Preparation method and application of chaenomeles speciosa extract

Abstract

The invention discloses a preparation method and application of a chaenomeles speciosa extract, and the preparation method comprises the following steps: S1, crushing a chaenomeles speciosa raw material, sieving, and uniformly mixing with water to obtain a first mixture; s2, adding compound lactic acid bacteria liquid into the first mixture, and performing heat-preservation fermentation to obtain a second mixture; s3, performing ultrasonic extraction on the second mixture, and then performing ceramic membrane filtration, vacuum concentration, sterilization and drying to obtain the chaenomeles speciosa extract. The extraction efficiency in the preparation process of the chaenomeles speciosa extract is remarkably improved by optimizing the technological process, the extracted chaenomeles speciosa extract shows excellent anti-oxidation, anti-aging, hypoglycemic, anti-allergic and anti-inflammatory effects, and a novel functional component source is provided for the fields of food and skin care products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and more specifically, relates to a preparation method of a sour papaya extract and application thereof. Background Art

[0002] Sour papaya ( Chaenomeles speciosa Sweet Nakai, also known as the stem-sticking papaya, stem-sticking crabapple, and wrinkled papaya, is the fruit of the genus Malus in the Rosaceae family. Li Shizhen's "Compendium of Materia Medica" records: "Papaya is warm in nature and sour in taste, soothing the liver and stomach", and has the effects of "relaxing muscles and tendons, harmonizing the stomach and removing dampness", and is a traditional Chinese medicine for preventing and treating spleen and stomach diseases. The fresh fruit of sour papaya is sour and astringent, and it is not suitable for raw consumption. It can be made into preserved fruit, fruit wine, and papaya milk. Sour papaya is rich in polyphenols, flavonoids, polysaccharides, vitamin C, papain, organic acids and other nutrients and bioactive substances, and has excellent antioxidant, antibacterial, anti-tumor, blood lipid-lowering and blood vessel softening effects. As a plant that is both a medicine and a food, the study of its bioactive components is of great significance for the development of new functional foods. Yunnan is the main production area of ​​sour papaya. Currently, a large amount of sour papaya is supplied to the local market for direct sales as fresh fruit. A small part is used for medicinal material processing. It has not yet been used for mass industrial production of food with antioxidant, anti-aging, blood sugar lowering, anti-allergic and anti-inflammatory effects, let alone in-depth research and development.

[0003] At present, the commonly used methods for extracting plant active ingredients, such as hot leaching, solvent extraction, enzymatic extraction, etc., have their own advantages, but also have limitations. For example, heating reflux extraction requires long-term heating, which easily leads to the destruction of thermally unstable components during the heating process; solvent extraction easily leads to solvent residues, affecting the quality and safety of the product; the cost of enzymes in enzymatic extraction is high, and the stability of enzymes as catalysts is affected by various factors such as temperature and pH value, and it is necessary to strictly control the operating conditions of enzymatic extraction, which is complicated to operate and difficult to control the extraction efficiency. Obviously, it is not favorable to use the above extraction methods for the extraction of effective ingredients in sour papaya, which will also cause the effective ingredients in sour papaya to be destroyed, or lead to the introduction of harmful substances during the extraction process, or due to improper control of parameters such as temperature and pressure during the extraction process, the extraction efficiency of effective ingredients is low, or it leads to increased equipment investment and production costs.

[0004] Therefore, it is urgent to propose a preparation method of a sour papaya extract and an application thereof, so as to solve the technical problems that the existing extraction method may cause the effective ingredients in the sour papaya to be destroyed, or cause harmful substances to be introduced during the extraction process, or cause low extraction efficiency of the effective ingredients due to improper control of parameters such as temperature and pressure during the extraction process, or cause increased equipment investment, and the sour papaya extract is still in a technical gap in the application of food with antioxidant, anti-aging, hypoglycemic, anti-allergic and anti-inflammatory effects. Summary of the invention

[0005] In view of the deficiencies in the prior art, the main purpose of the present invention is to propose a preparation method and application of a sour papaya extract, so as to solve at least one of the technical problems that the existing extraction method may cause the effective ingredients in the sour papaya to be destroyed, or cause harmful substances to be introduced during the extraction process, or cause low extraction efficiency of the effective ingredients due to improper control of parameters such as temperature and pressure during the extraction process, or cause increased equipment investment and production costs, and the sour papaya extract is still in a technical gap in the application of food with antioxidant, anti-aging, hypoglycemic, anti-allergic and anti-inflammatory effects.

[0006] To achieve this object, the present invention adopts the following technical solutions: In one aspect, the present invention provides a method for preparing a sour papaya extract, the preparation method comprising the following steps: Step S1: crushing the sour papaya raw material, sieving it and mixing it with water to obtain a first mixture; Step S2: inoculating the composite lactic acid bacteria liquid into the first mixture, and obtaining a second mixture after heat preservation and fermentation; Step S3: subjecting the second mixture to ultrasonic extraction, filtering through a ceramic membrane, concentrating under reduced pressure, sterilizing, and drying to obtain the sour papaya extract.

[0007] Preferably, in the step S1, before crushing the sour papaya raw material, the step of drying the sour papaya raw material is also included.

[0008] It should be noted that the purpose of the drying operation is to remove water or other moisture from the sour papaya raw material, for example, one or more techniques including natural air drying, freeze drying or microwave drying can be used.

[0009] Preferably, in step S2, the composite lactic acid bacteria in the composite lactic acid bacteria liquid includes fused Weissella BTN-HB-C1 isolated from the symbiotic bacteria of the utilitarian fruit plant, which is classified and named Weissella sp. ， It is deposited in the Guangdong Provincial Microbiological Culture Collection Center, the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the deposit date is May 13, 2024, and the deposit number is GDMCC No.64619.

[0010] Preferably, in step S2, the composite lactic acid bacteria also include Leuconostoc mesenteroides, and the Leuconostoc mesenteroides is classified and named as Leuconostoc mesenteroides ( Leuconostoc mesenteroides ), purchased from China Industrial Microbiological Culture Collection Center with the collection number CICC 6055.

