's pomegranate planting area has reached 1.75 million mu (approximately 116,667 hectares), with an annual output of 1 million tons, ranking first in the world. Well-known pomegranate producing areas include Lintong in Shaanxi, Huaiyuan in Anhui, Zaozhuang in Shandong, Yecheng in Xinjiang Uygur Autonomous Region, Mengzi in Yunnan, Xingyang in Henan, and Huili in Sichuan. In recent years, due to the promotion of seedless pomegranates, the national pomegranate cultivation area continues to increase. A portion of pomegranates is sold as fresh fruit, and a large portion is used to produce pomegranate beverages, resulting in a significant waste of resources as a large amount of pomegranate peel is not fully utilized. Pomegranate peel has a relatively high polyphenol content, and multiple studies have shown that pomegranate peel polyphenols possess various biological activities such as antioxidant, anti-aging, antibacterial, blood pressure lowering, and prevention of cardiovascular and cerebrovascular diseases. Therefore, developing and producing pomegranate peel polyphenols and applying them in the food industry has broad prospects.

[0004] Currently, there are numerous reports on the extraction of polyphenols from pomegranate peel, primarily using ethanol extraction. While conventional ethanol extraction is simple, the yield is often low. Supplementing ethanol extraction with microwave, ultrasonic, or enzymatic treatments can promote polyphenol dissolution and improve the extraction yield to some extent. Different extraction methods have their own advantages and disadvantages. For example, microwave-assisted extraction is quick and efficient, but polyphenols are easily degraded by microwaves; ultrasonic-assisted extraction requires less equipment and does not significantly increase extraction costs, but its effect on improving the extraction yield is not significant; enzymatic extraction can significantly improve the extraction yield, and the conditions are mild, resulting in high-activity polyphenols, but the disadvantage is increased enzyme usage costs.

[0005] Among the enzymatic methods for extracting effective components from plants, cellulase is the most commonly used, followed by pectinase and protease, or two or three enzymes may be used simultaneously. Plant cell walls consist of substances such as cellulose, hemicellulose, lignin, and pectin, so using a single enzyme has very limited effect on improving the extraction yield. Many studies use multiple enzymes (compound enzymes) simultaneously. For example, Wang Huabin et al. used cellulase and pectinase (mass ratio 2:1) to enzymatically hydrolyze pomegranate peel to extract polyphenols, achieving a yield of 23.87%, which is 16.84% higher than the non-enzymatic extraction method [Wang Huabin, Wang Shan, Fu Li. Research on enzymatic extraction process of polyphenols from pomegranate peel. China Food Journal, 2012, 12(6):56-65]. Using compound enzymes to improve the polyphenol extraction yield undoubtedly increases the extraction cost.

[0006] Microorganisms can produce a variety of hydrolytic enzymes that break down plant tissues, including cellulase, hemicellulase, ligninase, and pectinase, especially some molds and actinomycetes. Therefore, if a certain mold is cultured under suitable conditions, the fermentation broth will contain a large number of hydrolytic enzymes. Using the fermentation broth (crude enzyme solution) to directly hydrolyze plant raw materials, these enzymes can synergistically decompose cell wall components, thereby facilitating the release of active ingredients and improving the extraction yield. Compared to using pure enzymes, the cost can be significantly reduced.

[0007] To improve the extraction yield of polyphenols from pomegranate peel, this invention uses crude enzyme solution prepared by microbial fermentation to hydrolyze pomegranate peel, followed by extraction of polyphenols using an ultrasonic ethanol method, which can significantly improve the extraction yield. (III) Summary of the Invention

[0008] The purpose of this invention is to provide a novel microbial strain—Aspergillus foetidus SLP7-15—and its application in the extraction of polyphenols from pomegranate peel. Enzymatic hydrolysis of pomegranate peel with crude enzyme solution prepared by fermentation using this strain can significantly improve the polyphenol extraction yield.

[0009] The technical solution adopted in this invention is:

[0010] This invention provides a novel microbial strain—Aspergillus foetidus SLP7-15, deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No:63213, deposit date March 1, 2023, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province; postcode 510070.

[0011] The *Aspergillus sulphureus* SLP7-15 described in this invention is an excellent strain isolated from microbial enrichment cultures of pomegranate peel and obtained through screening. The morphological characteristics of *Aspergillus sulphureus* SLP7-15 are as follows: When cultured on potato dextrose agar (PDA) plates at 28°C, the initial mycelium is light yellow. After 48 hours of culture, the colony edges have irregular radial wrinkles, and the surface has numerous black granules; the reverse side of the colony is grayish-white. Conidiophores occur in the substrate and are 8–15 μm in diameter. The conidia are typically chrysanthemum-shaped, 50–150 μm in diameter, with spherical or nearly spherical vesicles, 20–35 μm in diameter. A double layer of conidiophores of varying lengths surrounds the vesicles, with conidia produced on the outer layer. The conidia are dark brown, spherical, 3.5–5.0 μm in diameter, and have a slightly rough surface. See the colony photos of Aspergillus smut SLP7-15 cultured on PDA plates at 28°C for 3 days. Figure 1 .

[0012] The nucleotide sequence of the ribosomal DNA internal transcribed spacer (rDNA-ITS) of Aspergillus spp. SLP7-15 is shown in SEQ ID NO.1.

