Method for producing pecticoligosaccharides by microbial degradation of pomace and application thereof

Abstract

The application discloses a method for producing pectin oligosaccharides by microbial degradation of pomace and application thereof. The method comprises the following steps: adding microbacterium A5 to a culture medium containing pomace to perform fermentation to obtain a fermentation liquor, performing fractional pressing filtration and filter sterilization on the fermentation liquor, performing ethanol precipitation to extract a pectin crude product, removing protein, performing ion exchange chromatography, dialysis desalting, freeze drying and the like to purify two pectin oligosaccharides POS-1 and POS-2 with a purity greater than 95%; the pectin oligosaccharides POS-1 and POS-2 have strong scavenging capacity on hydroxyl radicals and DPPH free radicals and the capacity of inhibiting the activity of respiratory RNA viruses such as influenza virus H1N1 and respiratory syncytial virus RSV, and can be applied to medicines or health foods.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fruit peel resource recycling and pectin oligosaccharide preparation, and more particularly to a method for producing pectin oligosaccharide by microbial degradation of pomelo peel and application, and production of pectin oligosaccharide POS-1 and POS-1 and application thereof through aerobic fermentation, separation and purification of pomelo peel. BACKGROUND

[0002] Pomelo belongs to Rutaceae and Citrus. It is one of the largest fruits in the south of the Yangtze River in China, especially in Guangdong, Guangxi and Fujian provinces, with an annual output of more than 5 million tons. In the process of pomelo planting, in addition to the edible fruit, an equal amount of inferior fruit is produced. Because of the thick structure of pomelo peel, it is not easy to degrade, becomes agricultural waste, causes great environmental burden, and also affects the healthy growth of pomelo. The thick white pulp of pomelo peel has the highest pectin content among all peels. Pectin has good gelation and thickening effects and is widely used as a food additive. Low molecular weight pectin (pectin oligosaccharide) can be obtained by chemical, enzymatic or physical methods to partially depolymerize pectin in the peel. It has been proved to have the effects of improving intestinal microflora, regulating lipid metabolism, reducing sugar metabolism and cholesterol levels in blood, and has the properties of anti-cancer, antibacterial, antioxidant and immunology. Anti-inflammatory, anti-tumor, anti-coagulation, antibacterial and antioxidant effects are applied to food and medicine abroad. These biological activities are related to the structure of oligomeric pectin, including molecular weight distribution (Mw), monosaccharide composition, chemical bond and branching degree (DB). The current degradation methods of pomelo peel, such as acid treatment, high pressure, ultrasonic and hydrogen peroxide, are difficult to obtain low molecular weight pectin molecules with stable quality and structure. Biological degradation can achieve directional depolymerization of pectin glycosidic bond and obtain low molecular weight pectin with stable structure and molecular weight. However, the technology of producing low molecular weight pectin by biological method in China develops slowly, especially there is a lack of industrialized production process of biological degradation method, which cannot meet the growing demand of the market of nutritional and healthy food. SUMMARY

[0003] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art, and provides a method for producing pectin oligosaccharide by microbial degradation of pomelo peel.

[0004] The second object of the present application is to provide pectin oligosaccharide prepared by the method.

[0005] The third object of the present application is to provide the application of the pectin oligosaccharide.

[0006] The above objects of the present application are achieved by the following technical solutions:

[0007] A method for producing pectin oligosaccharide from microbial degradation of pomelo peel, comprising the following steps:

[0008] S1. Adding the microbacterium A5 to the culture medium containing pomelo peel for fermentation to obtain a fermentation liquor;

[0009] S2. Squeezing and filtering the fermentation liquor of step S1 to obtain a filtrate and a residue;

[0010] S3. Ethanol precipitating the filtrate of step S2, centrifuging to obtain supernatant and precipitate, freeze-drying the precipitate to obtain a crude pectin oligosaccharide product;

[0011] S4. Removing impurities from the crude pectin oligosaccharide product of step S3, ethanol precipitating, freeze-drying the precipitate, and then performing ion exchange chromatography, dialysis desalting, concentration, and freeze-drying to obtain a pure pectin oligosaccharide product;

[0012] The microbacterium A5 was preserved in the China Center for Type Culture Collection on August 7, 2009, and the preservation number is CCTCC NO: M209174.

[0013] The present application utilizes the microbacterium A5 isolated by the inventor in the early stage for fermentation and degradation of pomelo peel, which can efficiently release the pectin components in the pomelo peel tissue cells, directionally degrade the pectin macromolecules, and produce a crude active pectin oligosaccharide product with low molecular weight. Compared with the traditional acid method, alkali method, and physical method for degradation, the microbial fermentation method of the present application does not need to perform heat treatment and non-directional degradation treatment by the acid and alkali method, the product molecular weight range is stable, no by-products caused by acid and alkali modification are brought, and no acid and alkali wastewater is discharged. The utilization rate of the pomelo young fruit, defective fruit, and peel is improved, the production cost of the pomelo peel extraction is reduced, and the industrialized scale-up production can be performed.

[0014] Preferably, the fermentation of step S1 is a step-by-step scale-up fermentation for degrading the pomelo peel. The microbacterium A5 is activated, and the activated bacterial liquid is added to the culture medium containing pomelo peel for step-by-step domestication and scale-up fermentation. The scale-up fermentation liquor is added to the complete pomelo peel culture medium for fermentation to obtain a fermentation liquor. The step-by-step domestication and scale-up fermentation refers to that the activated microbacterium A5 liquid is first cultured in the culture medium with low pomelo peel content to obtain the fermentation liquor in the middle and late logarithmic growth phase, the fermentation liquor is added to the culture medium with high pomelo peel content as the seed for scale-up culture, and finally added to the complete pomelo peel culture medium for fermentation culture. Through the step-by-step scale-up process of the pomelo fermentation by the microbacterium, on the one hand, the degradation activity of the strain to the pomelo peel is gradually enhanced, and on the other hand, the processing amount is increased and the fermentation period is shortened.

[0015] Preferably, the low-grapefruit-peel-content medium is LB medium added with 25% w / v grapefruit peel dry powder; the high-grapefruit-peel-content medium is LB medium added with 80% w / v grapefruit peel dry powder; and the complete grapefruit peel medium is 100% grapefruit peel dry powder added medium.