[0011] Preferably, in the step S2, before inoculating the composite lactic acid bacteria liquid into the first mixture, a step of preparing the composite lactic acid bacteria liquid is also included, and the step of preparing the composite lactic acid bacteria liquid includes: Step S21: activating and culturing the fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides respectively, and determining the number of live bacteria in each bacterial solution; Step S22: compounding the activated and cultured fusion Weissella BTN-HB-C1 and the activated and cultured Leuconostoc mesenteroides.

[0012] Preferably, in step S21, the activation culture of the fusion of Weissella BTN-HB-C1 and Leuconostoc mesenteroides comprises the following steps: Step S211: inoculating the fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides into MRS liquid culture medium respectively; Step S212: The fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides inoculated into the MRS liquid culture medium were statically cultured for 24 h at 37°C to activate for 2 to 3 generations.

[0013] Preferably, in step S211, the formula of the MRS liquid culture medium is that per 1 L of culture medium contains 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 5 g of sodium acetate, 2 g of diammonium citrate, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, and 0.05 g of manganese sulfate.

[0014] Preferably, in step S22, the mass ratio of Weissella bisporus BTN-HB-C1 and Leuconostoc mesenteroides fused in the composite lactic acid bacteria liquid is (2-6): (1-4).

[0015] It should be noted that the composite mass ratio of Weissella BTN-HB-C1 and Leuconostoc mesenteroides in the composite lactic acid bacteria liquid can also be preferably 2:2, can also be preferably 2:3, can also be preferably 2:4, can also be preferably 3:1, can also be preferably 3:2, can also be preferably 3:4, can also be preferably 4:1, can also be preferably 4:3, can also be preferably 5:1, can also be preferably 5:2, can also be preferably 5:3, can also be preferably 5:4, can also be preferably 6:1.

[0016] Preferably, in step S22, the mass ratio of Weissella BTN-HB-C1 and Leuconostoc mesenteroides fused in the composite lactic acid bacteria liquid is 2:1.

[0017] Preferably, in step S2, the temperature of the heat preservation fermentation is 25-37° C., and the fermentation time is 18-48 h.

[0018] It should be noted that, in the step S2, the heat preservation fermentation temperature may also be preferably 25-36 ℃, may also be preferably 25-35 ℃, may also be preferably 25-34 ℃, may also be preferably 25-33 ℃, may also be preferably 25-32 ℃, may also be preferably 25-31 ℃, may also be preferably 25-30 ℃, may also be preferably 25-29 ℃, may also be preferably 25-28 ℃, may also be preferably 25-27 ℃, may also be preferably 25-26 ℃.

[0019] It should be noted that in the step S2, the fermentation time may preferably be 18-46 h, may preferably be 18-44 h, may preferably be 18-42 h, may preferably be 18-40 h, may preferably be 18-38 h, may preferably be 18-36 h, may preferably be 18-34 h, may preferably be 18-32 h, may preferably be 18-30 h, may preferably be 18-28 h, may preferably be 18-26 h, may preferably be 18-24 h, may preferably be 18-22 h, may preferably be 18-20 h.

[0020] Preferably, in step S2, the volume percentage of the composite lactic acid bacteria inoculation amount to the first mixture is 1%-5%.

[0021] It should be noted that the volume percentage of the composite lactic acid bacteria inoculation amount of the first mixture may also be preferably 1%-4%, may also be preferably 1%-3%, may also be preferably 1%-2%, may also be preferably 2%-5%, may also be preferably 2%-4%, may also be preferably 2%-3%, may also be preferably 3%-5%, may also be preferably 3%-4%, may also be preferably 4%-5%.

[0022] Preferably, in step S3, the temperature of the ultrasonic extraction is 50-60°C, the extraction time is 30-60min, and the number of extractions is 1-2 times.

[0023] It should be noted that the temperature of the ultrasonic extraction may also be preferably 51°C, may also be preferably 52°C, may also be preferably 53°C, may also be preferably 54°C, may also be preferably 55°C, may also be preferably 56°C, may also be preferably 57°C, may also be preferably 58°C, may also be preferably 59°C, may also be preferably 60°C.

[0024] The extraction time may also be preferably 35-60 min, may also be preferably 40-60 min, may also be preferably 45-60min, may also be preferably 50-60 min, may also be preferably 55-60 min, may also be preferably 30-55min, may also be preferably 35-55 min, may also be preferably 40-55 min, may also be preferably 50-55 min, may also be preferably 30-50 min, may also be preferably 35-50 min, may also be preferably 40-50 min, may also be preferably 45-50 min.

[0025] In a second aspect, the present invention provides an application of a sour papaya extract in food, wherein the sour papaya extract in the food is prepared by any of the aforementioned preparation methods.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The present invention provides a method for preparing a sour papaya extract and its application. The process is optimized at the level of the preparation method of the sour papaya extract. By optimizing the process flow, especially by fermenting a composite lactic acid bacteria liquid obtained by combining a specific mass ratio of Weissella BTN-HB-C1 and Leuconostoc mesenteroides, the release of effective ingredients (total polyphenol content and total flavonoids content) in the sour papaya can be further promoted compared with fermentation of a single strain of bacteria. Combined with the high efficiency of ultrasonic extraction, the extraction efficiency in the preparation process of the sour papaya extract is significantly improved, while ensuring the superiority and stability of the product quality, and avoiding the defects of the traditional extraction process such as the inability to achieve green environmental protection during operation, harsh extraction conditions, low extraction efficiency, complex operation and high equipment investment cost.

[0027] (2) The present invention provides a method for preparing a sour papaya extract and its application. In step S22, the composite lactic acid bacteria liquid containing BTN-HB-C1 and Leuconostoc mesenteroides can produce a synergistic effect, so that the sour papaya extract has excellent IC50 values ​​in scavenging DPPH free radicals, IC50 values ​​in inhibiting AGEs, and hyaluronidase inhibition rate, showing excellent antioxidant, anti-glycation and anti-allergic abilities, and provides a new source of functional ingredients for the food and skin care products field.