[0013] The present invention also provides an application of the aforementioned Aspergillus oryzae SLP7-15 in the extraction of polyphenols from pomegranate peel. The method of application is as follows: (1) the fermentation broth obtained by culturing Aspergillus oryzae SLP7-15 is filtered, and the collected filtrate is crude enzyme solution; (2) the crude enzyme solution is mixed with pomegranate peel powder and enzymatically hydrolyzed at 35–40℃ for 4–6 h to obtain pomegranate peel enzymatic hydrolysate; (3) after adding deionized water and anhydrous ethanol to the pomegranate peel enzymatic hydrolysate, ultrasonic extraction is performed, and the mixture is filtered to obtain polyphenol extract; (4) the polyphenol extract is evaporated under reduced pressure, dissolved in anhydrous ethanol, and vacuum dried to obtain polyphenol extract.

[0014] Further, the method for preparing the crude enzyme solution in step (1) is as follows: Aspergillus spores SLP7-15 are inoculated into the enzyme-producing medium and cultured at 30℃ and 160–200 r / min for 56–64 h with shaking. The fermentation broth is filtered, and the filtrate is the crude enzyme solution. The final concentration composition of the enzyme-producing medium is: wheat bran 40–50 g / L, (NH4)2SO4 5–6 g / L, peptone 3–4 g / L, KH2PO4 1–2 g / L, MgSO4·7H2O 0.5–1 g / L, CaCl2 0.4–0.5 g / L, the solvent is tap water, and the pH is 6.0–6.5.

[0015] Furthermore, the preferred composition of the enzyme-producing culture medium is: 50 g / L wheat bran, 6 g / L (NH4)2SO4, 4 g / L peptone, 2 g / L KH2PO4, 1 g / L MgSO4·7H2O, 0.5 g / L CaCl2, with tap water as the solvent and pH 6.0.

[0016] Before the enzyme-producing culture of Aspergillus oryzae SLP7-15 described in this invention, it needs to be cultured on a plate medium to produce spores. Then, the spores are suspended in physiological saline to obtain Aspergillus oryzae SLP7-15 spore solution, which is then inoculated into the enzyme-producing medium at a volume fraction of 4%–5% for further culture. The specific enzyme-producing culture method is as follows:

[0017] ①Spore suspension preparation: Aspergillus smut SLP7-15 was inoculated onto potato dextrose agar (PDA) plates and cultured at 28℃ for 60–72 h to obtain plate cultures; sterile physiological saline was added to the plate cultures, and the spores were suspended by stirring with an inoculation loop to obtain Aspergillus smut SLP7-15 spore suspension; the PDA plate culture medium was a commercially available potato dextrose agar medium produced by Qingdao Haibo Biotechnology Co., Ltd., prepared with tap water at a concentration of 46 g / L, with natural pH, and sterilized by high-pressure steam at 121℃ for 20 min.

[0018] ② Enzyme production culture: The Aspergillus spore solution prepared in step ① was inoculated into the enzyme production culture medium at an inoculation rate of 4%–5% by volume, and cultured at 30℃ and shaking at 160–200 r / min for 56–64 h to obtain a fermentation broth with a dry cell concentration of 4.15–4.32 g / L and a cellulase activity of 76.2–82.7 U / mL.

[0019] Furthermore, the volume of crude enzyme solution used in step (2) is 4–6 mL / g based on the mass of pomegranate peel powder [i.e., the material-to-enzyme ratio is 1:4–1:6 (g:mL)]; the pomegranate peel powder is the peel of the plant pomegranate (Punica granatum), which is dried at 85°C and then pulverized through a 60-mesh sieve.

[0020] Further, the preparation method of the polyphenol extract in step (3) is as follows: After the enzymatic hydrolysis of pomegranate peel is completed, deionized water and anhydrous ethanol are added to the pomegranate peel hydrolysate to make the material-to-liquid ratio of the system reach 1:20–1:25 (g:mL) and the volume fraction of ethanol reach 50%–60%. After stirring evenly, it is extracted for 50–60 min in an ultrasonic cleaner with a water temperature of 60–70℃ and a power of 150W; after the ultrasonic ethanol extraction is completed, it is filtered through a Buchner funnel while hot to obtain the polyphenol extract.

[0021] Further, the method for recovering polyphenols from the polyphenol extract in step (4) is as follows: the polyphenol extract is distilled at 45°C and –0.1mPa until no liquid flows out, anhydrous ethanol with a volume of 1–1.5 mL / g based on the weight of the raw pomegranate peel is added, and after thorough shaking, it is centrifuged at 8000 r / min for 5–10 min. The supernatant is transferred to a clean petri dish and vacuum dried at 50°C and –0.1mPa to obtain the polyphenol extract.

[0022] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following: before the ultrasonic extraction of pomegranate peel polyphenols with ethanol, a crude enzyme solution prepared by microbial fermentation is added for enzymatic hydrolysis. The enzyme-producing microorganism used, *Aspergillus smutica* SLP7-15, was specifically isolated and screened for its ability to hydrolyze the cell walls of pomegranate peel and obtained through mutagenesis. The crude enzyme solution prepared by enzyme-producing fermentation contains highly active cellulase and other hydrolytic enzymes, which can synergistically hydrolyze substances such as cellulose, hemicellulose, and pectin in pomegranate peel, causing cell wall damage and facilitating the dissolution of polyphenols, thus significantly improving the polyphenol extraction yield. The microbial enzymatic hydrolysis-assisted extraction method for pomegranate peel polyphenols provided by this invention can increase the polyphenol extraction yield by 26.6% compared with the direct ultrasonic extraction method with ethanol. (iv) Description of the attached drawings

[0023] Figure 1 This is a colony photo of Aspergillus smut SLP7-15 cultured on a PDA at 28°C for 3 days.

