Penicillium pulvinus EJ2-16 and its application in extracting polysaccharides from loquat leaves

Abstract

The invention discloses a kind of cushion-shaped Penicillium EJ2-16 and its application in extracting loquat leaf polysaccharide, add the spore liquid of cushion-shaped Penicillium EJ2-16 to loquat leaf powder, stir evenly and ferment at 30 DEG C for 64-72h, obtain loquat leaf fermentation product; add deionized water to loquat leaf fermentation product, stir evenly and keep warm at 40-45 DEG C for 2-3h, concentrate after ultrasonic water extraction, obtain loquat leaf water extract concentrate; add ethanol to loquat leaf water extract concentrate to precipitate polysaccharide, wash and dry the precipitate with ethanol, obtain loquat leaf polysaccharide. The present invention adds microbial fermentation pretreatment before extracting polysaccharide from loquat leaf, and the fermented loquat leaf powder is enzymolyzed by adding water and keeping warm, which helps the bound loquat leaf polysaccharide to dissolve during ultrasonic water extraction, thereby significantly improving the extraction yield of polysaccharide. Compared with the conventional method without fermentation pretreatment, the extraction rate of loquat leaf polysaccharides can be increased by 62.4%.

Description

(I) Technical field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a strain of Penicillium pulvinus EJ2-16 and application thereof in extracting loquat leaf polysaccharide. (II) Background technology

[0002] Loquat (Eriobotrya japonica), also known as Luju, Luju, Jinwan, is a small evergreen tree of the Rosaceae family. It is distributed in most parts of my country, and is mainly produced in Fujian, Zhejiang, Jiangsu, Hubei and other places. Loquat fruit is a popular fruit. In recent years, the area of ​​loquat planting in the country has gradually expanded. The flowers, leaves, kernels and fruit stalks of loquat can be used as medicine. The medicinal materials in the previous editions of the Pharmacopoeia of the People's Republic of China are all included in loquat leaves. Loquat leaves are bitter in nature and slightly cold, and belong to the lung and stomach meridians. It has the functions of clearing the lungs and relieving cough, descending the adverse and relieving vomiting. It is mainly used to treat lung heat cough, qi reverse wheezing, stomach heat vomiting, and restlessness and thirst. The Chinese patent medicine loquat leaf paste is a concentrated extract after boiling loquat leaves in boiling water, which is used to treat lung heat dry cough, less sputum and dry throat. In recent years, there have been many studies on the chemical composition and pharmacological effects of loquat leaves at home and abroad. It mainly contains volatile oils, triterpenes, sesquiterpenes, flavonoids, polyphenols and polysaccharides. Polysaccharides are one of the active ingredients of loquat leaves, which have anti-tumor, antioxidant, anti-aging, immune enhancement, and blood sugar lowering effects. my country has a large area of ​​loquat trees, and a large number of waste leaves are produced every year. Extracting polysaccharides from loquat leaves and applying them to medicines, functional foods and feeds has good economic significance.

[0003] The basic method for extracting polysaccharides from plants is hot water extraction and ethanol precipitation (abbreviated as "water extraction and alcohol precipitation method"). On this basis, there are auxiliary extraction methods such as acid and alkali, ultrasound, microwave, high pressure and enzymatic hydrolysis. Different extraction methods have their own advantages and disadvantages. For example, the conventional water extraction and alcohol precipitation method is simple to operate, but has disadvantages such as low extraction yield and long time consumption; microwave-assisted extraction method has short extraction time and high efficiency, but polysaccharides are easily destroyed; ultrasound-assisted extraction method has low equipment requirements and will not significantly increase the extraction cost, but the effect of improving the extraction yield is not significant enough; enzymatic hydrolysis-assisted extraction method can significantly improve the extraction yield, and the conditions are mild, and the extracted polysaccharides have good activity. The disadvantage is that the cost of enzyme use is increased and the extraction process is time-consuming.

[0004] In recent years, enzymatic hydrolysis technology has been widely used in the extraction of plant active ingredients. It uses cellulase, pectinase or protease to hydrolyze plant raw materials under appropriate conditions. The cell wall of the plant is hydrolyzed, which is conducive to the dissolution of intracellular active ingredients during extraction. There are also reports on the application of enzymatic hydrolysis technology in the extraction of loquat leaf polysaccharides. For example, Xu Tianhao et al. used pectinase [Xu Tianhao, Sun Yaqian, Huang Yachun, et al. Ultrasonic-assisted enzymatic extraction of loquat leaf polysaccharides. Agricultural Products Processing, 2018, (5): 29-36], and He Chuanbo et al. used cellulase [He Chuanbo, Wei Haocheng, Xiong Hejian, et al. Optimization of enzymatic extraction of loquat leaf polysaccharides and their ion exchange chromatography purification. Food Science, 2016, 37 (8): 52-57]. After enzymatic hydrolysis of loquat leaves, polysaccharides were extracted with hot water. The results showed that enzymatic extraction can significantly improve the extraction rate of polysaccharides.

[0005] At present, the most widely used enzymes in the extraction of plant active ingredients are cellulase and pectinase. The cell wall of plants consists of substances such as cellulose, hemicellulose, lignin, pectin and protein. The use of cellulase or pectinase alone has very limited effect on improving the extraction rate of products. In order to improve the effect of enzymatic extraction, many studies use two or more enzymes such as cellulase, pectinase and protease at the same time. Although the use of multiple enzymes can effectively improve the extraction rate of products, it undoubtedly increases the cost of enzyme use.