[0016] Preferably, the pressing and filtering in step S2 is fractional pressing and filtering, and plate-and-frame filtering sterilization. Further, 0.45 μm filter membrane plate-and-frame filtering sterilization is used.

[0017] Preferably, the ethanol precipitation in step S3 uses 80% ethanol; and the effect of using 80% concentration ethanol for precipitation is the best.

[0018] Preferably, the filtrate is concentrated before the ethanol precipitation in step S3. Concentration of the fermentation liquor before ethanol precipitation not only saves the amount of ethanol used, but also improves the recovery rate of the pectin oligosaccharide crude extract. Further preferably, the fermentation liquor is concentrated to one third of the original volume.

[0019] Preferably, the removal of impurities in step S4 uses TCA method for protein removal. Compared with hydrochloric acid method and Sevage method, the TCA method has good effect on removal of impurities in the pectin crude product and low polysaccharide loss rate, and can remove 74.67% of the protein and replace the Sevage method which may bring about residual organic solvent.

[0020] Preferably, the ion exchange chromatography in step S4 is DEAE cellulose ion exchange chromatography; specifically, first, phosphate buffer is used to wash away alkaline, neutral polysaccharides and other impurities, and then different concentration gradients of NaCl solution are set for gradient elution, and when 0.3M and 0.4M sodium chloride solution is used for elution, two high-purity grapefruit pectin oligosaccharide components POS-1 and POS-2 can be separated.

[0021] The application also provides grapefruit pectin oligosaccharides prepared by any of the above-mentioned methods, wherein the grapefruit pectin oligosaccharides have a molecular weight distribution in the range of 1.7×10 4 ~ 2.0×10 4 , are composed of glucose and galactose, and have a molar ratio of glucose to galactose of 0.88~0.89:0.11~0.12.

[0022] Specifically, the grapefruit pectin oligosaccharides are POS-1 and POS-2; the weight average molecular weight of POS-1 is 19921, POS-1 is composed of glucose and galactose, and has a molar ratio of glucose to galactose of 0.887:0.113; and the weight average molecular weight of POS-2 is 17307, POS-2 is composed of glucose and galactose, and has a molar ratio of glucose to galactose of 0.883:0.117.

[0023] The research of the present application shows that the pectin oligosaccharide has strong scavenging ability on hydroxyl radical and DPPH radical and the ability to inhibit the activity of respiratory RNA viruses such as influenza virus H1N1 and respiratory syncytial virus RSV. Therefore, the present application also provides the use of the pectin oligosaccharide in preparing products with antioxidant activity, inhibiting influenza virus H1N1 and / or respiratory syncytial virus RSV.

[0024] Based on the above-mentioned method for producing pectin oligosaccharide by using microbacterium A5 to aerobically ferment shaddock peel, the present application also protects the use of microbacterium A5 in fermenting and degrading shaddock peel to produce pectin oligosaccharide.

[0025] Preferably, the pectin oligosaccharide has a molecular weight distribution in the range of 1.7*10 4 ~ 2.0*10 4 , and is composed of glucose and galactose, with the molar ratio of glucose to galactose being 0.88~0.89:0.11~0.12.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] (1) The present application uses microbacterium A5 to ferment and degrade shaddock peel, which can efficiently release the pectin components in the shaddock peel tissue cells, and directionally degrade the pectin macromolecules to produce low-molecular-weight active oligomeric pectin. After purification and refining processes, medicinal-grade pectin oligosaccharide can be obtained. Compared with the traditional acid method, alkali method and physical method of degradation, the microbial fermentation method of microbacterium A5 does not need to be subjected to heat treatment and non-directional degradation treatment by acid and alkali method, the molecular weight range of the product is stable, no by-products caused by acid and alkali modification are brought, and no acid and alkali wastewater is discharged. The utilization rate of shaddock young fruits, defective fruits and peels is improved, the production cost of shaddock peel extraction is reduced, and the industrialized scale-up production can be carried out.

[0028] (2) The present application establishes a purification process for extracting crude pectin product from the fermentation broth of microbacterium A5 fermenting and degrading shaddock peel, and further establishes the refining process conditions of two kinds of oligomeric pectin molecules. Two pectin oligosaccharides POS-1 and POS-2 with a purity greater than 95% are obtained through the steps of fractional pressing filtration and filter sterilization of the fermentation broth, ethanol precipitation to extract crude pectin, protein removal, DEAE ion exchange chromatography, dialysis desalting and freeze-drying. The pectin oligosaccharides POS-1 and POS-2 have strong scavenging ability on hydroxyl radical and DPPH radical and the ability to inhibit the activity of respiratory RNA viruses such as influenza virus H1N1 and respiratory syncytial virus RSV. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The figure is the colony morphology of microbacterium A5.

[0030] Figure 2 Growth curve of Microbacterium in 25% naringin medium.

[0031] Figure 3 Growth curve of Microbacterium in 80% naringin medium.

[0032] Figure 4 Flow chart of oligogalacturonan extraction process.

[0033] Figure 5 Standard curve of glucose.

[0034] Figure 6 Standard curve of protein content.

[0035] Figure 7 Separation profile of pectin on DEAE-cellulose ion exchange column chromatography.

[0036] Figure 8 Scanning profile of DPPH.

[0037] Figure 9 DPPH scavenging rate of different concentrations of pectin solution.

[0038] Figure 10 Standard curve of GPC for detecting pullulan.

[0039] Figure 11 Molecular weight and purity GPC profile of pectin oligosaccharide POS-1.

[0040] Figure 12 Molecular weight and purity GPC profile of pectin oligosaccharide POS-2.

[0041] Figure 13 Ion chromatogram of mixed standard 16 sugars. Mixed standard: solvent peak: 2.0 min for sodium hydroxide peak, 40 min for sodium acetate peak.

[0042] Figure 14 Gel permeation chromatography result of POS-1 pectin oligosaccharide.

[0043] Figure 15 Gel permeation chromatography result of POS-2 pectin oligosaccharide.