[0024] Figure 2 A standard curve for spectrophotometric determination of polyphenols (gallic acid).

[0025] Figure 3 This is the standard curve for glucose determination using the DNS method. (V) Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0027] The pomegranate peel described in this embodiment of the invention is the pericarp of the pomegranate (Punica granatum) plant, and the pomegranate peel powder is the fine powder obtained by drying the pomegranate peel at 85°C and then pulverizing it through a 60-mesh sieve.

[0028] Example 1: Isolation and screening of enzyme-producing strains

[0029] Microbial strains that produce enzymes to hydrolyze pomegranate peel were isolated and screened using the following steps:

[0030] (1) 5g of pomegranate peel powder was placed in a 50-mL Erlenmeyer flask, along with 1g of potting soil and 10mL of sterile physiological saline. The mixture was stirred thoroughly and incubated at 28℃ for 72h. The enriched culture contaminated with mold was then diluted with sterile physiological saline to a concentration of 1×10⁻⁶.-6 1×10 -7 1×10 -8 After dilution, 0.1 mL of the diluted solution was spread onto potato dextrose agar (PDA) plates and incubated at 28°C for 48 h. Colonies of different colors and morphologies were then transferred to fresh PDA plates and incubated at 28°C for 72 h to obtain 11 pure cultured mold strains. The strain numbers are shown in Table 1.

[0031] (2) 10 mL of sterile physiological saline was added to each of the 11 strains of fresh plate culture, and the spores were suspended by stirring with an inoculation loop to obtain the spore solution of each strain.

[0032] (3) Take 2 mL of the spore liquid of each strain prepared in step (2) and inoculate it into 50 mL of enzyme production medium (the inoculation amount is 4% by volume). After culturing for 64 h under the conditions of 30℃ and 160 r / min shaking, filter all the fermentation broth with a Buchner funnel. The collected filtrate is the crude enzyme solution.

[0033] (4) Add 1g of pomegranate peel powder to each of the 11 50-mL centrifuge tubes, add 6mL of crude enzyme solution of each strain prepared in step (3) [i.e., the material-enzyme ratio is 1:6 (g:mL)], stir evenly, and then enzymatically hydrolyze in a 35℃ water bath for 6h to obtain pomegranate peel hydrolysate.

[0034] (5) In step (4), 4 mL of deionized water and 10 mL of anhydrous ethanol were added to all the pomegranate peel hydrolysates obtained from the crude enzyme solutions of each strain [the system ratio of material to liquid is 1:20 (g:mL), and the ethanol volume fraction is 50%]. After shaking well, the mixture was extracted for 50 min in an ultrasonic cleaner at 60℃ and 150W. The mixture was then filtered through a Buchner funnel while hot, and the filtrate was collected and the polyphenol content was determined by spectrophotometry.

[0035] Starting from step (4), 6 mL of uninoculated enzyme-producing culture medium was used as a control instead of the crude enzyme solution; 6 mL of cellulase phosphate buffer (pH 6.0, 0.2 mol / L) with an activity of 2000 U / mL was used as a control instead of the crude enzyme solution for cellulase digestion. The polyphenol extraction yields of pomegranate peel digested with crude enzyme solutions of different strains and the control are shown in Table 1.

[0036] Table 1. Polyphenol extraction yield from pomegranate peel after enzymatic hydrolysis with crude enzyme solutions from different strains and the control.

[0037]

[0038] As shown in Table 1, the polyphenol extraction rate of pomegranate peel after enzymatic hydrolysis with crude enzyme solution prepared by SLP7 strain was 21.3%, which was 14.5% higher than the 18.6% of the un-hydrolyzed control. The polyphenol extraction rate of pomegranate peel hydrolyzed with cellulase was 20.2%, which was 8.60% higher than the un-hydrolyzed control, but still far lower than that of the crude enzyme solution prepared by SLP7 strain. Enzymatic hydrolysis of pomegranate peel with crude enzyme solution by some microbial strains did not improve the polyphenol extraction rate, and even significantly reduced it, such as SLP3 and SLP10 strains. This indicates that the microbial strains used for enzymatic hydrolysis of pomegranate peel to improve polyphenol extraction rate are selective; they must not only produce enzymes to hydrolyze pomegranate peel cell wall components, but also not produce enzymes to degrade polyphenols. In this invention, SLP7 strain was selected as the enzyme-producing strain for improving the polyphenol extraction rate of pomegranate peel.

[0039] The PDA plate culture medium is a ready-made potato dextrose agar medium produced by Qingdao Haibo Biotechnology Co., Ltd. It is prepared with tap water at a concentration of 46g / L, with natural pH, and sterilized by high-pressure steam at 121℃ for 20min. Before solidification, it is poured into sterile petri dishes with a diameter of 9cm, 15-20mL per dish.

[0040] The final concentration composition and preparation method of the enzyme-producing culture medium are as follows: 50 g / L wheat bran, 6 g / L (NH4)2SO4, 4 g / L peptone, 2 g / L KH2PO4, 1 g / L MgSO4·7H2O, 0.5 g / L CaCl2, with tap water as the solvent and pH 6.0. 50 mL of the enzyme-producing culture medium is placed in a 250 mL Erlenmeyer flask, sealed with 8 layers of gauze, and sterilized by autoclaving at 121°C for 20 min.