[0028] (3) The preparation method of the sour papaya extract and its application provided by the present invention can reduce the production cost by improving the production efficiency and the yield of finished products, which is conducive to the mass production of the sour papaya extract for commercial application in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The corresponding steps of a method for preparing a sour papaya extract of the present application are shown; Figure 2 The corresponding steps included in step S2 of a method for preparing a sour papaya extract of the present application are shown; Figure 3 The corresponding steps included in step S21 of the method for preparing a sour papaya extract of the present application are shown; Figure 4 The blood sugar lowering effects of comparative example 9 and example 1 on zebrafish are shown. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0031] The source information of the relevant raw materials and materials involved in the following examples, comparative examples and test examples are as follows: The 16S rDNA sequence of the fused Weissella BTN-HB-C1, the 16S rDNA sequence of the plantarum-like Lactobacillus BTN-HB-E1, the 16S rDNA sequence of the sinusoidal Weissella BTN-HB-F1, the 16S rDNA sequence of the Velezac BTN-HB-F4, and the 16S rDNA sequence of the enterocolitica 6055 involved in the present invention have submitted a sequence listing that complies with the WIPO ST.26 standard.

[0032] Fusion Weiss bacteria BTN-HB-C1: Take the prickle from Haba Snow Mountain in Yunnan, wash it with running water, rinse it with sterile water for 3 times, crush it with a sterilized mortar, weigh an appropriate amount of prickle sample in sterile water (add an appropriate amount of glass beads) as the mother liquor, shake it for 10 min, mix it thoroughly, make gradient dilutions with sterile water, select three appropriate gradients and then draw 100 μL to spread on MRS culture medium, each dilution gradient is repeated 3 times, and culture it in a 37 ℃ incubator for 24-48 hours. After separation and purification, a lactic acid bacteria strain with a smooth surface, white opaque, and neat edges was obtained. It was identified as fusion Weiss bacteria and named Weissella sp. 。