[0006] Microorganisms have a very strong ability to produce enzymes, especially some actinomycetes and molds, which can produce a variety of hydrolytic enzymes that decompose plant tissues, including cellulase, hemicellulase, ligninase, pectinase and protease, etc. Therefore, if a certain microorganism is used to directly ferment plant raw materials, the various hydrolytic enzymes produced by the bacteria during the growth process can synergistically decompose the cell wall components, thereby facilitating the release of effective ingredients in the cells and significantly improving the extraction rate.

[0007] In order to improve the extraction rate of loquat leaf polysaccharides, the present invention improves the conventional water extraction and alcohol precipitation extraction method, adds a microbial fermentation pretreatment step, and can greatly improve the extraction rate of loquat leaf polysaccharides. (III) Summary of the invention

[0008] The present invention aims to provide a new microbial strain, Penicillium pulvillorum EJ2-16, and its application in extracting loquat leaf polysaccharides. After loquat leaves are fermented by the strain, the extraction rate of loquat leaf polysaccharides can be significantly improved.

[0009] The technical solution adopted by the present invention is:

[0010] The present invention provides a new microbial strain - Penicillium pulvillorum EJ2-16, which is deposited in Guangdong Microbial Culture Collection Center, with a deposit number of GDMCC No: 62907, a deposit date of October 20, 2022, and an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; Postal Code 510070.

[0011] The cushion-shaped Penicillium EJ2-16 described in the present invention is an excellent strain isolated from the microbial enrichment culture of loquat leaves and obtained through screening and mutagenesis breeding. The colony morphology characteristics of the cushion-shaped Penicillium EJ2-16 are as follows: the strain EJ2-16 is streaked on the PDA plate culture medium and cultured at 30°C for 2 days. The front of the colony is grayish white with a thick flocculent mycelium layer. After culturing for 3 days, the colony gradually turns into khaki, has wrinkles, and has pink patches on the surface. The back is dark yellow and has no odor. Conidiophores extend from the matrix hyphae, have symmetrical branches, and have typical broom-like conidial heads of Penicillium molds; the conidia are spherical, 2.5-3.0μm in diameter, and have smooth walls. A photo of the colony of the strain EJ2-16 cultured on the PDA plate culture medium at 30°C for 4 days is shown in Figure 1 .

[0012] The nucleotide sequence of the ribosomal DNA internal transcribed spacer (rDNA-ITS) of Penicillium pulvinus EJ2-16 is shown in SEQ ID NO.1.

[0013] The present invention also provides an application of the cushion mold EJ2-16 in extracting loquat leaf polysaccharides, and the application method is as follows: (1) adding spore liquid of cushion mold EJ2-16 to loquat leaf powder, stirring evenly and fermenting at 30°C for 64-72 hours to obtain loquat leaf fermentation product; (2) adding deionized water to the loquat leaf fermentation product, stirring evenly and keeping warm at 40-45°C for 2-3 hours, concentrating after ultrasonic water extraction, and obtaining loquat leaf water extract concentrate; (3) adding ethanol to the loquat leaf water extract concentrate to precipitate polysaccharides, washing the precipitate with ethanol and drying, and obtaining a loquat leaf polysaccharide extract.

[0014] Furthermore, the loquat leaf powder in step (1) is fresh loquat leaves, which are washed with tap water, dried at 85°C, crushed and sieved through a 40-mesh sieve to form fine powder.

[0015] Furthermore, the preparation method of the spore liquid of Penicillium cushion mold EJ2-16 in step (1) is as follows: the spores of Penicillium cushion mold EJ2-16 stored at low temperature are inoculated on a potato dextrose agar (PDA) plate culture medium, and after constant temperature culture at 30°C for 72-80 hours, a sterile maltose aqueous solution is added to the culture, and the spores are suspended by stirring with an inoculation loop to obtain a spore liquid; preferably, the spore liquid is adjusted to a spore concentration of 6×10 6 – 9×10 6 The sterile maltose aqueous solution has a maltose concentration of 25-35 g / L and is sterilized with high pressure steam at 115°C for 20 min. The PDA plate medium is a finished potato glucose agar medium purchased from Qingdao Haibo Biotechnology Co., Ltd., prepared with tap water at a mass concentration of 46 g / L, with a natural pH, and sterilized with high pressure steam at 121°C for 20 min.

[0016] Furthermore, in the step (1), the volume of the spore liquid of Penicillium pulvinus EJ2-16 is 1.5-1.8 mL / g based on the mass of loquat leaf powder.

[0017] Furthermore, the preparation method of the loquat leaf water extract concentrate in the step (2) is as follows: deionized water is added to the loquat leaf fermentation product, the product is stirred evenly and kept warm at 40-45°C for 2-3h, then transferred to an ultrasonic cleaning machine with a water temperature of 80-85°C, ultrasonically extracted at 100-200W for 30-50min, and then the pH is adjusted to 9.0-10.0 with a saturated Ca(OH)2 aqueous solution, filtered with a Buchner funnel, the pH of the collected filtrate is adjusted to 4.0-5.0 with acetic acid, filtered with a Buchner funnel again, and the filtrate is concentrated under reduced pressure at 60°C and -0.1Mpa to 1 / 10 of the original volume to obtain a loquat leaf water extract concentrate; the volume of deionized water added is 20-25mL / g based on the mass of the raw loquat leaf powder [the solid-liquid ratio is 1:20-1:25 (g:mL)].