[0041] The polyphenol content was determined using the Folin-Ciocalteu colorimetric method. The specific method was as follows: 1 mL of the sample solution was added to a 10 mL stoppered test tube, followed by the addition of 0.5 mL of Folin-Ciocalteu reagent and 1.5 mL of a 15% sodium carbonate (Na₂CO₃) aqueous solution. The volume was then adjusted to 10 mL with deionized water. After standing at 25°C in the dark for 2 hours, the absorbance (A) was measured at 765 nm. 765 The measured absorbance is determined by the gallic acid concentration – A 765 Standard curve ( Figure 2 The concentration of gallic acid in the sample was calculated.

[0042] Gallic acid concentration – A 765Construction of the standard curve: Transfer 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of 100 mg / L gallic acid standard solution to 10⁻⁵ mL stoppered test tubes. Add 0.5 mL of Folin-Ciocalteu reagent and 1.5 mL of 15% sodium carbonate aqueous solution sequentially. Then, dilute to 10 mL with deionized water. Incubate at 25°C in the dark for 2 hours, and measure the absorbance (A) at 765 nm. 765 ), with gallic acid concentration as the x-axis, A 765 Plot a standard curve for the ordinate ( Figure 2 ).

[0043] Preparation of a 100 mg / L gallic acid standard solution: Accurately weigh 10 mg of gallic acid standard, dissolve it in 5 mL of anhydrous ethanol, and then dilute to 100 mL with deionized water.

[0044] The yield of polyphenols extracted from pomegranate peel is calculated according to Formula 1:

[0045]

[0046] Example 2: Mutagenesis and selection of enzyme-producing strain SLP7

[0047] Mutagenesis breeding was performed on strain SLP7 to screen for strains with excellent enzyme production performance. The specific method is as follows:

[0048] (1) Preparation of spore suspension: After SLP7 strain was activated and cultured on PDA agar at 28°C for 48 h, 5 mL of sterile physiological saline was added, and the spores were suspended by stirring with an inoculation loop. 1 mL of the spore suspension was transferred to an Erlenmeyer flask containing 50 mL of sterile physiological saline (with 20–30 glass beads added), and shaken at room temperature for 15 min. The spore suspension was filtered to remove mycelia (a small wad of fluffy cotton was placed at the bottom of the Erlenmeyer funnel). The spores in the spore suspension were counted under a microscope using a hemocytometer. The spores were then diluted appropriately with sterile physiological saline to adjust the spore count to 1.55 × 10⁻⁶. 7 per mL.

[0049] (2) Mutagenesis: Under red light illumination, 1.5 mL of the above spore solution and a sterile paperclip were placed in six 6 cm diameter petri dishes. The petri dishes were placed on magnetic stirrers and irradiated for 1, 2, 3, 4, 5, and 6 mins respectively at a distance of 30 cm from a 15W UV lamp that had been preheated for 30 min. 0.5 mL of the irradiated spore solution was taken, diluted appropriately, and 0.1 mL was transferred to PDA agar plates. The same procedure was performed to dilute and plate the spore solution without UV irradiation as a control to calculate the lethality rate. After inoculation, the PDA plates were wrapped in black cloth, inverted, and incubated at 28℃ for 48 h. The colonies on the plates were counted, and the lethality rate was calculated.

[0050] (3) Screening: Colonies with a lethality rate of over 90% on PDA plates were selected and transferred to fresh PDA plates to obtain 35 strains. 10 mL of sterile physiological saline was added to each strain's fresh plate culture after 56 h of incubation at 28°C, and the spores were suspended by stirring with an inoculation loop to obtain the spore solution for each strain. 2 mL of the spore solution from each strain was inoculated into 50 mL of enzyme-producing medium and cultured at 30°C and 200 r / min for 72 h. The fermentation broth was then filtered using a Buchner funnel, and the filtrate (i.e., crude enzyme solution) was collected. The cellulase activity of the fermentation filtrate from each strain was first determined. Twelve strains with relatively higher enzyme activity than the original strain SLP7 were selected. Following the method in Example 1, the crude enzyme solution from these strains was used to enzymatically hydrolyze pomegranate peel, and polyphenols were extracted by ultrasonic extraction with ethanol. The polyphenol extraction yields from pomegranate peel hydrolyzed by the crude enzyme solution of the mutant strain and the control are shown in Table 2.

[0051] Table 2. Polyphenol extraction yield from pomegranate peel hydrolyzed with crude enzyme solution of mutant strain and control strain.

[0052]

[0053] As shown in Table 2, among the 12 strains screened, strain SLP7-15 exhibited a cellulase activity of 81.4 U / mL, which was 28.6% higher than the 63.3 U / mL of the wild-type strain SLP7. The crude enzyme solution prepared by fermentation using this strain yielded a polyphenol extraction rate of 23.4% after hydrolyzing pomegranate peel, which was 9.86% higher than the 21.3% of the wild-type strain SLP7 and 25.8% higher than the 18.6% of the unhydrolyzed control. Therefore, strain SLP7-15 was selected in this invention as the enzyme-producing strain for improving the polyphenol extraction rate from pomegranate peel.

[0054] The final concentration composition and preparation method of the enzyme-producing culture medium are the same as in Example 1.

[0055] The cellulase activity assay was performed as follows: 1.5 mL of 10 g / L sodium carboxymethyl cellulose solution (pH 6.0, prepared with 0.2 mol / L phosphate buffer) and 0.5 mL of crude enzyme solution were added to 10 mL graduated test tubes. The tubes were incubated in a 50°C water bath for 30 min, then 3 mL of DNS reagent was added, and the mixture was boiled for 5 min. After cooling under running water, deionized water was added to bring the volume to 10 mL, and the mixture was shaken well. A crude enzyme solution inactivated by boiling at 100°C for 10 min was used as a reference. The absorbance (A) was measured at 540 nm using a spectrophotometer. 540 ), from the glucose standard curve ( Figure 3Calculate the glucose concentration in the sample, and then calculate the cellulase activity (U / mL). Cellulase activity is defined as the amount of enzyme required to hydrolyze sodium carboxymethyl cellulose to produce 1 μg of glucose per minute under pH 6.0 and 50℃ conditions; one unit of enzyme activity (U) is defined as this amount of enzyme.