[0033] The 16S rDNA sequence of the fused Weissella BTN-HB-C1 is shown in SEQ ID NO.1, and is as follows: ggctcaggat gaacgctggc ggcgtgccta atacatgcaa gtcgaacgct ttgtggttca actgatttga agagcttgct cagatatgac gatggacatt gcaaagagtg gcgaacgggt gagtaacacg tgggaaacct acctcttagc aggggataac atttggaaac agatgctaat accgtataac aatgacaacc gcatggttgt tatttaaaag atggttctgc tatcactaag agatggtccc gcggtgcatt agctagttgg taaggtaatg gcttaccaag gcgatgatgc atagccgagt tgagagactg atcggccaca atgggactga gacacggccc atactcctac gggaggcagc agtagggaat cttccacaat gggcgaaagc ctgatggagc aacgccgcgt gtgtgatgaa gggttcggc tcgtaaaaca ctgttgtaag agaagaatga cattgagagt aactgttcaa tgtgtgacgg tatcttacca gaaaggaacg gctaaatacg tgccagcagc cgcggtaata cgtatgttcc aagcgttatc cggatttatt gggcgtaaag cgagcgcaga cggttatta agtctgaagt gaaagccctc agctcaactg aggaattgct ttggaaactg gatgacttga gtgcagtaga ggaaagtgga actccatgtg tagcggtgaa atgcgtagat atatggaaga acaccagtgg cgaaggcggc tttctggact gtaactgacg ttgaggctcg aaagtgtggg tagcaaacag gattagatac cctggtagtc cacaccgtaa acgatgagtg ctaggtgttt gagggtttcc gcccttaagt gccgcagcta acgcattaag cactccgcct ggggagtacg accgcaaggt tgaaactcaa aggaattgac ggggacccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga catcccttga caactccaga gatggagtgt tcccttcggg gacaaggtga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttattactag ttgccagcat tcagttgggc actctagtga gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggcgt atacaacgag ttgccaaccc gcgagggtga gctaatctct taaagtacgt ctcagttcgg attgtaggct gcaactcgcc tacatgaagt cggaatcgct agtaatcgcg gatcagcacg ccgcggtgaa tacgttcccg ggtcttgtac acaccgcccg tcacaccatg agagtttgta acacccaaag ccggtggggt aaccttcggg agccagccgt ctaaggtggg acagatgatt agggtgaagt cgt Lactobacillus plantarum BTN-HB-E1: Obtained from the pricklyash fruit sourced from Haba Snow Mountain in Yunnan, and its 16S rDNA sequence is shown in SEQ ID NO.2 as follows: ggctcaggac gaacgctggc ggcgtgccta atacatgcaa gtcgaacgaa ctctggtaat gattggtgct tgcatcatga attgcatttg agtgagtggc gaactggtga gtaacacgtg ggaaacctgc ccagaagcgg gggataacac ctggaaacag atgctaatac cgcataacaa cttggaccgc atggtccgag tttgaaagat ggcttcggct atcacttttg gatggtcccg cggcgtatta gctagatggt gaggtaacgg ctcaccatgg caatgatacg tagccgacct gagagggtaa tcggccacat tgggactgag acacggccca aactcctacg ggaggcagca gtagggaatc ttccacaatg gacgaaagtc tgatggagca acgccgcgtg agtgaagaag ggttcggct cgtaaaactc tgttgttaaa gaagaacata tctgagagta actgttcagg tattgacggt atttaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc gagcgcaggc ggttttttaa gtctgatgtg aaagccttcg gctcaaccga agaagtgcat cggaaactgg gaaacttgag tgcagaagag gacagtggaa ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa caccagtggc gaaggcggct gtctggtctg taactgacgc tgaggctcga aagtatgggt agcaaacagg attagatacc ctggtagtcc ataccgtaaa cgatgaatgc taagtgttgg agggttccg cccttcagtg ctgcagctaa cgcattaagc attccgcctg gggagtacgg ccgcaaggct gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac atactatgca aatctaagag attagacgtt cccttcgggg acatggatac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tattatcagt tgccagcatt aagttgggca ctctggtgag actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta caatggatgg tacaacgagt tgcgaactcg cgagagtaag ctaatctctt aaagccattc tcagttcgga ttgtaggctg caactcgcct acatgaagtc ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatga gagtttgtaa cacccaaagt cggtggggta accttttagg aaccagccgc ctaaggtggg acagatgatt agggtgaagt cgt Weissella sinusoidalis BTN-HB-F1: The fruit of the green thorn fruit from Haba Snow Mountain in Yunnan was obtained, and its 16S rDNA sequence is shown in SEQ ID NO.3, which is as follows: ggctcaggat gaacgctggc ggcgtgccta atacatgcaa gtcgaacgct ttgtggttca actgatttga agagcttgct cagatatgac gatggacatt gcaaagagtg gcgaacgggt gagtaacacg tgggaaacct acctcttagc aggggataac atttggaaac agatgctaat accgtataac aatagcaacc gcatggttgc tacttaaaag atggttctgc tatcactaag agatggtccc gcggtgcatt agttagttgg tgaggtaatg gctcaccaag acgatgatgc atagccgagt tgagagactg atcggccaca atgggactga gacacggccc atactcctac gggaggcagc agtagggaat cttccacaat gggcgaaagc ctgatggagc aacgccgcgt gtgtgatgaa gggtttcggc tcgtaaaaca ctgttgtaag agaagaatga cattgagagt aactgttcaa tgtgtgacgg tatcttacca gaaaggaacg gctaaatacg tgccagcagc cgcggtaata cgtatgttcc aagcgttatc cggatttatt gggcgtaaag cgagcgcaga cggttattta agtctgaagt gaaagccctc agctcaactg aggaattgct ttggaaactg gatgacttga gtgcagtaga ggaaagtgga actccatgtg tagcggtgaa atgcgtagat atatggaaga acaccagtgg cgaaggcggc tttctggact gtaactgacg ttgaggctcg aaagtgtggg tagcaaacag gattagatac cctggtagtc cacaccgtaa acgatgagtg ctaggtgttt gagggtttcc gcccttaagt gccgcagcta acgcattaag cactccgcct ggggagtacg accgcaaggt tgaaactcaa aggaattgac ggggacccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga catcccttga caactccaga gatggagcgt tcccttcggg gacaaggtga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttattactag ttgccagcat ttagttgggc actctagtga gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggcgt atacaacgag ttgccaaccc gcgagggtga gctaatctct taaagtacgt ctcagttcgg attgtaggct gcaactcgcc tacatgaagt cggaatcgct agtaatcgcg gatcagcacg ccgcggtgaa tacgttcccg ggtcttgtac acaccgcccg tcacaccatg agagtttgta acacccaaag ccggtggggt aaccttcggg agccagccgt ctaaggtggg acagatgatt agggtgaagt cgt Bacillus velezensis BTN-HB-F4: Obtained from Prinsepia utilis Royle sourced from Haba Snow Mountain in Yunnan, and its 16S rDNA sequence is as shown in SEQ ID NO.4, specifically as follows: ggctcaggac gaacgctggc ggcgtgccta atacatgcaa gtcgagcgga cagatgggag cttgctccct gatgttagcg gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac cggggctaat accggatggt tgtttgaacc gcatggttca gacataaaag gtggcttcgg ctaccactta cagatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtgccgttc aaatagggcg gcaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaac tgggaactt gagtgcagaa gaggagagtg gattccacg tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtactga cgctgaggag cgaagcgtg gggagcgac aggattagat accctggtag tccacgccgt aaacgatgag tgctaagtgt taggggtttccgcccctta gtgctgcagc taacccatta agcactccgc ctggggagta cggtcgcaag actgaactc aaaggaattg acggggcccc gcacaagcgg tggagtgt ggttaattc gaagcaacccc gagaacctt accaggcttt gataccctct gagataggac gtccccttcg ggggcagagt gandaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccacc attcagttgg gcactctaag gtgactgccg gtgacaacc ggaggaggt ggggatgacg tcaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga cagacaaag ggcagcgaaa ccgcgaggtt aagccaatcc cacaatctg ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg cggatcagca tgccgcggtg atacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg taacacccga agtcggtgag gtaaccttta tggagccagc cgccgaaggt gggacagatg attggggtga agtcgt Leuconostoc mesenteroides 6055: purchased from China Industrial Microbiological Culture Collection Center, with the collection number CICC6055. Its 16S rDNA sequence is shown in SEQ ID NO.5, and is as follows: ggctcaggat gaacgctggc ggcgtgccta atacatgcaa gtcgaacgca cagcgaaagg tgcttgcacc tttcaagtga gtggcgaacg ggtgagtaac acgtggacaa cctgcctcaa ggctggggat aacatttgga aacagatgct aataccgaat aaaacttagt gtcgcatgac acaaagttaa aaggcgcttc ggcgtcacct agagatggat ccgcggtgca ttagttagtt ggtggggtaa aggcctacca agacaatgat gcatagccga gttgagagac tgatcggcca cattgggact gagacacggc ccaaactcct acgggaggct gcagtaggga atcttccaca atgggcgaaa gcctgatgga gcaacgccgc gtgtgtgatg aaggctttcg ggtcgtaaag cactgttgta tgggaagaac agctagaata ggaaatgatt ttagtttgac ggtaccatac cagaaaggga cggctaaata cgtgccagca gccgcggtaa tacgtatgtc ccgagcgtta tccggattta ttgggcgtaa agcgagcgca gacggtttat taagtctgat gtgaaagccc ggagctcaac tccggaatgg cattgggaac tggttaactt gagtgcagta gaggtaagtg gaactccatg tgtagcggtg gaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gcttactgga ctgcaactga cgttgaggct cgaaagtgtg ggtagcaaac aggattagat accctggtag tccacaccgt aaacgatgaa cactaggtgt taggaggttt ccgcctctta gtgccgaagc taacgcatta agtgttccgc ctggggagta cgaccgcaag gttgaaactc aaaggaattg acggggaccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatccttt gaagctttta gagatagaag tgttctcttc ggagacaaag tgacaggtgg tgcatggtcg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttattgt tagttgccag cattcagatg ggcactctag cgagactgcc ggtgacaaac cggaggaagg cggggacgac gtcagatcat catgcccctt atgacctggg ctacacacgt gctacaatgg cgtatacaac gagttgccaa cccgcgaggg tgagctaatc tcttaaagt cgtctcagtt cggattgtag tctgcaactc gactacatga agtcggaatc gctagtaatc gcggatcagc acgccgcggt gaatacgttc ccgggtcttg tacacaccgc ccgtcacacc atgggagttt gtaatgccca aagccggtgg cctaaccttt taggaaggag ccgtctaagg caggacagat gactggggtg aagtcgt Terminology Note: DPPH: DPPH is a stable free radical, which is usually used to evaluate the activity of antioxidants. DPPH-IC50 refers to the concentration of antioxidant required to remove 50% of the initial DPPH free radicals within a specific time. The lower the DPPH-IC50 value, the stronger the scavenging ability of the antioxidant.