[0018] Furthermore, the preparation method of loquat leaf polysaccharide in step (3) is: adding 3-4 times the volume of anhydrous ethanol to the loquat leaf water extract concentrate so that the ethanol volume fraction of the solution reaches 75%-80%, standing at 4°C for 14-18h, centrifuging at 8000r / min for 5-10min, discarding the supernatant, adding 1-2mL / g of anhydrous ethanol based on the mass of the raw loquat leaf powder, washing once, centrifuging again, and drying the precipitate in a vacuum at 50°C and -0.1Mpa to constant weight to obtain a loquat leaf polysaccharide extract.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: before extracting polysaccharides from loquat leaves, microbial fermentation pretreatment is added, the microbial strain Penicillium pulvinus EJ2-16 used is purposefully screened for the ability to hydrolyze loquat leaf cell walls, it grows moderately in loquat leaf powder added with maltose, produces a variety of hydrolases, and then the loquat leaf powder is heated and enzymolyzed with water, and substances such as cellulose, hemicellulose and lignin in the cell wall are hydrolyzed, which helps the bound loquat leaf polysaccharide to dissolve during ultrasonic water extraction, thereby significantly improving the extraction rate of polysaccharides. Compared with the conventional method without fermentation pretreatment, the extraction rate of loquat leaf polysaccharides can be increased by 62.4%. (IV) Description of the drawings

[0020] Figure 1 This is a photo of the colonies of Penicillium pulvinus EJ2-16 cultured on PDA at 30°C for 4 days.

[0021] Figure 2 This is the standard curve for the determination of polysaccharides using the phenol-sulfuric acid method.

[0022] Figure 3 This is the standard curve for determining glucose using the DNS method. (V) Specific implementation methods

[0023] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0024] The loquat leaf of the invention is the leaf of the loquat tree (Eriobotrya japonica) of the Rosaceae family, which is collected from the Moganshan campus of Zhejiang University of Technology. The loquat leaf powder is fresh loquat leaves, which are washed with tap water, dried at 85°C, crushed and sieved through a 40-mesh sieve to form fine powder.

[0025] Example 1: Isolation and screening of loquat leaf fermentation microbial strains

[0026] The microbial strain for fermenting loquat leaves is isolated and screened according to the following steps:

[0027] (1) Add 10 g of loquat leaf powder to a 250-mL conical flask, add 18 mL of sterile saline, stir evenly, and culture at 30°C for 72 h. Dilute the enriched culture full of mold with sterile saline at 1×10 -6 , 1×10 -7 , 1×10 -8 After dilution, 0.1 mL of the dilution was spread on potato dextrose agar (PDA) plate medium and cultured at 30°C for 48 h. Fungal colonies with different colors and morphologies were picked and transferred to fresh PDA plate medium and cultured at 30°C for 72 h to obtain 9 pure culture strains. The numbers of the strains are shown in Table 1.

[0028] (2) Add 10 mL of sterile maltose aqueous solution to each of the 9 fresh plate cultures, stir with an inoculating loop to suspend the spores, transfer the spore solution to a sterile test tube, and adjust the spore concentration with sterile maltose aqueous solution so that the spore solution of different strains is between 5.0 × 10 6 –9.0×10 6 The concentration of the sterile maltose aqueous solution is 35 g / L, and the high-pressure steam is sterilized at 115° C. for 20 min.

[0029] (3) Add 5 g of loquat leaf powder to 9 100-mL flasks sterilized by dry heat at 160°C for 2 h, and then add 9 mL of each mold spore solution prepared in step (2) (the volume dosage is 1.8 mL / g based on the mass of loquat leaf powder). After stirring evenly, the flasks are sealed with 8 layers of gauze and cultured at 30°C for 72 h to obtain loquat leaf fermentation products.

[0030] (4) Take 1 / 5 of the total weight of the loquat leaf fermentation product fermented by each strain in step (3) (equivalent to 1 g of raw loquat leaf powder), put it in a 50-mL centrifuge tube, add 25 mL of deionized water, stir evenly, keep warm in a 40°C water bath for 3 h, then transfer to an ultrasonic cleaner at 80°C, extract at 100W for 50 min, adjust the pH to 9.0 with a saturated Ca(OH)2 aqueous solution, filter with a Buchner funnel while hot, adjust the pH of the filtrate to 5.0 with acetic acid, and filter with a Buchner funnel again. Take 1 mL of the filtrate in a 10-mL centrifuge tube, add 3 mL of anhydrous ethanol (the ethanol volume fraction of the system is 75%), shake thoroughly, let stand at 4 ° C for 14 h, centrifuge at 8000 r / min for 5 min, discard the supernatant, add 2 mL of anhydrous ethanol to wash once, centrifuge again and discard the supernatant, add 5 mL of deionized water to dissolve the precipitate, and use the phenol-sulfuric acid method to determine the soluble polysaccharide content in the solution.

[0031] Starting from step (3), 9 mL of sterile maltose aqueous solution (concentration 35 g / L) was added to 5 g of loquat leaf powder as a blank fermentation control; according to the method of step (4), 25 mL of phosphate buffer (pH 6.0, 0.2 mol / L) containing cellulase (activity 2000 U / mL) was added to 1 g of loquat leaf powder as a cellulase extraction control. The polysaccharide extraction yields of loquat leaves fermented by different strains and the control are shown in Table 1.

[0032] Table 1 Extraction rate of polysaccharides from loquat leaves fermented by different strains and the control

[0033]

[0034] As can be seen from the data in Table 1, the extraction rate of loquat leaf polysaccharides did not significantly increase in the blank fermentation control with sterile maltose aqueous solution, and in the fermentation of most strains, and even decreased, such as EJ4 and EJ7 strains, indicating that the microbial strains used to ferment loquat leaves to improve the polysaccharide extraction rate have species selectivity. The extraction rate of polysaccharides can also be improved by hydrolyzing loquat leaves with cellulose, which is 11.3% higher than the control without enzymatic hydrolysis, but far less than the fermentation of EJ2 strain. After loquat leaves were fermented by EJ2 strain, the polysaccharide extraction rate was 4.33%, which was 25.1% higher than the 3.46% of the unfermented control. The present invention selects EJ2 strain as the fermentation strain to improve the extraction rate of loquat leaf polysaccharides.