[0056] Cellulase activity is calculated according to Formula 2.

[0057]

[0058] In Formula 2, C: glucose concentration (μg / mL) calculated from the standard curve; V1: enzyme reaction system volume, i.e., 2 mL; T: reaction time, i.e., 30 min; V2: crude enzyme solution volume, i.e., 0.5 mL.

[0059] Construction of the glucose standard curve: Add 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of 1 g / L standard glucose aqueous solution to seven 10-mL graduated test tubes, respectively. Then add 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, and 0.8 mL of pH 6.0, 0.2 mol / L phosphate buffer to each tube, respectively. Add 3.0 mL of DNS solution to each tube. Boil the mixture in a water bath for 5 minutes, cool under running water, and then dilute to 10 mL with deionized water. Shake well and measure A using a spectrophotometer. 540 Plotting glucose concentration on the x-axis, A 540 Plot a standard curve for the ordinate ( Figure 3 ).

[0060] Preparation of DNS reagent: Add 6.3g of 3,5-dinitrosalicylic acid and 262mL of 2mol / L NaOH aqueous solution to 500mL of hot aqueous solution containing 182g of sodium tartrate, then add 5g of redistilled phenol and 5g of sodium sulfite, stir to dissolve, cool, and add deionized water to make up to 1L. Store in a brown bottle and use after 7 days.

[0061] Example 3: Classification and identification of strain SLP7-15

[0062] Strain SLP7-15 was streaked onto PDA plates and cultured at 28°C. Initially, the mycelia were pale yellow. After 48 hours of culture, the colony edges developed irregular radial wrinkles, and the surface contained numerous black granules. The reverse side of the colony was grayish-white. Conidiophores developed in the substrate, with a diameter of 8–15 μm. The conidia heads were typically chrysanthemum-shaped, with a diameter of 50–150 μm. The vesicles were spherical or nearly spherical, with a diameter of 20–35 μm. A double layer of conidiophores of varying lengths was present around the vesicles, with conidia produced on the outer layer. The conidia were dark brown, spherical, with a diameter of 3.5–5.0 μm and a slightly rough surface. A photograph of Aspergillus spp. SLP7-15 colonies cultured on PDA plates at 28°C for 3 days is shown below. Figure 1 .

[0063] The rDNA-ITS nucleotide sequence of strain SLP7-15 was determined as shown in SEQ ID NO.1. This sequence was BLAST-aligned with typical Aspergillus foetidus strains CBS121.28, Aspergillus welwitschiae CBS139.54, and Aspergillus niger strains. The rDNA-ITS sequences of strain SLP7-15 (niger)ATCC16888 showed 100% homology, but the colony morphology of strain SLP7-15 was more consistent with the characteristics of Aspergillus foetidus. Therefore, the biological taxonomic position of strain SLP7-15 can be determined as follows (refer to NCBI, http: / / www.ncbi.nlm.nih.gov): Kingdom Fungi, Phylum Ascomycota, Subphylum Pezizomycotina, Class Eurotiomycetes, Subclass Eurotiomycetidae, Order Eurotiales, Family Aspergillaceae, Genus Aspergillus, Species Aspergillus foetidus.

[0064] The rDNA-ITS nucleotide sequence of the strain SLP7-15 is as follows:

[0065] .

[0066] In summary, strain SLP7 was isolated from the microbial enrichment of pomegranate peel powder. After UV mutagenesis, strain SLP7-15, namely Aspergillus foetidus SLP7-15, was obtained through screening. This strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No:63213, deposit date March 1, 2022, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China.

[0067] Example 4: Application of Aspergillus oryzae SLP7-15 enzymatic hydrolysis-assisted extraction of pomegranate peel polyphenols

[0068] The following steps can be taken to extract polyphenols from pomegranate peel using enzymatic hydrolysis assisted by Aspergillus oryzae SLP7-15:

[0069] (1) Aspergillus spore powder SLP7-15 preserved in freeze-dried tubes was inoculated onto fresh PDA plate medium and cultured at 28°C for 68 h. 10 mL of sterile physiological saline was added to the plate culture, and the spores were suspended by stirring with an inoculation loop to obtain a spore solution of Aspergillus spores SLP7-15. The composition and preparation method of the PDA plate medium were the same as in Example 1.

[0070] (2) Inoculate 4 mL of the spore solution prepared in step (1) into 100 mL of enzyme-producing medium (inoculation amount of 4% by volume), and culture at 30℃ and 160 r / min for 64 h to obtain a fermentation broth with a dry cell concentration of 4.32 g / L. Filter all fermentation broth using a Buchner funnel, and the collected filtrate is the crude enzyme solution, with a cellulase activity of 82.7 U / mL. The final concentration composition of the enzyme-producing medium is the same as in Example 1. 100 mL of enzyme-producing medium is placed in a 250-mL Erlenmeyer flask, sealed with 8 layers of gauze, and sterilized by high-pressure steam at 121℃ for 20 min.