[0034] AGEs: AGEs are Advanced Glycation End-products (AGEs), which are stable end-products produced by a series of reactions between the free amino groups of macromolecules such as proteins, amino acids, lipids or nucleic acids and the aldehyde groups of reducing sugars under non-enzymatic conditions. AGEs-IC50 refers to the inhibitor concentration that can inhibit 50% of AGEs formation in in vitro experiments. The lower the AGEs-IC50 value, the stronger the anti-glycation activity of the test substance.

[0035] Hyaluronidase: Hyaluronidase is an enzyme that can degrade hyaluronic acid. It is widely used in medicine, cosmetics and other fields. The inhibition rate of hyaluronidase refers to the ability of a substance to inhibit the activity of hyaluronidase. It is usually used to evaluate the moisturizing and anti-allergic activity of cosmetic raw materials. The higher the hyaluronidase inhibition rate, the stronger the anti-allergic activity of the substance.

[0036] Interleukin-6 (IL-6): IL-6 is mainly produced by multiple cell types, including fibroblasts, monocytes / macrophages, T lymphocytes, B lymphocytes, epithelial cells and keratinocytes. It is the most typical cytokine associated with inflammation. It plays an important role in host defense by regulating immune and inflammatory responses.

[0037] Nitric Oxide (NO): NO is an important signaling molecule that transmits intracellular and intercellular signals and is involved in a variety of inflammatory signal transductions. The amount of NO secreted in cells is an important indicator for judging anti-inflammatory activity.

[0038] See attached Figure 1-3 The present invention provides a method for preparing a sour papaya extract, the preparation method comprising the following steps: Step S1: crushing the sour papaya raw material, sieving it and mixing it with water to obtain a first mixture; Step S2: inoculating the composite lactic acid bacteria liquid into the first mixture, and obtaining a second mixture after heat preservation and fermentation; Step S3: subjecting the second mixture to ultrasonic extraction, filtering through a ceramic membrane, concentrating under reduced pressure, sterilizing, and drying to obtain the sour papaya extract.

[0039] Furthermore, in the step S2, before inoculating the composite lactic acid bacteria liquid into the first mixture, a step of preparing the composite lactic acid bacteria liquid is also included, and the step of preparing the composite lactic acid bacteria liquid includes: Step S21: activating and culturing the fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides respectively, and determining the number of live bacteria in each bacterial solution; Step S22: compounding the activated and cultured fusion Weissella BTN-HB-C1 and the activated and cultured Leuconostoc mesenteroides.

[0040] Furthermore, in the step S21, the activation culture of the fusion of Weissella BTN-HB-C1 and Leuconostoc mesenteroides comprises the following steps: Step S211: inoculating the fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides into MRS liquid culture medium respectively; Step S212: The fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides inoculated into the MRS liquid culture medium were statically cultured for 24 h at 37°C to activate for 2 to 3 generations.

[0041] Example 1

[0042] This embodiment 1 provides a method for preparing a sour papaya extract, the preparation method comprising: Step S1: crushing the sour papaya, passing through a 40-mesh sieve, and mixing with water at a mass ratio of 1:20 to obtain a first mixture; Step S2: sterilizing the first mixture and then inoculating and fermenting it. The microorganism used for inoculating and fermenting is a composite lactic acid bacteria liquid of Weissella BTN-HB-C1 and Leuconostoc mesenteroides, the live bacteria ratio is 2:1, the fermentation temperature is 37°C, the fermentation time is 24 h, and the bacterial inoculation amount is 3% (v / v) of the first mixture. After the fermentation is completed, a second mixture is obtained; Step S3: After the fermentation is completed, the second mixture is subjected to ultrasonic-assisted extraction, the ultrasonic time is 30 min, the ultrasonic temperature is 50°C, the solid-liquid ratio is 1:20, and the extraction is performed twice, and then filtered through a 220 nm ceramic membrane, concentrated under reduced pressure, sterilized, and dried to obtain the sour papaya extract.

[0043] The total polyphenol content of the sour papaya extract is 17.75wt%, the total flavonoid content is 29.91wt%, the IC50 value of the sour papaya extract in scavenging DPPH free radicals is 45.38 μg / mL, the IC50 value in inhibiting AGEs is 191.93 μg / mL, and the hyaluronidase inhibition rate is 98.70%.

[0044] Example 2

[0045] This embodiment 2 provides a method for preparing a sour papaya extract, and the steps included in the preparation method are similar to those in embodiment 1, except that the ratio of live bacteria of Weissella BTN-HB-C1 and Leuconostoc mesenteroides in the composite bacterial liquid is 6:1.

[0046] The total polyphenol content of the sour papaya extract is 16.93wt%, the total flavonoid content is 28.87wt%, the IC50 value of the sour papaya extract in scavenging DPPH free radicals is 47.22 μg / mL, the IC50 value in inhibiting AGEs is 201.68 μg / mL, and the hyaluronidase inhibition rate is 97.47%.

[0047] Example 3

[0048] This embodiment 3 provides a method for preparing a sour papaya extract, and the steps included in the preparation method are similar to those in embodiment 1, except that the ratio of live bacteria of Weissella BTN-HB-C1 and Leuconostoc mesenteroides in the composite bacterial liquid is 1:2.

[0049] The total polyphenol content of the sour papaya extract is 17.14wt%, the total flavonoid content is 28.82wt%, the IC50 value of the sour papaya extract in scavenging DPPH free radicals is 49.47 μg / mL, the IC50 value in inhibiting AGEs is 213.45 μg / mL, and the hyaluronidase inhibition rate is 94.65%.