[0035] The PDA plate culture medium is a finished potato dextrose agar culture medium purchased from Qingdao Haibo Biotechnology Co., Ltd. It is prepared with tap water at a mass concentration of 46 g / L, with a natural pH, placed in a conical flask, tied with 8 layers of gauze, sterilized at 121°C by high-pressure steam for 20 minutes, and poured into a sterile culture dish with a diameter of 9 cm before solidification, with 15-20 mL per dish.

[0036] The loquat leaf polysaccharide content is determined by the phenol-sulfuric acid method, and the specific method is: the loquat leaf polysaccharide extract is diluted with deionized water in appropriate multiples (the concentration of polysaccharides in the sample is estimated to be within the determination range of the standard curve); the solid polysaccharide sample is prepared into a solution with a mass concentration of 0.1 mg / mL. Pipette 1 mL of the sample solution to be tested into a test tube, add 1 mL of a 5% phenol aqueous solution by volume, shake well and quickly add 5 mL of concentrated sulfuric acid (mass concentration 98%), shake well and place at room temperature for 10 minutes, heat in a boiling water bath for 15 minutes, and cool to room temperature with running water. Take 1 mL of deionized water as a blank control and use it as a reference to measure the absorbance (A 490 ). The A of glucose samples with different mass concentrations was determined by the same method. 490 , plot the glucose mass concentration—A 490 Standard curve ( Figure 2 ), and the regression equation was obtained, and the polysaccharide content in the loquat leaf polysaccharide sample was determined by calculation using the regression equation.

[0037] The extraction rate of loquat leaf polysaccharide is calculated according to the following formula:

[0038]

[0039] Example 2: Mutation breeding of fermentation strain EJ2

[0040] The strain EJ2 was subjected to mutation breeding to screen strains with better fermentation performance. The specific method is as follows:

[0041] (1) Preparation of spore solution: After strain EJ2 was activated and cultured on PDA plate medium at 30°C for 64 hours, 5 mL of sterile saline was added and stirred with an inoculation loop to suspend the spores. 1 mL of spore solution was transferred to a conical flask containing 50 mL of sterile saline (with 20-30 glass beads) and shaken at room temperature for 15 minutes. The spore solution was filtered to remove the hyphae (a small ball of cotton wool was plugged at the bottom of the triangular funnel), and the spores in the spore solution were counted under a microscope using a hemocytometer. The spores were appropriately diluted with sterile saline to adjust the spore count to 1.82×10 7 Pieces / mL.

[0042] (2) Mutagenesis: Under red light illumination, take 1.5 mL of the above spore solution and a sterile paper clip and place them in 5 culture dishes with a diameter of 6 cm. The culture dishes are placed on magnetic stirrers and irradiated at a distance of 30 cm from a 15W ultraviolet lamp that has been preheated for 30 minutes for 1, 2, 3, 4, 5, and 6 minutes. Take 0.5 mL of the spore solution after the above irradiation treatment, dilute it appropriately, and transfer 0.1 mL to spread on PDA plate culture medium. In the same way, dilute the spore solution that has not been irradiated with ultraviolet light and spread it on the plate as a control to calculate the lethality. The inoculated PDA plate is wrapped with black cloth and inverted at 30°C for 48 hours. The colonies on the plate are counted and the lethality is calculated.

[0043] (3) Screening: The colonies on the PDA plate with a lethality rate of more than 90% were picked and transferred to fresh PDA plate culture medium to obtain 45 strains. 10 mL of sterile physiological saline was added to the fresh plate culture of each strain after culturing at 30°C for 72 hours, and the spores were suspended by stirring with an inoculation loop to obtain the spore liquid of each strain. 2 mL of the spore liquid of each strain was taken and inoculated into 50 mL of enzyme production culture medium. After 72 hours of shaking culture at 30°C and 200 r / min, the fermentation liquid was filtered with a Buchner funnel, the filtrate was collected, and the cellulase activity of the fermentation filtrate of each strain was determined. 15 strains with a relatively higher enzyme activity than that of the wild strain were selected, and then the spore liquid of these 15 strains was inoculated with loquat leaf powder for fermentation according to the method of Example 1, and polysaccharides were extracted by ultrasonic water extraction and alcohol precipitation. The extraction rate of loquat leaf polysaccharides by the mutant strains screened again is shown in Table 2.

[0044] Table 2 Polysaccharide extraction rate after fermentation of loquat leaves by the mutant strains screened multiple times

[0045]

[0046] From the data in Table 2, it can be seen that among the 15 strains screened, the strain numbered EJ2-16 has a cellulase production activity of 94.2U / mL, which is 29.4% higher than the 72.8U / mL of the wild strain EJ2. After fermenting loquat leaf powder with this strain, the polysaccharide extraction rate is 5.58%, which is 28.9% higher than the 4.33% of the wild strain EJ2 and 61.3% higher than the 3.46% of the unfermented control.

[0047] The enzyme production culture medium is composed of: 40 g / L corn flour, 6 g / L peptone, 4 g / L (NH4)2SO4, 5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L CaCl2, 0.1 g / L FeSO4·7H2O, and the solvent is tap water with a pH value of 6.0. A 250-mL conical flask is filled with 50 mL of the enzyme production culture medium, the mouth of the flask is tied with 8 layers of gauze, and the flask is sterilized with high-pressure steam at 121°C for 20 minutes.