[0071] (3) Put 10g of pomegranate peel powder into a 250-mL beaker, add 60mL of crude enzyme solution prepared in step (2) [i.e., the material-to-enzyme ratio is 1:6 (g:mL)], stir evenly, and then enzymatically hydrolyze in a 35℃ water bath for 6h to obtain pomegranate peel hydrolysate.

[0072] (4) In step (3), add 40 mL of deionized water and 100 mL of anhydrous ethanol to all the pomegranate peel enzymatic hydrolysate [system material-to-liquid ratio 1:20 (g:mL), ethanol volume fraction 50%]. Shake well and extract for 50 min in an ultrasonic cleaner at 60℃ and 150W. After ultrasonic extraction, filter through a Buchner funnel while hot to obtain the polyphenol extract.

[0073] (5) The polyphenol extract prepared in step (4) was distilled under reduced pressure at 45°C and -0.1 mPa until no liquid flowed out. 10 mL of anhydrous ethanol (1 mL / g based on the mass of the raw pomegranate peel powder) was added, and the mixture was shaken thoroughly and transferred to a centrifuge tube. The mixture was centrifuged at 8000 r / min for 5 min. The supernatant was transferred to a clean petri dish and dried under vacuum at 50°C and -0.1 mPa to obtain the polyphenol extract.

[0074] Following the steps above, 4.87g of polyphenol extract was obtained from 10g of pomegranate peel, with a polyphenol content of 47.9%, yielding 2.33g of polyphenols, representing an extraction yield of 23.3%.

[0075] Comparative Example 1: Extraction of polyphenols from pomegranate peel without enzymatic hydrolysis (compared to Example 4)

[0076] (1) 10g of pomegranate peel powder was placed in a 250-mL beaker, and 60mL of deionized water was added. After stirring evenly, the mixture was kept in a 35℃ water bath for 6 hours. Then, 40mL of deionized water and 100mL of anhydrous ethanol were added [the system material-liquid ratio was 1:20 (g:mL), and the ethanol volume fraction was 50%]. After shaking evenly, the mixture was extracted for 50 minutes in an ultrasonic cleaner at 60℃ and 150W. After the ultrasonic ethanol extraction was completed, the mixture was filtered through a Buchner funnel while still hot to obtain the polyphenol extract.

[0077] (2) The polyphenol extract prepared in step (1) was distilled under reduced pressure at 45°C and -0.1 mPa until no liquid flowed out. 10 mL of anhydrous ethanol (1 mL / g based on the mass of the raw pomegranate peel powder) was added, and the mixture was shaken thoroughly and transferred to a centrifuge tube. The mixture was centrifuged at 8000 r / min for 5 min, and the supernatant was transferred to a clean petri dish and dried under vacuum at 50°C and -0.1 mPa to obtain the polyphenol extract.

[0078] Following the steps above, 4.05g of polyphenol extract was obtained from 10g of pomegranate peel, with a polyphenol content of 45.7%, resulting in 1.85g of polyphenols and an extraction yield of 18.5%.

[0079] Comparing the results of Example 4 and Comparative Example 1, it can be seen that before extracting pomegranate peel polyphenols, adding the crude enzyme solution prepared by Aspergillus oryzae SLP7-15 fermentation to hydrolyze the polyphenols increased the polyphenol extraction yield from 18.5% to 23.3%, an increase of 25.9%.

[0080] Example 5: Application of using Aspergillus oryzae SLP7-15 enzymatic hydrolysis to assist in the extraction of pomegranate peel polyphenols

[0081] The polyphenols extracted from pomegranate peel using Aspergillus oryzae SLP7-15 enzymatic hydrolysis can be performed according to the following steps:

[0082] (1) Aspergillus spores SLP7-15 PDA colonies stored at 4℃ were inoculated onto fresh PDA agar plates and cultured at 28℃ for 64 h. 10 mL of sterile physiological saline was added to the agar plate culture, and the spores were suspended by stirring with an inoculation loop to obtain a spore suspension of Aspergillus spores SLP7-15. The composition and preparation method of the PDA agar plate culture medium were the same as in Example 1.

[0083] (2) Inoculate 5 mL of the spore solution prepared in step (1) into 100 mL of enzyme-producing medium (inoculation amount of 5% by volume), and culture at 30℃ and 180 r / min for 60 h to obtain a fermentation broth with a dry cell concentration of 4.18 g / L. Filter all the fermentation broth using a Buchner funnel, and the collected filtrate is the crude enzyme solution. The cellulase activity of the filtrate is 78.4 U / mL. The final concentration composition and preparation method of the enzyme-producing medium are the same as in Example 4.

[0084] (3) Put 10g of pomegranate peel powder into a 250-mL beaker, add 50mL of crude enzyme solution prepared in step (2) [i.e., the material-to-enzyme ratio is 1:5 (g:mL)], stir evenly, and then enzymatically hydrolyze in a 40℃ water bath for 5h to obtain pomegranate peel hydrolysate.

[0085] (4) In step (3), add 55 mL of deionized water and 125 mL of anhydrous ethanol to all the pomegranate peel enzymatic hydrolysate [system material-to-liquid ratio 1:23 (g:mL), ethanol volume fraction 54.3%]. Shake well and extract for 55 min in an ultrasonic cleaner at 65℃ and 150W. After ultrasonic extraction, filter through a Buchner funnel while hot to obtain the polyphenol extract.