[0044] Figure 16 Effect of POS-1 and POS-2 pectin oligosaccharides on lung mucosal histopathology of H1N1 infected mice. (A) normal control group; (B) H1N1 virus infection group; (C) Rib administration group; (D) POSSD administration group; (E) POS-1 administration group; (F) POS-2 administration group.

[0045] Figure 17 Effects of POS-1 and POS-2 pectin oligosaccharides on lung mucosal histopathology of RSV-infected mice. (A) normal control group; (B) RSV virus infection group; (C) Rib administration group; (D) POSSD administration group; (E) POS-1 administration group; (F) POS-2 administration group. DETAILED DESCRIPTION

[0046] The present application is further illustrated by the following description with reference to the accompanying drawings and specific examples. Unless otherwise defined, the reagents, methods and apparatuses used in the present application are conventional reagents, methods and apparatuses in the art.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0048] Microbacterium A5, which was deposited with the China Center for Type Culture Collection on August 7, 2009, has a deposit number of CCTCC NO: M209174.

[0049] Liquid LB medium: 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 1 mol / L NaOH to adjust pH to 7.4 with deionized water to 1 L.

[0050] Solid LB plate medium: per liter: 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 15 g of agar or agarose, 1 mol / L NaOH to adjust pH to 7.4 with deionized water to 1 L, 121 °C, sterilization for 20 min.

[0051] Example 1 Gradual domestication of Microbacterium A5 pummelo peel degradation activity and optimization of scale-up fermentation process conditions

[0052] I. Methods

[0053] 1. Pummelo medium preparation

[0054] Pummelo peel pretreatment: wash the pummelo with water to remove dirt and impurities on the surface of the pummelo peel. Stir with a high-speed blender, dry at 65 °C, and obtain pummelo dry powder.

[0055] (1) 25% pummelo peel medium, per liter: 500 mL of LB medium + 250 g of pummelo peel powder, add water to 1 L, 0.2 mol / L NaOH to adjust pH to 4. Dispense into 250 mL conical flasks, 150 mL / flask, 121 °C, sterilization for 20 min.

[0056] (2) 80% grapefruit peel culture medium: 200mL LB medium + 400g grapefruit peel powder, add water to make up to 1L, adjust pH=4 with 0.2mol / L HCl, dispense into 1L shake flasks, 500mL / flask, sterilize at 121℃ for 20min.

[0057] (3) Complete grapefruit peel culture medium: 500g grapefruit peel powder, add water to make up to 1L, adjust pH to 4 with 1mL 1mol / L NaOH, sterilize at 121℃ for 20min.

[0058] 2. Domestication and scale-up fermentation

[0059] (1) Streak the Microbacterium A5 strain stored at 4℃ on an LB solid medium plate and incubate at 30℃ for 24h. Pick a single colony and inoculate it into a 4mL LB liquid medium test tube for activation, and incubate at 30℃ and 150rpm / min for 12h with shaking.

[0060] (2) Take 3 mL of bacterial culture and inoculate it into 150 mL of 25% grapefruit peel medium. Incubate at 30℃ and 150 rpm for 3 days with shaking. After the grapefruit peel particles in the 150 mL medium / 250 mL Erlenmeyer flask have basically degraded and there are no large particles, transfer 100 mL of the culture medium to a 500 mL (80% grapefruit peel medium) / 1 L Erlenmeyer flask and incubate at 30℃ and 150 rpm for 5-7 days with shaking. During the fermentation process, take samples every 4 hours and measure OD600. Finally, plot the growth curve of Microbacterium A5. (Vertical axis: OD600, horizontal axis: shaking fermentation time).

[0061] (3) After the grapefruit peel particles in the 1L conical flask disappear and become a paste, take 500mL of the degradation liquid into a 3L complete grapefruit peel culture medium / 5L aerated and stirred fermentation tank, 30℃, control the dissolved oxygen value ≥40%, automatically adjust the pH to 4 with 0.2mol / L HCl and 1mol / L NaOH, and automatically stir the fermentation mechanically.

[0062] II. Results

[0063] (1) Colony morphology of Microbacterium A5

[0064] like Figure 1 As shown, the starting colonies of Microbacterium A5 used for grapefruit peel degradation are round, pale yellow, thick in the middle and thin at the edges, and relatively moist.

[0065] (2) Domestication of the degradation activity of microbes on grapefruit peel and growth curves during scale-up fermentation.

[0066] The scale-up process of microbial fermentation for grapefruit degradation involves two aspects: firstly, gradually enhancing the degradation activity of the microbial strain on grapefruit peel; and secondly, increasing the processing capacity and shortening the fermentation cycle. After this scale-up process, as...Figure 2 As shown, under the condition of 30℃, 150 rpm shaking culture, the microbacteria entered the logarithmic growth phase at 30h-36h, and the fermentation broth at 35-36 hours of logarithmic growth was determined as the seed liquid for the next step of 500mL / 1L scale-up fermentation. As shown in Figure 3 As shown, under the condition of 30℃, 150 rpm shaking culture, the microbacteria entered the logarithmic growth phase at 30h-36h, and the fermentation broth at 35-36 hours of logarithmic growth was determined as the seed liquid for the next step of 500mL / 1L scale-up fermentation. As shown in

[0067] Example 2 Optimization of the process of extracting pectin oligosaccharide crude extract from fermentation broth

[0068] The process route of pectin oligosaccharide extraction is as shown in Figure 4 As shown: The fermentation broth after fermentation treatment of microbacteria A5 in Example 1 was filtered by a plate frame filter, and the filtrate and residue were separated; the filtrate was precipitated by ethanol, and the supernatant and precipitate were obtained by centrifugation; the precipitate was freeze-dried to obtain pectin crude product; the pectin crude product was deproteinized by TCA or Sevage method, and then precipitated by ethanol; the precipitate was freeze-dried and then separated by DEAE cellulose ion exchange chromatography to obtain MCP-1 and MCP-2; the separated product was desalted by dialysis, concentrated by PEG8000, and freeze-dried to obtain pure oligosaccharide. Among them:

[0069] 1. Filtration and sterilization of fermentation broth

[0070] (1) The 3L fermentation broth in the fermentation tank was loaded into a 100 mesh nylon filter bag, and the filtrate was taken by a presser, and then loaded into a 200 mesh nylon filter bag, and the filtrate was taken by a presser.