[0048] The cellulase activity assay is 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 are added to a 10-mL graduated test tube, respectively, and the mixture is kept warm in a 50°C water bath for 30 min. Then, 3 mL of DNS reagent is added, and the mixture is boiled for 5 min. After cooling with running water, the mixture is fixed to 10 mL with deionized water and shaken well. The crude enzyme solution inactivated by boiling at 100°C for 10 min is used as a reference and the absorbance (A) at a wavelength of 540 nm is measured by a spectrophotometer. 540 ). Based on the glucose standard curve ( Figure 3 ) Calculate the glucose concentration in the sample, and then calculate the cellulase activity (U / mL). Definition of cellulase activity: Under the conditions of pH 6.0 and 50°C, the amount of enzyme required to hydrolyze sodium carboxymethyl cellulose to produce 1 μg of glucose per minute is 1 enzyme activity unit (U).

[0049] The cellulase activity was calculated according to the following formula (1).

[0050]

[0051] In formula (1), C: glucose concentration (mg / mL) calculated from the standard curve; V1: volume of enzyme reaction system, i.e. 2 mL; T: reaction time, i.e. 30 min; V2: volume of crude enzyme solution, i.e. 0.5 mL.

[0052] Preparation of glucose standard curve: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL of standard glucose solution with a concentration of 1 mg / mL were added to 6 10-mL graduated test tubes, and then 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, 0.8 mL of pH 6.0, 0.2 mol / L phosphate buffer were added, and then 3.0 mL of DNS solution was added to each tube. The mixture was boiled in a boiling water bath for 5 min, cooled in running water, and then diluted to 10 mL with deionized water. Shake well and measure A on a spectrophotometer. 540 , with glucose concentration as the horizontal axis, A 540 Draw a standard curve for the ordinate ( Figure 3 ).

[0053] 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, add deionized water to make up to 1L, store in a brown bottle, and use after 7 days.

[0054] Example 3: Classification and Identification of Strain EJ2-16

[0055] Strain EJ2-16 was streaked on a PDA plate medium and cultured at 30°C for 2 days. The front of the colony was grayish white with a thick flocculent hyphae layer. After 3 days of culture, the colony gradually turned into a yellowish brown color with wrinkles and pink patches on the surface. The back was dark yellow and odorless. Conidiophores extended from the matrix hyphae, with symmetrical branches and typical broom-like conidial heads of Penicillium molds; conidia were spherical, 2.5–3.0 μm in diameter, and had smooth walls. See the colony photos of strain EJ2-16 cultured on a PDA plate medium at 30°C for 4 days. Figure 1 .

[0056] The rDNA-ITS nucleotide sequence of strain EJ2-16 was measured as shown in SEQ ID NO. 1. The sequence was compared with the typical strain CBS of Penicillium pulvillorum by BLAST comparison at NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov). The rDNA-ITS sequence of 280.39 has 99.04% homology, and the colony morphology of strain EJ2-16 also conforms to the characteristics of Penicillium pulvillorum. Therefore, the biological classification position of strain EJ2-16 can be determined as follows (refer to Mycobank, http: / / www.mycobank.org): Fungi, Ascomycota, Pezizomycotina, Eurotiomycetes, Eurotiomycetidae, Eurotiales, Aspergillaceae, Penicillium, Penicillium pulvillorum.

[0057] The rDNA sequence of the ITS region is:

[0058] .

[0059] In summary, strain EJ2 was isolated from the microbial enrichment of loquat leaf powder, and after ultraviolet mutagenesis, strain EJ2-16 was screened, namely Penicillium pulvillorum EJ2-16. The strain is deposited in Guangdong Microbiological Culture Collection Center, with the deposit number: GDMCC No: 62907, the deposit date is October 20, 2022, and the address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; Postal Code 510070.

[0060] Example 4: Application of Penicillium pulvinus EJ2-16 in the extraction of loquat leaf polysaccharides

[0061] Penicillium pulvinus EJ2-16 is used to extract polysaccharides from loquat leaves. The following steps can be followed:

[0062] (1) Freeze-dried spore powder of Penicillium pulvinus EJ2-16 was inoculated on a fresh PDA plate medium and cultured at 30°C for 80 h. 10 mL of sterile maltose aqueous solution was added to the culture dish and stirred with an inoculation loop to suspend the spores. The spore solution was transferred to a sterile test tube and the spore concentration was adjusted to 6.55 × 10 6The composition and preparation method of the PDA plate medium are the same as those in Example 1; the concentration of the sterile maltose aqueous solution is 35 g / L, and the high-pressure steam is sterilized at 115° C. for 20 min.

[0063] (2) 10 g loquat leaf powder was placed in a 150-mL Erlenmeyer flask sterilized by dry heat at 160°C for 2 h, and 18 mL of the spore solution of Penicillium pulvinus EJ2-16 prepared in step (1) was added (the volume dosage was 1.8 mL / g based on the mass of loquat leaf powder), and stirred evenly. The flask was sealed with 8 layers of gauze and cultured at 30°C for 72 h to obtain loquat leaf fermentation product.

[0064] (3) All the loquat leaf fermentation products of step (2) were transferred to a 500-mL Erlenmeyer flask, and 250 mL of deionized water [solid-liquid ratio of 1:25 (g:mL)] was added. After stirring evenly, the mixture was placed in a 40°C water bath for 3 h. Afterwards, the Erlenmeyer flask was transferred to an ultrasonic cleaner at 80°C, and ultrasonic extraction was performed at 100W for 50 min. The pH was then adjusted to 9.0 with a saturated Ca(OH)2 aqueous solution, and the mixture was filtered through a Buchner funnel while hot. The pH of the filtrate was adjusted to 5.0 with acetic acid, and the mixture was filtered through a Buchner funnel again. All the filtrate was concentrated to 25 mL under reduced pressure at 60°C and -0.1 MPa to obtain a concentrated solution.