[0086] (5) The polyphenol extract prepared in step (4) was distilled under reduced pressure at 45°C and -0.1 mPa until no liquid flowed out. 15 mL of anhydrous ethanol (1.5 mL / g based on the mass of the raw pomegranate peel powder) was added, and after thorough shaking, it was transferred to a centrifuge tube and centrifuged at 8000 r / min for 8 min. The supernatant was transferred to a clean petri dish and dried under vacuum at 50°C and -0.1 mPa to obtain the polyphenol extract.

[0087] Following the steps above, 5.03g of polyphenol extract was obtained from 10g of pomegranate peel, with a polyphenol content of 48.8%, yielding 2.46g of polyphenols, with an extraction rate of 24.6%.

[0088] Comparative Example 2: Extraction of polyphenols from pomegranate peel without enzymatic hydrolysis (compared to Example 5)

[0089] (1) 10g of pomegranate peel powder was placed in a 250-mL beaker, and 50mL of deionized water was added. After stirring evenly, the mixture was kept in a 40℃ water bath for 5 hours. Then, 55mL of deionized water and 125mL of anhydrous ethanol were added [the system material-liquid ratio was 1:23 (g:mL), and the ethanol volume fraction was 54.3%]. After shaking evenly, the mixture was extracted for 55 minutes in an ultrasonic cleaner at 65℃ and 150W. After the ultrasonic ethanol extraction was completed, the mixture was filtered through a Buchner funnel while still hot to obtain the polyphenol extract.

[0090] (2) The polyphenol extract prepared in step (1) was distilled under reduced pressure at 45°C and -0.1 mPa until no liquid flowed out. 15 mL of anhydrous ethanol (1.5 mL / g based on the mass of the raw pomegranate peel powder) was added. After thorough shaking, the mixture was transferred to a centrifuge tube and centrifuged at 8000 r / min for 8 min. The supernatant was transferred to a clean petri dish and dried under vacuum at 50°C and -0.1 mPa to obtain the polyphenol extract.

[0091] Following the steps above, 4.20g of polyphenol extract was obtained from 10g of pomegranate peel, with a polyphenol content of 46.2%, yielding 1.94g of polyphenols, representing an extraction yield of 19.4%.

[0092] Comparing the results of Example 5 and Comparative Example 2, it can be seen that before extracting pomegranate peel polyphenols, adding the crude enzyme solution prepared by Aspergillus oryzae SLP7-15 fermentation to hydrolyze the polyphenols increased the polyphenol extraction yield from 19.4% to 24.6%, an increase of 26.8%.

[0093] Example 6: Application of using Aspergillus oryzae SLP7-15 enzymatic hydrolysis to assist in the extraction of pomegranate peel polyphenols

[0094] The polyphenols extracted from pomegranate peel using Aspergillus oryzae SLP7-15 enzymatic hydrolysis can be performed according to the following steps:

[0095] (1) Aspergillus spores SLP7-15 PDA colonies stored at 4℃ were inoculated onto fresh PDA agar plates and cultured at 28℃ for 60 h. 10 mL of sterile physiological saline was added to the agar plate culture, and the spores were suspended by stirring with an inoculation loop to obtain a spore suspension of Aspergillus spores SLP7-15. The composition and preparation method of the PDA agar plate culture medium were the same as in Example 1.

[0096] (2) Inoculate 5 mL of the spore solution prepared in step (1) into 100 mL of enzyme-producing medium (inoculation amount of 5% by volume), and culture at 30℃ and 200 r / min for 56 h to obtain a fermentation broth with a dry cell concentration of 4.15 g / L. Filter all the fermentation broth using a Buchner funnel, and the collected filtrate is the crude enzyme solution. The cellulase activity of the filtrate is 76.2 U / mL. The final concentration composition and preparation method of the enzyme-producing medium are the same as in Example 4.

[0097] (3) Put 10g of pomegranate peel powder into a 250-mL beaker, add 40mL of crude enzyme solution prepared in step (2) [i.e., the material-to-enzyme ratio is 1:4 (g:mL)], stir evenly, and then enzymatically hydrolyze in a 40℃ water bath for 4h to obtain pomegranate peel hydrolysate.

[0098] (4) In step (3), add 60 mL of deionized water and 150 mL of anhydrous ethanol to all the pomegranate peel hydrolysate [system ratio 1:25 (g:mL), ethanol volume fraction 60%]. Shake well and extract for 60 min in an ultrasonic cleaner at 70℃ and 150W. After ultrasonic extraction, filter through a Buchner funnel while hot to obtain the polyphenol extract.

[0099] (5) The polyphenol extract prepared in step (4) was distilled under reduced pressure at 45°C and -0.1 mPa until no liquid flowed out. 15 mL of anhydrous ethanol (1.5 mL / g based on the mass of the raw pomegranate peel powder) was added, and after thorough shaking, it was transferred to a centrifuge tube and centrifuged at 8000 r / min for 10 min. The supernatant was transferred to a clean petri dish and dried under vacuum at 50°C and -0.1 mPa to obtain the polyphenol extract.

[0100] Following the steps above, 5.21g of polyphenol extract was obtained from 10g of pomegranate peel, with a polyphenol content of 49.3%, yielding 2.57g of polyphenols, representing an extraction yield of 25.7%.

[0101] Comparative Example 3: Extraction of polyphenols from pomegranate peel without enzymatic hydrolysis (compared to Example 6)

[0102] (1) 10g of pomegranate peel powder was placed in a 250-mL beaker, and 40mL of deionized water was added. After stirring evenly, the mixture was kept in a 40℃ water bath for 4 hours. Then, 60mL of deionized water and 150mL of anhydrous ethanol were added [the system ratio of material to liquid was 1:25 (g:mL), with an ethanol volume fraction of 60%]. After shaking evenly, the mixture was extracted for 60 minutes in an ultrasonic cleaner at 70℃ and 150W. After the ultrasonic extraction was completed, the mixture was filtered through a Buchner funnel while still hot to obtain the polyphenol extract.