[0071] (2) The filtrate was first filtered by a plate frame filter with a 1.0 μM filter membrane to remove the incompletely degraded pomelo peel particles, and then a 0.45 μM filter membrane was used to filter the bacteria by a plate frame filter.

[0072] (3) The filtrate was concentrated by a vacuum rotary evaporator (concentration ratio 3:1) or not concentrated for comparison of the extraction rate of pectin oligosaccharide crude extract by ethanol precipitation method.

[0073] 2. Selection of the optimal ethanol precipitation concentration of pectin oligosaccharide crude extract

[0074] Effect of different ethanol concentration on the recovery of oligogalacturonide: 175 mL, 225 mL, 300 mL, 425 mL, 675 mL of absolute ethanol were added into 75 mL of unconcentrated fermentation broth to get 70%, 75%, 80%, 85%, 90% ethanol precipitation system, respectively. The fermentation broth was concentrated to one third of the original volume, and 58 mL, 75 mL, 100 mL, 142 mL, 225 mL of absolute ethanol were added into 25 mL of the concentrated fermentation broth to get 70%, 75%, 80%, 85%, 90% ethanol precipitation system, respectively. The above treatment was placed in a chromatography refrigerator at 4°C overnight for sufficient precipitation. After complete precipitation, the fermentation broth with different ethanol concentrations was centrifuged at 6000 RPM for 20 min. The precipitate was collected and freeze-dried to calculate the recovery rate of oligogalacturonide at different ethanol concentrations.

[0075] Recovery rate = (weight of precipitate) / (weight of oligogalacturonide in fermentation broth) × 100%

[0076] The results are shown in Table 1. Concentrating the fermentation broth by rotary evaporation before ethanol precipitation not only saves the amount of ethanol used, but also improves the recovery rate of pectin oligosaccharide crude extract. The recovery rate of pectin oligosaccharide crude extract from three-fold concentrated fermentation broth with 80% ethanol is the highest, reaching 87%.

[0077] Table 1 Precipitate weight and recovery rate of pectin oligosaccharide crude extract obtained by different ethanol concentrations

[0078]

[0079] 3. Optimization of protein removal method for oligogalacturonide crude extract

[0080] Comparison of hydrochloric acid, TCA method and Sevage method for protein removal rate in crude extract

[0081] (1) Prepare a 10 mg / mL pectin crude extract solution.

[0082] (2) Effect of different hydrochloric acid addition amounts on protein removal rate: adjust the pH of the pectin crude extract solution to 2, 2.5, 3, 3.5 with 0.5 mol / L hydrochloric acid, and place it at 4°C for 12 h.

[0083] Effect of different TCA addition amounts on protein removal rate: adjust the pH of the pectin crude extract solution to 2, 2.5, 3, 3.5 with 10% TCA, and place it at 4°C for 12 h.

[0084] Sevage method for protein removal: mix the pectin crude extract solution with Sevage reagent at a ratio of 4:1. Shake well for 30 min, and centrifuge at 4000 RPM for 10 min. Take the supernatant and discard the middle layer protein precipitate. Repeat multiple times until no protein precipitate is produced.

[0085] Each group of 3 parallel repeats. Centrifuge at 3000 rpm / min for 5 min, discard the precipitate, and take the supernatant. The oligosaccharide content in the supernatant is determined by the phenol-sulfuric acid method, and the residual amount of protein is determined by the Coomassie brilliant blue method. The supernatant is precipitated with 80% ethanol, placed at 4°C overnight, centrifuged at 7000 RPM for 20 min, and the precipitate is collected to detect the oligosaccharide mass.

[0086] (3) Test method:

[0087] ① Phenol-sulfuric acid method for determining the content of pectin oligosaccharides

[0088] Concentrated sulfuric acid can hydrolyze naringin peel pectin oligosaccharides into reducing monosaccharides and rapidly dehydrate. After hydrolysis and dehydration of naringin peel polysaccharides by concentrated sulfuric acid, sugar aldehyde derivatives are generated. The addition of phenol will show an orange yellow color and have a characteristic absorption peak at a wavelength of 490 nm. The content of oligomeric pectin in the sample is detected by the sulfuric acid method using glucose as a standard. A standard curve of the relationship between glucose content and OD490 is established. Dry the glucose in an oven. Weigh 0.0010 g of dried glucose in a 10 mL centrifuge tube, dissolve it with deionized water, and dilute it to 10 mL. A 0.1 mg / mL glucose standard solution is prepared. nm

[0089] Sample treatment according to the conditions in Table 2, three replicates for each concentration. Then use the enzyme marker to detect OD 490 . Finally, draw the standard curve (ordinate: OD 490 , abscissa: glucose concentration). Figure 5 For the glucose standard curve determined by the phenol-sulfuric acid method, the results show that there is a good linear relationship between the glucose concentration and OD490. The linear equation is: y = 20.942x - 0.0278, and the goodness of fit is 0.9960.

[0090] Table 2 Sample treatment conditions for determining the glucose concentration standard curve by the phenol-sulfuric acid method

[0091]

[0092] When determining the polysaccharide content of the sample, mix the sample and phenol 2:1, then add 5 times the amount of concentrated sulfuric acid to the phenol. After mixing, stand for 10 min. 40°C water bath for 10 min. Cool to room temperature and measure OD 490 . Substitute the glucose standard curve to calculate the polysaccharide concentration of the sample.

[0093] Polysaccharide loss rate = ((oligosaccharide content in pectin extract before deproteinization - oligosaccharide content in supernatant after deproteinization) / oligosaccharide content in pectin extract before deproteinization x 100%)

[0094] ② Coomassie brilliant blue method for determining protein content ​

[0095] Coomassie Brilliant Blue G-250 dye binds to proteins under acidic conditions. The dye's maximum absorption peak, originally located at 465 nm, changes to 595 nm after binding. Simultaneously, the solution color changes from brownish-black to blue. This is confirmed by measuring the OD (Organization Discharge). 595 This allows you to calculate the amount of protein.