[0065] (4) To all the concentrated solution obtained in step (3), add 75 mL of anhydrous ethanol (3 times the volume of the concentrated solution, the ethanol volume fraction of the system is 75%), let it stand at 4°C for 14 h, centrifuge at 8000 r / min for 5 min, discard the supernatant, add 20 mL of anhydrous ethanol (2.0 mL / g based on the mass of the raw loquat leaf powder) to wash once, centrifuge again, and dry the precipitate at 50°C and -0.1 MPa in a vacuum dryer to constant weight to obtain a loquat leaf polysaccharide extract.

[0066] According to the above steps, 0.994 g of polysaccharide extract was extracted from 10 g of loquat leaves, and the polysaccharide content was 56.7%, that is, 0.564 g of loquat leaf polysaccharide was obtained, and the extraction yield was 5.64%.

[0067] Comparative Example 1: Extraction of loquat leaf polysaccharides by conventional water extraction and alcohol precipitation method (compared with Example 4)

[0068] (1) 10 g loquat leaf powder was placed in a 500-mL Erlenmeyer flask, and 250 mL of deionized water (solid-liquid ratio was 1:25 (g:mL)) was added. After stirring, the mixture was placed in a 40°C water bath for 3 h. The flask was then placed in an ultrasonic cleaner at 80°C, and ultrasonic extraction was performed at 100 W for 50 min. The pH was then adjusted to 9.0 with a saturated Ca(OH)2 aqueous solution, and the mixture was filtered through a Buchner funnel while hot. The pH of the filtrate was adjusted to 5.0 with acetic acid, and the mixture was filtered through a Buchner funnel again. The entire filtrate was concentrated to 25 mL at 60°C and -0.1 MPa under reduced pressure to obtain a concentrated solution.

[0069] (2) To all the concentrated solution obtained in step (1), add 75 mL of anhydrous ethanol (3 times the volume of the concentrated solution, the ethanol volume fraction of the system is 75%), let it stand at 4°C for 14 h, centrifuge at 8000 r / min for 5 min, discard the supernatant, add 20 mL of anhydrous ethanol (2.0 mL / g based on the mass of the raw loquat leaf powder) to wash once, centrifuge again, and dry the precipitate at 50°C and -0.1 MPa in a vacuum dryer to constant weight to obtain a loquat leaf polysaccharide extract.

[0070] According to the above steps, 0.645 g of polysaccharide extract was extracted from 10 g of loquat leaves, and the polysaccharide content was 54.5%, that is, 0.352 g of loquat leaf polysaccharide was obtained, and the extraction yield was 3.52%.

[0071] By comparing the results of Example 4 and Comparative Example 1, it can be seen that before ultrasonic water extraction of polysaccharides from loquat leaves, fermentation pretreatment with Penicillium pulvinus EJ2-16 was added, and the extraction rate of polysaccharides was increased to 5.64%, which was 60.2% higher than the 3.52% of the conventional method without fermentation pretreatment.

[0072] Example 5: Application of Penicillium pulvinus EJ2-16 in the extraction of loquat leaf polysaccharides

[0073] Penicillium pulvinus EJ2-16 is used to extract polysaccharides from loquat leaves. The following steps can be followed:

[0074] (1) PDA plate spores of Penicillium pulvinus EJ2-16 stored at 4°C were inoculated on fresh PDA plate medium and cultured at 30°C for 76 h. 10 mL of sterile maltose aqueous solution was added to the culture dish and stirred with an inoculation loop to suspend the spores. The spore solution was transferred to a sterile test tube and the spore concentration was adjusted to 7.95 × 10 6 The composition and preparation method of the PDA plate medium are the same as those in Example 1; the concentration of the sterile maltose aqueous solution is 30 g / L, and the high-pressure steam is sterilized at 115° C. for 20 min.

[0075] (2) 25 g loquat leaf powder was placed in a 250-mL Erlenmeyer flask sterilized by dry heat at 160°C for 2 h, and 40 mL of the spore solution of Penicillium pulvinus EJ2-16 prepared in step (1) was added (the volume dosage was 1.6 mL / g based on the mass of loquat leaf powder), and stirred evenly. The flask was sealed with 8 layers of gauze and cultured at 30°C for 68 h to obtain loquat leaf fermentation product.

[0076] (3) All the loquat leaf fermentation products of step (2) were transferred to a 1-L Erlenmeyer flask, 550 mL of deionized water [solid-liquid ratio of 1:22 (g:mL)] was added, and after stirring evenly, it was placed in a 45°C water bath for 2.5 h. Afterwards, the Erlenmeyer flask was transferred to an ultrasonic cleaner at a water temperature of 85°C, and ultrasonic extraction was performed at 150W for 40 min. The pH was then adjusted to 9.5 with a saturated Ca(OH)2 aqueous solution, and the filtrate was filtered through a Buchner funnel while hot. The pH of the filtrate was adjusted to 4.5 with acetic acid, and the filtrate was filtered through a Buchner funnel again. All the filtrate was concentrated to 55 mL under reduced pressure at 60°C and -0.1 MPa to obtain a concentrated solution.