[0050] Comparative Example 1 This comparative example 1 provides a method for preparing a sour papaya extract, and the steps included in the preparation method are similar to those in Example 1, except that the Weissella fused BTN-HB-C1 in the composite bacterial liquid is replaced with Lactobacillus plantarum BTN-HB-E1.

[0051] The total polyphenol content of the sour papaya extract is 16.69wt%, the total flavonoid content is 27.18wt%, the IC50 value of the sour papaya extract in scavenging DPPH free radicals is 65.09 μg / mL, the IC50 value in inhibiting AGEs is 288.91 μg / mL, and the hyaluronidase inhibition rate is 80.52%.

[0052] Comparative Example 2 This comparative example 2 provides a method for preparing a sour papaya extract, and the steps included in the preparation method are similar to those in Example 1, except that the fused Weissella BTN-HB-C1 in the composite bacterial liquid is replaced with Weissella sinusoidalis BTN-HB-F1.

[0053] The total polyphenol content of the sour papaya extract is 15.11wt%, the total flavonoid content is 25.54wt%, the IC50 value of the sour papaya extract in scavenging DPPH free radicals is 71.62 μg / mL, the IC50 value in inhibiting AGEs is 331.37 μg / mL, and the hyaluronidase inhibition rate is 84.98%.

[0054] Comparative Example 3 This comparative example 3 provides a method for preparing a sour papaya extract, and the steps included in the preparation method are similar to those in Example 1, except that the Weissella fusion BTN-HB-C1 in the composite bacterial liquid is replaced with Bacillus velezensis BTN-HB-F4.

[0055] The total polyphenol content of the sour papaya extract is 17.06wt%, the total flavonoid content is 24.90wt%, the IC50 value of the sour papaya extract in scavenging DPPH free radicals is 75.36 μg / mL, the IC50 value in inhibiting AGEs is 270.31 μg / mL, and the hyaluronidase inhibition rate is 85.07%.

[0056] Comparative Example 4 This comparative example 4 provides a method for preparing a sour papaya extract, and the steps included in the preparation method are similar to those in Example 1, except that the composite bacterial solution of fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides is replaced by the bacterial solution of fused Weissella BTN-HB-C1.

[0057] The total polyphenol content of the sour papaya extract is 15.91wt%, the total flavonoid content is 27.85wt%, the IC50 value of the sour papaya extract in scavenging DPPH free radicals is 53.30 μg / mL, the IC50 value in inhibiting AGEs is 258.35 μg / mL, and the hyaluronidase inhibition rate is 92.81%.

[0058] Comparative Example 5 This comparative example 5 provides a method for preparing a sour papaya extract, and the steps included in the preparation method are similar to those in Example 1, except that the composite bacterial solution of fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides is replaced with Leuconostoc mesenteroides bacterial solution.

[0059] The total polyphenol content of the sour papaya extract is 14.86wt%, the total flavonoid content is 28.92wt%, the IC50 value of the sour papaya extract in scavenging DPPH free radicals is 58.07 μg / mL, the IC50 value in inhibiting AGEs is 231.79 μg / mL, and the hyaluronidase inhibition rate is 88.85%.

[0060] Comparative Example 6 This comparative example 6 provides a method for preparing a sour papaya extract, and the steps included in the preparation method are similar to those in Example 1, except that the composite bacterial solution of fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides is replaced by Lactobacillus plantarum BTN-HB-E1 bacterial solution.

[0061] The total polyphenol content of the sour papaya extract is 16.81wt%, the total flavonoid content is 27.01wt%, the IC50 value of the sour papaya extract in scavenging DPPH free radicals is 62.16 μg / mL, the IC50 value in inhibiting AGEs is 304.62 μg / mL, and the hyaluronidase inhibition rate is 80.81%.

[0062] Comparative Example 7 This comparative example 7 provides a method for preparing a sour papaya extract, and the steps included in the preparation method are similar to those in Example 1, except that the composite bacterial solution of fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides is replaced with Weissella sinusoidalis BTN-HB-F1 bacterial solution.

[0063] The total polyphenol content of the sour papaya extract is 14.09wt%, the total flavonoid content is 25.97wt%, the IC50 value of the sour papaya extract in scavenging DPPH free radicals is 78.44 μg / mL, the IC50 value in inhibiting AGEs is 309.56 μg / mL, and the hyaluronidase inhibition rate is 79.20%.

[0064] Comparative Example 8 This comparative example 8 provides a method for preparing a sour papaya extract, and the steps included in the preparation method are similar to those in Example 1, except that the composite bacterial solution of fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides is replaced by Bacillus velezensis BTN-HB-F4 bacterial solution.

[0065] The total polyphenol content of the sour papaya extract is 17.03wt%, the total flavonoid content is 24.02wt%, the IC50 value of the sour papaya extract in scavenging DPPH free radicals is 92.61 μg / mL, the IC50 value in inhibiting AGEs is 394.93 μg / mL, and the hyaluronidase inhibition rate is 73.58%.

[0066] Comparative Example 9 This comparative example 9 provides a method for preparing a sour papaya extract, which adopts a traditional heating reflux extraction process.

[0067] The total polyphenol content of the sour papaya extract is 11.21wt%, the total flavonoid content is 19.54wt%, the IC50 value of the sour papaya extract in scavenging DPPH free radicals is 79.84 μg / mL, the IC50 value in inhibiting AGEs is 361.86 μg / mL, and the hyaluronidase inhibition rate is 78.58%.

[0068] Analysis of the above embodiments and comparative examples shows that: (1) In the method for preparing a sour papaya extract of the present application, the mass ratio of the composite lactic acid bacteria liquid fused with Weissella BTN-HB-C1 and Leuconostoc mesenteroides in step S22 is in the range of (2-6): (1-4). Examples 1-3 respectively take the two end points and an intermediate value of the above mass ratio range, and it can be seen that: When the composite mass ratio of Weissella BTN-HB-C1 and Leuconostoc mesenteroides in the composite lactic acid bacteria liquid is 2:1, the total polyphenol content and total flavonoids content in the sour papaya extract are better than when the composite mass ratio of Weissella BTN-HB-C1 and Leuconostoc mesenteroides is the endpoint value; and the IC50 value in DPPH scavenging and the IC50 value in AGEs inhibition are smaller, showing excellent antioxidant and anti-glycation abilities; in addition, the hyaluronidase inhibition rate is higher, showing excellent anti-allergic ability.