[0096] Standard curve for protein content determination using bovine serum albumin: Weigh 1 mg of Coomassie Brilliant Blue G-250. Dissolve in 0.5 mL of 95% ethanol, then add 1 mL of 85% phosphate solution. Make up to 10 mL with deionized water. Weigh 1 mg of bovine serum albumin and make up to 10 mL (100 μg / mL). Prepare protein detection solutions of varying concentrations according to Table 3, mix well in EP tubes, and incubate for 5 min. Perform triplicate for each concentration. Measure OD using a microplate reader. 595 Finally, plot the standard curve (vertical axis: OD). 595 (x-axis: protein concentration). Figure 6 The standard curve for protein determination using the Coomassie Brilliant Blue method shows the relationship between protein and OD. 595 There is a good linear relationship between them, and the linear equation is: y=0.007x-0.0006, with a goodness of fit of 0.9958.

[0097] Table 3. Sample loading conditions for determining the protein standard curve.

[0098]

[0099] When determining the protein content of a sample, the sample is mixed with Coomassie Brilliant Blue at a 1:5 ratio. Then, the OD is measured. 595 The protein content of the sample was calculated by substituting the values ​​into the standard curve.

[0100] Protein removal rate = (Protein content in pectin extract before deproteinization - Residual protein content in supernatant after deproteinization) / Protein content in pectin extract before deproteinization × 100%

[0101] Results: The protein concentration of the crude pectin solution was measured to be 126.371 μg / mL, and the oligosaccharide content was 7.397 mg / mL. The effects of different pH values of hydrochloric acid on the protein removal rate and polysaccharide loss rate are shown in Table 4, the effects of TCA adjusting different pH values on the protein removal rate and polysaccharide loss rate are shown in Table 5, and the effects of different Sevage method processing times on the protein removal rate and polysaccharide loss are shown in Table 6. It can be seen that the polysaccharide loss after adjusting the pH of the protein by hydrochloric acid or TCA is not large. The maximum removal rate of hydrochloric acid for adjusting the pH of the protein is 26.45%, and the maximum removal rate of TCA for adjusting the pH of the protein is 74.67%. As shown in Table 6, with the increase of the number of Sevage method for removing protein, the protein content gradually decreases. After removing protein for 7 times, the protein removal rate reaches 92.70%, and the polysaccharide loss rate is 22.48%. Compared with the hydrochloric acid and TCA method, although the Sevage method has high protein removal rate, the polysaccharide loss rate is also high, and the Sevage method is complicated to operate, which may cause the emission of organic solvents.

[0102] By comparing the three methods of removing protein, the method of adjusting the pH of the low oligogalacturonate crude extract to 3 by 10% TCA was selected to remove protein. The protein removal solution was centrifuged at 3000 rpm to remove the precipitate, and the supernatant was placed at 4°C overnight with 80% ethanol solution. The precipitate was collected by centrifugation at 7000 rpm for 20 min and freeze-dried for storage.

[0103] Table 4 Effects of different pH values of hydrochloric acid on protein removal rate and polysaccharide loss rate

[0104]

[0105] Table 5 Effects of TCA adjusting different pH values on protein removal and polysaccharide loss rate

[0106]

[0107] Table 6 Effects of different Sevage method processing times on protein removal rate and polysaccharide loss

[0108]

[0109] 4. Purification of pectin by DEAE ion exchange chromatography

[0110] (1) Pretreatment of DEAE cellulose DE-52

[0111] Take 50 g DEAE cellulose powder DE-52, room temperature with 1 L of deionized water swelling overnight. Multiple pour the suspended fine particles. The first use of DEAE cellulose powder to boil 20 min degassing. After settling, filter out the deionized water. With 1 L 0.5 M NaOH-NaCl solution soak 4 h. After filtration with deionized water wash. Test the filtrate pH is neutral. After drying with 1 L 0.5 mol / L HCl soak 4 h. With deionized water to neutral after soaking in PBS at pH = 6.5. Degassing 20 min, placed in 4 ℃ chromatography refrigerator for standby.

[0112] (2) Column packing

[0113] After the column is fixed vertically on the iron stand, check for leaks with deionized water. Check if the bottom of the column is leaking. Leave about 3 cm high water column at the bottom of the column. The treated DEAE cellulose is stirred evenly, open the water clamp, and use a glass rod to drain the column. After natural settlement, close the water clamp, and seal the column with phosphate buffer.

[0114] (3) Equilibrium

[0115] Equilibrate 5 column volumes with pH = 6.5 phosphate buffer at a flow rate of 3 mL / min.

[0116] (4) Sample loading

[0117] Dissolve the sample in pH = 6.5 phosphate buffer to a concentration of 25 mg / mL. Centrifuge at 5000 RPM for 10 min. Take the supernatant and discard the precipitate. Filter the supernatant with a 0.22 μm filter membrane. Load 3 mL according to the sample volume of 1%-5% of the column bed volume. After the sample enters the stationary phase, add pH = 6.5 phosphate buffer along the inner wall of the column to seal the column, and set the flow rate to 1 mL / min.

[0118] (5) Washing

[0119] Wash away basic and neutral polysaccharides and other impurities with pH = 6.5 phosphate buffer. Collect 1 tube of effluent (5 mL / tube) every 5 min.

[0120] (6) Elution

[0121] Elute with 0.1 M, 0.2 M, 0.3 M, and 0.4 M NaCl solutions in sequence. The elution volume is 100 mL, and the flow rate is 1 mL / min. Collect 1 tube (5 mL / tube) every 5 min. Determine the polysaccharide content by the phenol-sulfuric acid method. Plot the elution curve with tube number as the abscissa and OD 490 as the ordinate.

[0122] (7) Regeneration and cleaning of DEAE cellulose anion exchanger

[0123] Wash the column with 5 column volumes of NaOH-NaCl solution at a flow rate of 3 mL / min. Wash with 5 column volumes of deionized water and store in a 4°C refrigerator.

[0124] (8) Dialysis and desalting of the eluted sample from ion exchange chromatography

[0125] According to the elution curve, the elution peak corresponding to the 0.3 M NaCl solution was collected as POS-1 solution, and the elution peak corresponding to the 0.4 M NaCl solution was collected as POS-2 solution. Dialysis and desalting were performed respectively.