[0077] (4) To the entire concentrated solution obtained in step (3), add 220 mL of anhydrous ethanol (4 times the volume of the concentrated solution, the ethanol volume fraction of the system is 80%), let it stand at 4°C for 16 h, centrifuge at 8000 r / min for 10 min, discard the supernatant, add 35 mL of anhydrous ethanol (1.4 mL / g based on the mass of the raw loquat leaf powder), wash once, centrifuge again, and dry the precipitate at 50°C and -0.1 MPa in a vacuum dryer to constant weight to obtain a loquat leaf polysaccharide extract.

[0078] According to the above steps, 2.56 g of polysaccharide extract was extracted from 25 g of loquat leaves, and the polysaccharide content was 57.4%, that is, 1.47 g of loquat leaf polysaccharide was obtained, and the extraction yield was 5.88%.

[0079] Comparative Example 2: Extraction of loquat leaf polysaccharides by conventional water extraction and alcohol precipitation method (compared with Example 5)

[0080] (1) 25 g loquat leaf powder was placed in a 1-L Erlenmeyer flask, and 550 mL of deionized water (solid-liquid ratio was 1:22 (g:mL)) was added. After stirring evenly, the mixture was placed in a 45°C water bath for 2.5 h. Afterwards, the flask was placed in an ultrasonic cleaner at 85°C, and ultrasonic extraction was performed at 150 W for 40 min. The pH was then adjusted to 9.5 with a saturated Ca(OH)2 aqueous solution, and the mixture was filtered through a Buchner funnel while hot. The pH of the filtrate was adjusted to 4.5 with acetic acid, and the mixture was filtered through a Buchner funnel again. The entire filtrate was concentrated to 55 mL under reduced pressure at 60°C and -0.1 MPa to obtain a concentrated solution.

[0081] (2) To the entire concentrated solution obtained in step (1), add 220 mL of anhydrous ethanol (4 times the volume of the concentrated solution, the ethanol volume fraction of the system is 80%), let it stand at 4°C for 16 h, centrifuge at 8000 r / min for 10 min, discard the supernatant, add 35 mL of anhydrous ethanol (1.4 mL / g based on the mass of the raw loquat leaf powder) to wash once, centrifuge again, and dry the precipitate at 50°C and -0.1 MPa in a vacuum dryer to constant weight to obtain a loquat leaf polysaccharide extract.

[0082] According to the above steps, 1.64 g of polysaccharide extract was extracted from 25 g of loquat leaves, and the polysaccharide content was 55.2%, that is, 0.905 g of loquat leaf polysaccharide was obtained, and the extraction yield was 3.62%.

[0083] By comparing the results of Example 5 and Comparative Example 2, it can be seen that before ultrasonic water extraction of polysaccharides from loquat leaves, fermentation pretreatment with Penicillium pulvinus EJ2-16 was added, and the extraction rate of polysaccharides was increased to 5.88%, which was 62.4% higher than the 3.62% of the conventional method without fermentation pretreatment.

[0084] Example 6: Application of Penicillium pulvinus EJ2-16 in the extraction of loquat leaf polysaccharides

[0085] Penicillium pulvinus EJ2-16 is used to extract polysaccharides from loquat leaves. The following steps can be followed:

[0086] (1) PDA plate spores of Penicillium pulvinus EJ2-16 stored at 4°C were inoculated on fresh PDA plate medium and cultured at 30°C for 72 h. 10 mL of sterile maltose aqueous solution was added to the culture dish and stirred with an inoculation loop to suspend the spores. The spore solution was transferred to a sterile test tube and the spore concentration was adjusted to 7.70 × 10 6 The components and preparation method of the PDA plate culture medium are the same as those in Example 1; the concentration of the sterile maltose aqueous solution is 25 g / L, and the high-pressure steam is sterilized at 115° C. for 20 min.

[0087] (2) 50 g loquat leaf powder was placed in a 250-mL Erlenmeyer flask sterilized by dry heat at 160°C for 2 h, and 75 mL of the spore solution of Penicillium pulvinus EJ2-16 prepared in step (1) was added (the volume dosage was 1.5 mL / g based on the mass of loquat leaf powder), and stirred evenly. The flask was sealed with 8 layers of gauze and cultured at 30°C for 64 h to obtain loquat leaf fermentation product.

[0088] (3) All the loquat leaf fermentation products of step (2) were evenly distributed by weight into two 1-L Erlenmeyer flasks, and 500 mL of deionized water [solid-liquid ratio of 1:20 (g:mL)] was added to each flask. After stirring evenly, the flask was placed in a 45°C water bath for 2 hours. After that, the flask was transferred to an ultrasonic cleaner at a water temperature of 85°C, and ultrasonic extraction was performed at 200W for 30 minutes. The pH was then adjusted to 10.0 with a saturated Ca(OH)2 aqueous solution, and the filtrate was filtered through a Buchner funnel while hot, and the filtrate was combined. The pH of the filtrate was adjusted to 4.0 with acetic acid, and the filtrate was filtered through a Buchner funnel again. All the filtrates were concentrated to 100 mL under reduced pressure at 60°C and -0.1Mpa to obtain a concentrated solution.

[0089] (4) To all the concentrated solution obtained in step (3), add 400 mL of anhydrous ethanol (4 times the volume of the concentrated solution, the ethanol volume fraction of the system is 80%), let it stand at 4°C for 18 h, centrifuge at 8000 r / min for 10 min, discard the supernatant, add 50 mL of anhydrous ethanol (1.0 mL / g based on the mass of the raw loquat leaf powder) to wash once, centrifuge again, and dry the precipitate at 50°C and -0.1 MPa in a vacuum dryer to constant weight to obtain a loquat leaf polysaccharide extract.

[0090] According to the above steps, 4.93 g of polysaccharide extract was extracted from 50 g of loquat leaves, and the polysaccharide content was 58.0%, that is, 2.86 g of loquat leaf polysaccharide was obtained, and the extraction yield was 5.72%.