[0069] In Comparative Examples 1-3, the fused Weissella BTN-HB-C1 in the composite lactic acid bacteria liquid is replaced with other bacterial species, namely, plantarum-like Lactobacillus BTN-HB-E1, Weissella sinusoidalis BTN-HB-F1, and Bacillus velezensis BTN-HB-F4. In Comparative Examples 4-8, one of the fused Weissella BTN-HB-C1 or Leuconostoc mesenteroides in the composite lactic acid bacteria liquid is removed or the composite lactic acid bacteria liquid is replaced with other single bacterial species liquid.

[0070] Comparative analysis shows that the results of the total polyphenol content and total flavonoid content of Comparative Examples 1-2, 4, 6-7 are all inferior to those of Examples 1-3; while the total polyphenol content of Comparative Examples 3 and 8 is slightly greater than that of Example 2 (but less than that of Examples 1 and 3), the total flavonoid content of Comparative Examples 3 and 8 is 3.97% and 4.85% lower than that of Example 2; although the total flavonoid content of Comparative Example 5 is slightly greater than that of Examples 2-3 (but less than that of Example 1), the total polyphenol content of Comparative Example 5 is 2.07% and 2.28% lower than that of Examples 2-3. In terms of the overall content level of total polyphenol content and total flavonoid content, Examples 1-3 are significantly better than Comparative Examples 1-8.

[0071] In addition, the IC50 values ​​of Comparative Examples 1-8 in scavenging DPPH free radicals and in inhibiting AGEs are greatly improved compared with Examples 1-3, that is, the antioxidant and anti-glycation abilities are greatly reduced compared with Examples 1-3; and in terms of hyaluronidase inhibition rate, the anti-allergic ability of Comparative Examples 1-8 is also greatly reduced compared with Examples 1-3.

[0072] It can be seen from this that the preparation method of the present application can produce a synergistic effect by fusing Weissella BTN-HB-C1 and Leuconostoc mesenteroides in the composite lactic acid bacteria liquid in step S22, so that the IC50 value of the sour papaya extract in scavenging DPPH free radicals, the IC50 value in inhibiting AGEs and the hyaluronidase inhibition rate are all excellent, showing excellent antioxidant, anti-glycation and anti-allergic abilities.

[0073] (2) The composite lactic acid bacteria liquid in the method for preparing a sour papaya extract of the present application contains a combination of Weissella BTN-HB-C1 and Leuconostoc mesenteroides, which is the first combination made by the inventors. The beneficial effect achieved by this combination is much better than the traditional heating reflux extraction process.

[0074] Test Example 1 Effects of sour papaya extract on LPS-induced RAW264.7 cell inflammation model: The cell viability was determined by CCK-8 method. The cells in good growth state and exponential growth phase were obtained, digested with trypsin, and then inoculated into 96-well plates after counting. 100 μL per well, about 1×10 4 Cells were placed in an incubator for about 24 h, and then replaced with complete medium containing different concentrations of the test substance and incubated in the incubator for a total of 24 h. The drug-containing medium was discarded, and a mixed solution of 100 μL DMEM medium and 10 μL CCK-8 was added to each well and incubated at 37 °C for 2 h. The OD value of each well was measured at a wavelength of 450 nm, and the cell viability was calculated according to formula (1): ; Where: A1 is the OD value of the sample group, A2 is the OD value of the control group, and A0 is the OD value of the blank group.

[0075] Sample group: A sour papaya extract (the sour papaya extract prepared in Examples 1-3 and Comparative Examples 1-9) diluted with complete culture medium and LPS were added to each well; Model group: complete medium and LPS were added to each well, but no extracts or drugs were added; Positive control group: 5 μM dexamethasone sodium acetate and LPS diluted in complete medium were added to each well; Blank control group: Complete culture medium was added to each well without adding LPS or any other treatment factors.

[0076] Test method: A blank control group, a model group, a positive control group and a sample group were set up. The sample group was the acid papaya extract prepared in Examples 1-3 and Comparative Examples 1-9. Complete medium was added to each well of the blank control group, 5 μM dexamethasone sodium acetate diluted with complete medium was added to each well of the positive control group, and acid papaya extract diluted with complete medium was added to each well of the sample group. Three replicate wells were set up for each group and incubated in a 37°C, 5% CO2 incubator for 2 h. Except for the blank control group, LPS with a final concentration of 1 μg / mL was added to each well and incubated in a 37°C, 5% CO2 incubator for 24 h. The cell supernatant was collected by centrifugation in a low-temperature centrifuge, and the ELISA kit and NO detection kit were used for detection according to the instructions. The contents of IL-6 and NO in the collected cell supernatant were detected respectively. The test data are shown in Table 1.

[0077] Table 1. Experimental data of relative expression of IL-6 and NO in Examples 1-3, Comparative Examples 1-9, and the positive control group, blank control group, and model group

[0078] As shown in Table 1, the sour papaya extracts prepared in Examples 1-3 can significantly inhibit NO production and IL-6 expression, and have excellent anti-inflammatory effects. The sour papaya extracts in Examples 1-3 are superior to the extracts in Comparative Examples 1-9 in terms of anti-inflammatory effects.

[0079] Test Example 2 Experimental Animals: Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish aged 5 days post fertilization (5 dpf) were used for the determination of the maximum detection concentration (MTC) of the sample-assisted hypoglycemic efficacy and efficacy evaluation.

[0080] MTC determination: 5 dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 30 zebrafish in each beaker. The water was soluble in sour papaya extract, and a normal control group and a model control group were set up with a volume of 25 mL. Except for the normal control group, the other experimental groups were soluble in 1.5 mg / mL egg yolk powder solution during the day and 30 mg / mL glucose solution at night to establish a high-sugar and high-fat model of zebrafish. During the sample treatment, the number of zebrafish deaths in each experimental group was counted every day and the dead zebrafish were removed in time. After 2 days of treatment at 28 ℃, the MTC of the samples on the model zebrafish was determined.