[0103] (2) The polyphenol extract prepared in step (1) was distilled under reduced pressure at 45°C and -0.1 mPa until no liquid flowed out. 15 mL of anhydrous ethanol (1.5 mL / g based on the mass of the raw pomegranate peel powder) was added. After thorough shaking, the mixture was transferred to a centrifuge tube and centrifuged at 8000 r / min for 10 min. The supernatant was transferred to a clean petri dish and dried under vacuum at 50°C and -0.1 mPa to obtain the polyphenol extract.

[0104] Following the steps above, 4.34g of polyphenol extract was obtained from 10g of pomegranate peel, with a polyphenol content of 46.7%, yielding 2.03g of polyphenols, with an extraction yield of 20.3%.

[0105] Comparing the results of Example 6 and Comparative Example 3, it can be seen that before extracting pomegranate peel polyphenols, adding the crude enzyme solution prepared by Aspergillus oryzae SLP7-15 fermentation to hydrolyze the polyphenols increased the polyphenol extraction yield from 20.3% to 25.7%, an increase of 26.6%.

Claims

1. Aspergillus smut ( Aspergillus foetidus SLP7-15, characterized in that, The Aspergillus oryzae SLP7-15 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63213.

2. The use of Aspergillus oryzae SLP7-15 as described in claim 1 in the extraction of polyphenols from pomegranate peel.

3. The application as described in claim 2, characterized in that, The method of application is as follows: (1) Filter the fermentation broth obtained by culturing Aspergillus SLP7-15, and collect the filtrate as crude enzyme solution; (2) Mix the crude enzyme solution with pomegranate peel powder, and enzymatically hydrolyze it at 35–40℃ for 4–6 h to obtain pomegranate peel hydrolysate; (3) Add deionized water and anhydrous ethanol to the pomegranate peel hydrolysate, and then perform ultrasonic extraction, filter, and obtain polyphenol extract; (4) Evaporate the polyphenol extract under reduced pressure, dissolve it in anhydrous ethanol, and vacuum dry it to obtain polyphenol extract.

4. The application as described in claim 3, characterized in that, The method for preparing the crude enzyme solution in step (1) is as follows: Aspergillus spores SLP7-15 are inoculated into the enzyme-producing medium and fermented at 30℃ and 160–200 r / min for 56–64 h with shaking. The fermentation broth is filtered, and the filtrate is the crude enzyme solution. The final concentration composition of the enzyme-producing medium is: wheat bran 40–50 g / L, (NH4)2SO4 5–6 g / L, peptone 3–4 g / L, KH2PO4 1–2 g / L, MgSO4·7H2O 0.5–1 g / L, CaCl2 0.4–0.5 g / L, the solvent is tap water, and the pH is 6.0–6.

5.

5. The application as described in claim 4, characterized in that, The enzyme-producing culture medium consisted of: 50 g / L wheat bran, 6 g / L (NH4)2SO4, 4 g / L peptone, 2 g / L KH2PO4, 1 g / L MgSO4·7H2O, 0.5 g / L CaCl2, with tap water as the solvent and a pH of 6.

0.

6. The application as described in claim 3, characterized in that, Before enzyme production culture, Aspergillus spp. SLP7-15 is first cultured on a plate medium to produce spores. Then, the spores are suspended in physiological saline to obtain Aspergillus spp. SLP7-15 spore solution. This solution is then inoculated into the enzyme production medium at a volume fraction of 4%–5%. The culture method for Aspergillus spp. SLP7-15 spore solution is as follows: Aspergillus spp. SLP7-15 is inoculated onto PDA plate medium and cultured at 28℃ for 60–72 h to obtain a plate culture. Sterile physiological saline is added to the plate culture, and the spores are suspended by stirring with an inoculation loop to obtain Aspergillus spp. SLP7-15 spore solution.

7. The application as described in claim 3, characterized in that, The volume of crude enzyme solution used in step (2) is 4–6 mL / g based on the mass of pomegranate peel powder.

8. The application as described in claim 3, characterized in that, The pomegranate peel powder mentioned is from the plant pomegranate (Pomegranate var. sarcodactylis Punica granatum The peel of the fruit is dried at 85℃ and then pulverized into fine powder through a 60-mesh sieve.

9. The application as described in claim 3, characterized in that, The preparation method of the polyphenol extract in step (3) is as follows: add deionized water and anhydrous ethanol to the pomegranate peel enzymatic hydrolysate to make the material-liquid ratio of the system reach 1 g:20 mL–1 g:25 mL and the volume fraction of ethanol reach 50%–60%; after stirring evenly, extract for 50–60 min in an ultrasonic cleaner with a water temperature of 60–70℃ and a power of 150 W; after ultrasonic ethanol extraction, filter through a hot Buchner funnel to obtain the polyphenol extract.

10. The application as described in claim 3, characterized in that, Step (4) The method for recovering polyphenols from the polyphenol extract is as follows: the polyphenol extract is distilled at 45°C and –0.1 mPa until no liquid flows out, anhydrous ethanol with a volume of 1–1.5 mL / g based on the weight of the raw pomegranate peel is added, and after thorough shaking, it is centrifuged at 8000 r / min for 5–10 min. The supernatant is transferred to a clean petri dish and vacuum dried at 50°C and –0.1 mPa to obtain the polyphenol extract.

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