[0091] Comparative Example 3: Extraction of loquat leaf polysaccharides by conventional water extraction and alcohol precipitation method (compared with Example 6)

[0092] (1) Add 25 g of loquat leaf powder and 500 mL of deionized water (solid-liquid ratio is 1:20 (g:mL)) to each of two 1-L Erlenmeyer flasks, stir evenly, and place in a 45°C water bath for 2 h. Then, transfer the flask to an ultrasonic cleaner at 85°C, perform ultrasonic extraction at 200W for 30 min, adjust the pH to 10.0 with a saturated Ca(OH)2 aqueous solution, filter with a Buchner funnel while hot, and combine the filtrates. Adjust the pH of the filtrate to 4.0 with acetic acid, filter with a Buchner funnel again, and concentrate all the filtrates to 100 mL under reduced pressure at 60°C and -0.1 MPa to obtain a concentrated solution.

[0093] (2) To all the concentrated solution obtained in step (1), add 400 mL of anhydrous % ethanol (4 times the volume of the concentrated solution, the ethanol volume fraction of the system is 80%), let it stand at 4°C for 18 hours, centrifuge at 8000 r / min for 10 minutes, discard the supernatant, add 50 mL of anhydrous ethanol (1.0 mL / g based on the mass of the raw loquat leaf powder) to wash once, centrifuge again, and dry the precipitate at 50°C and -0.1 MPa in a vacuum dryer to constant weight to obtain a loquat leaf polysaccharide extract.

[0094] According to the above steps, 3.12 g of polysaccharide extract was extracted from 50 g of loquat leaves, and the polysaccharide content was 56.6%, that is, 1.77 g of loquat leaf polysaccharide was obtained, and the extraction yield was 3.54%.

[0095] By comparing the results of Example 6 and Comparative Example 3, it can be seen that before ultrasonic water extraction of polysaccharides from loquat leaves, fermentation pretreatment with Penicillium pulvinus EJ2-16 was added, and the polysaccharide extraction rate was increased to 5.72%, which was 61.6% higher than the 3.54% of the conventional method without fermentation pretreatment.

Claims

1. Penicillium pulvillorum EJ2-16, deposited in Guangdong Microbiological Culture Collection Center, deposit number: GDMCC No:62907, deposit date October 20, 2022, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; Postal Code: 510070.

2. A use of Penicillium pulvinus EJ2-16 according to claim 1 in extracting loquat leaf polysaccharides, characterized in that: The application method is as follows: (1) adding spore liquid of Penicillium pulvinum EJ2-16 to loquat leaf powder, stirring evenly and fermenting at 30° C. for 64-72 hours to obtain loquat leaf fermentation product; the volume amount of spore liquid of Penicillium pulvinum EJ2-16 is 1.5-1.8 mL / g based on the mass of loquat leaf powder; The preparation method of the spore liquid of Penicillium cushion mold EJ2-16 is as follows: the spores of Penicillium cushion mold EJ2-16 stored at low temperature are inoculated on a PDA plate medium, and after constant temperature culture at 30°C for 72-80 hours, a sterile maltose aqueous solution is added to the culture, and the spores are suspended by stirring with an inoculation loop to obtain a spore liquid; the concentration of the sterile maltose aqueous solution is 25-35 g / L, and the concentration of the spore liquid is 6×10 6 -9×10 6 Pieces / mL; (2) adding deionized water to the loquat leaf fermentation product, stirring evenly, and keeping the mixture at 40-45° C. for 2-3 hours, and concentrating the mixture by ultrasonic water extraction to obtain a loquat leaf water extract concentrate; (3) Ethanol was added to the loquat leaf water extract concentrate to precipitate polysaccharides. The precipitate was washed with ethanol and dried to obtain loquat leaf polysaccharides.

3. The use according to claim 2, characterized in that The loquat leaf powder in step (1) is fresh loquat leaves, which are washed with tap water, dried at 85°C, crushed and sieved through a 40-mesh sieve to form fine powder.

4. The use according to claim 2, characterized in that The preparation method of the loquat leaf water extract concentrate in the step (2) is as follows: deionized water is added to the loquat leaf fermentation product, and the mixture is stirred evenly and kept warm at 40-45° C. for 2-3 hours, and then transferred to an ultrasonic cleaning machine with a water temperature of 80-85° C., and ultrasonically extracted at 100-200W for 30-50 minutes, and then the pH is adjusted to 9.0-10.0 with a saturated Ca(OH)2 aqueous solution, and filtered with a Buchner funnel. The collected filtrate is adjusted to a pH of 4.0-5.0 with acetic acid, and filtered with a Buchner funnel again. The filtrate is concentrated under reduced pressure at 60° C. and -0.1 MPa to 1 / 10 of the original volume to obtain the loquat leaf water extract concentrate.

5. The use according to claim 4, characterized in that The volumetric amount of deionized water added is 20-25 mL / g based on the mass of the raw loquat leaf powder.

6. The use according to claim 2, characterized in that The preparation method of loquat leaf polysaccharide in step (3) is as follows: add 3-4 times the volume of anhydrous ethanol to the loquat leaf water extract concentrate to make the ethanol volume fraction of the solution reach 75%-80%, stand at 4°C for 14-18h, centrifuge at 8000r / min for 5-10min, discard the supernatant, add 1-2mL / g of anhydrous ethanol based on the mass of the raw loquat leaf powder, wash once, centrifuge again, and vacuum dry the precipitate at 50°C and -0.1MPa to constant weight to obtain loquat leaf polysaccharide.

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