[0126] Preparation of the dialysis bag: The dialysis bag was cut to an appropriate length. The dialysis bag was boiled in 2% sodium bicarbonate and 1 mmol / L EDTA (pH 8.0) for 10 min. The dialysis bag was washed with deionized water.

[0127] Dialysis was performed using a dialysis bag with a 2000 cut-off. The dialysis bag was tested for leaks. The sample was added to the dialysis bag, and the amount of the sample should not exceed 2 / 3 of the volume of the bag. Deionized water was added to a 2 L beaker and stirred slowly on a magnetic stirrer. The solution was tested with 1% AgNO3 until no white precipitate was formed.

[0128] (9) Concentration of the eluted sample from ion exchange chromatography

[0129] PEG8000 has strong water absorption. The water in the POS-1 solution and the POS-2 solution was removed to concentrate the solutions by using the water absorption of PEG8000. The POS-1 solution and the POS-2 solution were first placed in a dialysis bag, and powdered PEG8000 was placed on the outer surface of the dialysis bag. The dialysis bag was placed at 4°C.

[0130] Results: The use of ion strength gradient elution during elution allowed the components to be separated to reach a limited adsorption equilibrium state one after another, and finally achieve the separation effect. The elution curve of DEAE ion exchange chromatography is shown in Figure 7 The elution peak I was the neutral polysaccharides and impurities such as impure proteins, which were removed by using a phosphate buffer solution with pH = 6.5 to elute tubes 1-40. The elution peak II (POS-1) appeared when a 0.3 M NaCl solution was used to elute tubes 40-60, and tubes 53-55 were collected for dialysis and desalting. The elution peak III (POS-2) appeared when a 0.4 M NaCl solution was used to elute tubes 60-80, and tubes 72-74 were collected for dialysis and desalting. No elution peak appeared after subsequent elution with 0.3 M NaCl solution and 0.4 M NaCl solution.

[0131] Example 3: Determination of the activity and molecular weight of shaddock pectin

[0132] I. Detection of the antioxidant activity of pectin by scavenging DPPH

[0133] 1. Method

[0134] (1) 2,2-diphenyl-1-picrylhydrazyl (DPPH) was prepared into a 0.4 mmol / L solution with 50% ethanol solution. Pectin solutions of 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 2.5 mg / mL and ascorbic acid solution were prepared. The pectin solutions included a crude pectin solution, a POS-1 solution eluted after DEAE cellulose ion exchange chromatography, and a POS-2 solution. Ascorbic acid is a recognized antioxidant with strong antioxidant properties, and thus was used as a positive control.

[0135] (2) 2 mL of the 2.5 mg / mL pectin sample solution, 2 mL of the 2.5 mg / mL ascorbic acid solution, and 2 mL of the 50% ethanol solution were each added to 2 mL of the DPPH solution, mixed rapidly, and left to stand at room temperature for 30 min before full wavelength scanning.

[0136] (3) 2 mL of each of the 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 2.5 mg / mL pectin solutions were each added to 2 mL of the DPPH solution. Three parallel repeats were performed for each. After mixing, the solutions were left to stand in the dark for 30 min. An enzyme marker was used to measure OD 522 . For the control group, 2 mL of the 50% ethanol solution was added to 2 mL of the DPPH solution to measure control OD 522 . For the blank group, 2 mL of the pectin solution was added to 2 mL of the 50% ethanol solution to measure blank OD 522 . The DPPH clearance rate was calculated as follows:

[0137] DPPH clearance rate = 1- × 100%

[0138] 2. Results

[0139] As shown in Figure 8 , DPPH has a characteristic absorption peak at 326 nm and 522 nm. After the addition of the pectin solution or ascorbic acid, the characteristic absorption peak at 522 nm decreased significantly, and especially after the addition of ascorbic acid, the absorption peak at 522 nm became significantly flatter. Therefore, when measuring the clearance rate of the pectin solution and ascorbic acid on DPPH, the change in absorbance at 522 nm was used to reflect the clearance rate of the sample solution on DPPH.

[0140] From Figure 9It can be seen that pectin crude product, POS-1 and POS-2 all have scavenging effect on organic free radical DPPH, and the antioxidant activity is in the order of POS-1 > POS-2 > pectin crude product. Especially, the antioxidant activity of POS-1 is almost similar to that of ascorbic acid, and has very strong antioxidant activity. The reason why the antioxidant activity of POS-1 and POS-2 is stronger than that of pectin crude product is that pectin crude product contains impurities such as protein and certain organic matter. POS-1 and POS-2 are small molecular pectin purified by ion exchange chromatography, and not only have higher polysaccharide purity, but also have smaller molecular weight. In addition, with the increase of the concentration of pectin solution, the antioxidant activity is also enhanced.

[0141] II. Gel permeation chromatography (GPC) for detecting the purity and molecular weight of pectin oligosaccharide

[0142] 1. Method

[0143] POS-1 and POS-2 were prepared into a solution of 2 mg / mL with mobile phase. The flow rate was set to 0.8 mL / min, and the methanol in the pipeline was cleaned with 0.2 g / L NaN3 solution. Check whether the pipeline is blocked or leaking. Install the column and observe whether the column interface is leaking. Replace the NaN3 solution with the mobile phase. First run the baseline at 0.4 mL / min, and then run the baseline at 0.8 mL / min. If the baseline is a straight line after 60 min, then sample.

[0144] 2. Results

[0145] The standard curve equation was obtained by GPC detection fitting with different molecular weight of pullulan as standard product: y = 0.0683x3-2.0164x2+18.779x-50.576, R2=0.9998 (as shown in Figure 10 ). The results of POS-1 pectin oligosaccharide purity and molecular weight are shown in Figure 11 , and the GPC detection results show that POS-1 is a single symmetrical elution peak, and the weight average molecular weight is 19921, indicating that the relative molecular mass distribution of oligosaccharide is uniform, and the purity is greater than 95%. The results of POS-2 pectin oligosaccharide are shown in Figure 12 , and the GPC detection results show that POS-1 is a single symmetrical elution peak, and the weight average molecular weight is 17307, indicating that the relative molecular mass distribution of oligosaccharide is uniform, and the purity is greater than 95%.