[0081] Test method: 5 dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 30 zebrafish in each beaker. The acid papaya extract was given in water, and the positive control group was given pioglitazone at a concentration of 15.0 μg / mL. The normal control group and the model control group were set up at the same time, and the capacity of each beaker was 25 mL. Except for the normal control group, the other experimental groups were given 1.5 mg / mL egg yolk powder solution in water during the day and 30 mg / mL glucose solution in water at night to establish a high-sugar and high-fat model of zebrafish. After 2 days of treatment at 28℃, the zebrafish were washed 3 times with standard dilution water, and the data were collected with a blood glucose meter. The glucose level (S) of the zebrafish was analyzed and statistically analyzed. The statistical analysis results of this indicator were used to evaluate the sample's auxiliary hypoglycemic efficacy. The formula for the sample's auxiliary hypoglycemic efficacy is as follows:

[0082] Sample group: water-soluble administration of 15.6 μg / mL of sour papaya extract, water-soluble administration of 1.5 mg / mL of egg yolk powder solution during the day, and water-soluble administration of 30 mg / mL of glucose solution at night; Model control group: no sour papaya extract was given, and the water-soluble solution was given with a mass concentration of 1.5 mg / mL of egg yolk powder during the day and a mass concentration of 30 mg / mL of glucose solution at night; Positive control group: pioglitazone (pioglitazone is a thiazolidinedione antidiabetic drug and an insulin sensitizer) with a mass concentration of 15.0 μg / mL, egg yolk powder solution with a mass concentration of 1.5 mg / mL in water during the day, and glucose solution with a mass concentration of 30 mg / mL in water at night; Normal control group: no experimental intervention factors (including egg yolk powder, glucose and sour papaya extract) were given.

[0083] Test data such as Figure 4As shown, the acid papaya extract (water-soluble mass concentration of 15.6 μg / mL) prepared by the preparation method of the acid papaya extract described in Example 1 can effectively reduce the blood glucose content of zebrafish and has an auxiliary blood glucose-lowering effect; while the acid papaya extract (water-soluble mass concentration of 15.6 μg / mL) prepared by the heating reflux extraction process of Comparative Example 9 is significantly inferior to the acid papaya extract prepared in Example 1 in terms of auxiliary blood glucose-lowering effect.

[0084] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing a sour papaya extract, characterized in that: The preparation method comprises the following steps: Step S1: crushing the sour papaya raw material, sieving it and mixing it with water to obtain a first mixture; Step S2: inoculating the composite lactic acid bacteria liquid into the first mixture, and obtaining a second mixture after heat preservation and fermentation; Step S3: subjecting the second mixture to ultrasonic extraction, filtering through a ceramic membrane, concentrating under reduced pressure, sterilizing, and drying to obtain the sour papaya extract.

2. The method for preparing a sour papaya extract according to claim 1, characterized in that: In the step S1, before the sour papaya raw material is crushed, the step of drying the sour papaya raw material is also included.

3. The method for preparing a sour papaya extract according to claim 1, characterized in that: In step S2, the composite lactic acid bacteria in the composite lactic acid bacteria liquid includes the fused Weissella BTN-HB-C1 isolated from the symbiotic bacteria of the prickle fruit plant from Haba Snow Mountain in Yunnan, which is classified and named Weissella sp. ， It is deposited in the Guangdong Provincial Microbiological Culture Collection Center, the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the deposit date is May 13, 2024, and the deposit number is GDMCC No.64619.

4. The method for preparing a sour papaya extract according to claim 3, characterized in that: In step S2, the composite lactic acid bacteria also include Leuconostoc mesenteroides, which is classified and named as Leuconostoc mesenteroides ( Leuconostoc mesenteroides ), purchased from China Industrial Microbiological Culture Collection Center, with the collection number CICC6055.

5. The method for preparing a sour papaya extract according to claim 4, characterized in that: In the step S2, before inoculating the composite lactic acid bacteria liquid into the first mixture, a step of preparing the composite lactic acid bacteria liquid is also included, and the step of preparing the composite lactic acid bacteria liquid includes: Step S21: activating and culturing the fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides respectively, and determining the number of live bacteria in each bacterial solution; Step S22: compounding the activated and cultured fusion Weissella BTN-HB-C1 and the activated and cultured Leuconostoc mesenteroides.

6. The method for preparing the sour papaya extract according to claim 5, characterized in that: In the step S21, the activation culture of the fusion of Weissella BTN-HB-C1 and Leuconostoc mesenteroides comprises the following steps: Step S211: inoculating the fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides into MRS liquid culture medium respectively; Step S212: The fused Weissella BTN-HB-C1 and Leuconostoc mesenteroides inoculated into the MRS liquid culture medium were statically cultured for 24 h at 37°C to activate for 2 to 3 generations.

7. The method for preparing the sour papaya extract according to claim 6, characterized in that: In step S211, the formula of the MRS liquid culture medium is that per 1 L of culture medium, it contains 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 5 g of sodium acetate, 2 g of diammonium citrate, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, and 0.05 g of manganese sulfate.

8. The method for preparing a sour papaya extract according to claim 5, characterized in that: In step S22, the mass ratio of Weissella bisporus BTN-HB-C1 and Leuconostoc mesenteroides fused in the composite lactic acid bacteria liquid is in the range of (2-6): (1-4).

9. The method for preparing the sour papaya extract according to claim 8, characterized in that: In step S22, the composite lactic acid bacteria liquid is fused with Weissella BTN-HB-C1 and Leuconostoc mesenteroides in a mass ratio of 2:

1.

10. The method for preparing the sour papaya extract according to claim 1, characterized in that: In step S2, the temperature of the heat preservation fermentation is 25-37°C, and the fermentation time is 18-48 hours.

11. The method for preparing the sour papaya extract according to claim 1, characterized in that: In step S2, the volume percentage of the composite lactic acid bacteria inoculation amount to the first mixture is 1%-5%.

12. The method for preparing the sour papaya extract according to claim 1, characterized in that: In step S3, the temperature of the ultrasonic extraction is 50-60°C, the extraction time is 30-60 min, and the number of extractions is 1-2 times.

13. Application of a sour papaya extract in food, characterized in that: The sour papaya extract in the food is prepared by the preparation method of the sour papaya extract according to any one of claims 1 to 12.

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