[0146] III. Ion chromatography for detecting the monosaccharide composition of two kinds of pectin oligosaccharide

[0147] 1. Method

[0148] (1) Preparation of reagents

[0149] 15 mM NaOH solution: 2.4 g 50% NaOH solution, 2 L water;

[0150] 15 mM NaOH & 100 mM NaOAC solution: 1.2 g 50% NaOH solution, 8.2 g NaOAC, 1 L water.

[0151] (2) Preparation of standard solution and calculation method

[0152] Take 16 kinds of monosaccharide standard (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid, mannuronic acid) to prepare standard stock solution.

[0153] Take each monosaccharide standard solution to precisely configure the concentration standard as a mixed standard. According to the absolute quantitative method, the mass of different monosaccharides is determined, and the molar ratio is calculated according to the molar mass of monosaccharide.

[0154] (3) Sample preparation

[0155] Precisely weigh 5 mg of sample into an ampoule, add 3 M TFA 2 mL, hydrolyze at 120 ℃ for 2 h. Accurately pipette the acid hydrolysis solution into a tube and dry it under nitrogen, add 10 mL of water and vortex to mix, pipette 40 uL and add 960 uL of deionized water, centrifuge at 12000 rpm for 5 min. Take the supernatant into IC analysis.

[0156] (4) Chromatographic method

[0157] Chromatographic column: Dionex Carbopac T PA20 (3*150); mobile phase: A: H2O; B: 15 mM NaOH C: 15 mM NaOH & 100 mM NaOAC; flow rate: 0.3 mL / min; injection volume: 5 uL; column temperature: 30 °C; detector: electrochemical detector.

[0158] 2. Results

[0159] The ion chromatography detection results show that the ion chromatogram of 16 kinds of monosaccharide standards (such as Figure 13 ), POS-1 monosaccharide is composed of glucose and galactose, and the molar ratio is 0.887:0.113 (such as Figure 14 ). POS-2 monosaccharide is composed of glucose and galactose, and the molar ratio is 0.883:0.117 (such as Figure 15 ). It is shown that POS-1 and POS-2 are both polygalactose.

[0160] Four, the influence of two pectin oligosaccharides on the lung mucosal histopathology of H1N1 infected mice

[0161] 1. Method

[0162] (1) Animal feeding and grouping: 30 BALB / c mice aged 2 weeks were fed in a SPF environment with 22±2 ℃, 55±15% relative air humidity and 12 h dark / light cycle, and were given conventional balanced diet and sterile water for 10 days of adaptation. Then, the mice were divided into 6 groups according to body weight matching method, 5 mice in each group, and ear tags were punched on the left ears of the mice using ear tag pliers for marking.

[0163] Among them, the respiratory tract H1N1 virus infected mouse model and the respiratory tract RSV virus mouse model were constructed as follows:

[0164] Method for infecting mice with respiratory tract H1N1 virus

[0165] Establishment of H1N1 virus infected mouse model: the mice were anesthetized by intraperitoneal injection, and then intranasally infected with IAV PR8 virus strain (2×10 3 PFU / mouse).

[0166] Method for infecting mice with respiratory tract RSV virus

[0167] The mice were anesthetized by intraperitoneal injection, and then intranasally infected with RSV A2 virus strain (6.9×10 5 PFU / mouse).

[0168] (2) Drug administration method: pectin oligosaccharide freeze-dried powder POS-1 and POS-2 were prepared into a 30 mg / mL solution with distilled water. The original pectin was prepared into a 40 mg / mL POSSD solution by hot water extraction. Ribavirin was prepared into a 10 mg / mL solution with distilled water. Then, the ribavirin administration group was given intragastrical administration at a dose of 50 mg / kg / d, and the pectin administration group was given intragastrical administration to the mice at a dose of 300 mg / kg / d, with a volume of 0.1-0.2 mL. The normal control group and the virus control group of mice were given intragastrical administration of an equal amount of distilled water. The mice were weighed every two days, and the intragastrical administration dose was adjusted according to the body weight change. The mice in each group were given intragastrical administration continuously for 3 weeks, and then were infected with respiratory virus, and the tissue sampling was performed after 5 days of continuous intragastrical administration.

[0169] Animal grouping and drug administration method:

[0170]

[0171] (3) Paraffin section preparation: the mice were killed by dissecting and taking heart blood after anesthesia, and the right upper lung tissue and colon were taken and fixed with 4% paraformaldehyde at 4 ℃, followed by dehydration, embedding, and sectioning (4 μm).

[0172] (4) Histopathology analysis: The sections were stained with hematoxylin-eosin and observed under a microscope to analyze the pathological changes of the lung tissue (200x).

[0173] 2. Results

[0174] (1) Effects of POS-1 and POS-2 on the lung histopathology of H1N1 infected mice. As shown in FIG. 1, the lung tissue of the mice was stained with hematoxylin-eosin to evaluate the effects of the two pectin oligosaccharides on the lung histopathology of H1N1 infected mice. The results showed that the bronchial epithelium of the normal control group was a single layer of ciliated columnar epithelium, with loose cell arrangement, thin wall, and incomplete ring-shaped smooth muscle. The epithelial cells of the terminal bronchioles were arranged tightly, with thick walls and thin folds (FIG. 1A). The mice in the H1N1 virus infection group had extensive bronchial epithelial cell necrosis and shedding, nuclear fragmentation, and enhanced cytoplasmic acidophilia. There was extensive inflammatory cell infiltration around the alveoli, bronchi, and blood vessels, with mild hemorrhage around a small range of blood vessels, and pus cells exuding from the lumen. The lung showed severe inflammatory damage (FIG. 1B). Compared with the H1N1 virus infection group, the ribavirin positive drug control group showed only a small amount of edema and inflammatory cell infiltration around the pulmonary arterioles, with widened interstitial space and no other obvious abnormalities. This indicated that ribavirin had a significant recovery effect on the lung tissue damage caused by H1N1 virus infection (FIG. 1C). The POSSD administration group showed extensive bronchial epithelial cell necrosis and shedding, nuclear fragmentation, and enhanced cytoplasmic acidophilia, with extensive inflammatory cell infiltration around the alveoli, bronchi, and blood vessels. There was mild hemorrhage around a small range of blood vessels, pus cells exuding from the lumen, and edema around the pulmonary arterioles, showing severe lung inflammatory damage (FIG. 1D). The POS-1 administration group showed some bronchial epithelial damage, with thickened mucosal muscle layer, inflammatory cell infiltration in the alveoli and alveolar walls, and inflammatory cell infiltration around some bronchi and blood vessels, accompanied by edema around the arterioles and widened interstitial space (FIG. 1E). The POS-2 administration group showed reduced bronchial epithelial cell necrosis and cytoplasmic acidophilia, with only a small amount of inflammatory cell infiltration around some bronchi and blood vessels, and more edema around the pulmonary arterioles. However, the alveoli were clean, and there were no other obvious abnormalities (FIG. 1F). This indicated that POS-2 had a significant therapeutic and preventive effect on the lung tissue damage caused by H1N1 infection. Figure 16 Figure 16 Figure 16 Figure 16 Figure 16 Figure 16 Figure 16

[0175] (2) Effects of POS-1 and POS-2 on the lung histopathology of RSV infected mice.​​​​​​​Figure 17 The effects of two pectin oligosaccharides on the lung mucosal histopathology of RSV infected mice were evaluated by detecting the location and degree of cell infiltration in the lung tissue of mice by hematoxylin-eosin staining. The results showed that the lung bronchial epithelial cells of the normal control group of mice were closely arranged, normal in shape, and the alveolar wall thickness and alveolar size were uniform (such as Figure 17 -A). The alveolar wall and alveoli of the RSV virus infected group of mice had more inflammatory cell exudation, there were inflammatory cell infiltrations around the blood vessels and formed a vascular cuff, the bronchial epithelial cells were damaged, the cells were swollen, and the cytoplasm was vacuolated, showing a more serious lung inflammatory injury (such as Figure 17 -B). Compared with the RSV virus infected group, the ribavirin positive drug control group had only a small amount of inflammatory cells infiltrating around the blood vessels, and there was no obvious other lung damage (such as Figure 17 -C). The POSSD administration group had extensive bronchial epithelial cell necrosis and shedding, nuclear fragmentation, enhanced cytoplasmic acidophilia, extensive inflammatory cell infiltration around the alveoli and bronchi, and blood vessels, mild hemorrhage around a small range of blood vessels, pus cell exudation in the lumen, and edema around the pulmonary arterioles, showing a more serious lung inflammatory injury (such as Figure 17 -D). The bronchial epithelial cells of the POS-1 administration group of mice were normal in shape, the inflammatory cell infiltration and exudation around the blood vessels and alveoli were reduced, and the alveolar wall thickness was uniform (such as Figure 17 -E). The POS-2 administration group of mice had only a small amount of inflammatory cell focal infiltration around the bronchial and blood vessels, and a small amount of inflammatory cell exudation and infiltration in the alveoli and alveolar wall, and no other lung damage (such as Figure 17 -F). It was shown that the two pectin oligosaccharides had a certain recovery effect on the lung mucosal tissue damage caused by RSV infection.

Claims

1. A method for producing pecticoligosaccharides from citrus peel by microbial degradation, characterized by, It comprises the following steps: S1. Adding the microbacterium A5 to the culture medium containing the grapefruit peel to carry out fermentation to obtain a fermentation liquor; S2. Carrying out pressing and filtering on the fermentation liquor of step S1 to obtain a filtrate and a residue; S3. Carrying out ethanol precipitation on the filtrate of step S2, centrifuging to obtain a supernatant and a precipitate, freeze-drying the precipitate to obtain a crude oligogalacturonide product; S4. Removing the impurities from the crude oligogalacturonide product of step S3, carrying out ethanol precipitation, freeze-drying the precipitate, and then carrying out ion exchange chromatography, dialysis desalting, concentration, and freeze-drying to obtain a pure oligogalacturonide product; The microbacterium A5 is preserved in the China Center for Type Culture Collection on August 7, 2009, and the preservation number is CCTCC NO: M209174; The fermentation of step S1 is to first culture the activated microbacterium A5 in a culture medium with a low content of grapefruit peel to obtain a fermentation liquor in the middle and late logarithmic growth phase, then add the fermentation liquor as a seed to a culture medium with a high content of grapefruit peel to carry out amplification culture, and finally add it to a complete grapefruit peel culture medium to carry out fermentation culture; the culture medium with a low content of grapefruit peel is an LB culture medium added with 25% w / v grapefruit peel dry powder; the culture medium with a high content of grapefruit peel is an LB culture medium added with 80% w / v grapefruit peel dry powder; and the complete grapefruit peel culture medium is a culture medium added with 100% grapefruit peel dry powder; The ethanol precipitation of step S3 uses 80% ethanol; and the filtrate is concentrated before ethanol precipitation; The removal of impurities of step S4 uses the TCA method to remove proteins; The ion exchange chromatography of step S4 is DEAE cellulose ion exchange chromatography, and 0.3M and 0.4M sodium chloride solutions are used for elution; The pectic oligosaccharide has a molecular weight distribution in the range of 1.7 x 10 4 ~ 2.0 x 10 4 consisting of glucose and galactose in a molar ratio of 0.88-0.89:0.11-0.

12.

2. The method of claim 1, wherein, The pressing and filtering of step S2 is fractional pressing and filtering and plate-frame filtering to remove bacteria.

3. Pectic oligosaccharides obtainable by the process according to any one of claims 1 or 2, having a molecular weight distribution in the range of 1.7 x 10 4 ~ 2.0 x 10 4 consisting of glucose and galactose in a molar ratio of 0.88-0.89 : 0.11-0.

12.

4. The use of the oligogalacturonide product of claim 3 in the preparation of a product for resisting oxidation, inhibiting influenza virus H1N1, and / or respiratory syncytial virus RSV.

5. Use of Microbacterium sp. A5 in the production of pecticoligosaccharides from fermented pomace, characterized in that, The microbacterium A5 was preserved in China Center for Type Culture Collection on August 7, 2009, and the preservation number is CCTCC NO: M209174; the pecticoligosaccharide has a molecular weight distribution in the range of 1.7 x 10 4 ~ 2.0 x 10 4 , and is composed of glucose and galactose, and the molar ratio of glucose and galactose is 0.88 ~ 0.89: 0.11 ~ 0.12.

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