WO2019114159A1 - 一种油茶粕多肽、多糖及其制备方法、应用和油茶粕脱毒方法 - Google Patents

一种油茶粕多肽、多糖及其制备方法、应用和油茶粕脱毒方法 Download PDF

Info

Publication number
WO2019114159A1
WO2019114159A1 PCT/CN2018/082479 CN2018082479W WO2019114159A1 WO 2019114159 A1 WO2019114159 A1 WO 2019114159A1 CN 2018082479 W CN2018082479 W CN 2018082479W WO 2019114159 A1 WO2019114159 A1 WO 2019114159A1
Authority
WO
WIPO (PCT)
Prior art keywords
oil tea
camellia
preparation
powder
molecular weight
Prior art date
Application number
PCT/CN2018/082479
Other languages
English (en)
French (fr)
Inventor
盛蓓蓓
Original Assignee
盛蓓蓓
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201711304360.0A external-priority patent/CN108060196B/zh
Priority claimed from CN201711304376.1A external-priority patent/CN107964033B/zh
Priority claimed from CN201711308451.1A external-priority patent/CN108003253B/zh
Priority claimed from CN201711304358.3A external-priority patent/CN108041282B/zh
Application filed by 盛蓓蓓 filed Critical 盛蓓蓓
Publication of WO2019114159A1 publication Critical patent/WO2019114159A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

  • the object of the present invention is to overcome the deficiencies of the prior art, and provide a Camellia oleracea polypeptide, a polysaccharide, a preparation method thereof, an application method and a method for detoxification of Camellia oleifera.
  • Step S3 filtering the supernatant with absorbent cotton, collecting the filtrate, and lyophilizing to obtain a crude lyophilized powder
  • step S4 the crude lyophilized powder is dissolved in ultrapure water, separated by an ultrafiltration membrane of different molecular weight cut off, and the peptide having a molecular weight in the range of (5000 u, 3000 u) is collected and freeze-dried.
  • the trypsin is added in an amount of from 2.5 to 3.5 g/100 g based on the weight of the tantalum powder.
  • Step S2 adding alkaline protease to enzymatic hydrolysis, the enzymatic hydrolysis conditions are: pH value, 8.3-8.7; enzymatic hydrolysis temperature, 53-57 ° C; enzymatic hydrolysis time, 9-11 hours; after the end of enzymatic hydrolysis, heating to make alkaline protease Inactivated, cooled to room temperature, and allowed to stand for supernatant;
  • Step S3 filtering the supernatant with absorbent cotton, collecting the filtrate, and lyophilizing to obtain a crude lyophilized powder
  • the ultrafiltration membranes of different molecular weight cutoffs comprise ultrafiltration membranes having molecular weight cutoffs of 5000 u and 3000 u, respectively.
  • the method of inactivating the alkaline protease by heating is: high temperature treatment at 90 ° C for 10 minutes.
  • the alkaline protease is added in an amount of from 2.5 to 3.5 g/100 g based on the weight of the niobium powder.
  • the enzymatic hydrolysis conditions of the alkaline protease are preferably pH 8.5, temperature 55 ° C, and time 10 hours.
  • camellia polypeptide is used for the preparation of a feed or a medicament for promoting estrus and conception of a cow.
  • the small fruit oil tea stalk is used as a raw material, and is obtained by water extraction and alcohol precipitation, DEAE-52 cellulose column chromatography and Sephadex G-l00 column chromatography.
  • the average molecular weight is 75.3, 85.6 or 94.7 kDa.
  • the small fruit oil tea refers to a solid residue obtained by removing the oil after the fruit of the small fruit oil tea is subjected to a pressing treatment or a leaching method.
  • the preparation method of the above-mentioned camellia polysaccharide comprises the following steps:
  • Step S1 pulverizing the small fruit oil tea stalk to 80-100 mesh, extracting by boiling water, filtering, combining the filtrate, and concentrating;
  • Step S2 adding 4-5 volumes of absolute ethanol to the concentrated solution, precipitating at a low temperature, and collecting the precipitate by centrifugation;
  • step S3 the precipitate is dissolved in deionized water, applied to a DEAE-52 chromatography column, and sequentially eluted with a gradient of pure water and different concentrations of sodium chloride solution, respectively, and a pure water eluate is collected, and 0.3 mol/L of chlorination is separately collected.
  • step S4 the content of polysaccharides in each tube is detected by a sulfuric acid-phenol method, and the number of tubes collected is plotted on the abscissa and the ultraviolet absorbance is plotted on the ordinate, and a chromatographic elution curve of Sephadex G-100 is drawn, and each concentrate is passed through Sephadex G- After separation of 100 columns, one main elution peak was obtained, and the eluted peaks were collected and lyophilized.
  • Camellia oleracea polysaccharide antagonizes the use of foot-and-mouth disease vaccine for bull semen damage.
  • Camellia oleracea detoxification method
  • a method for detoxifying a small fruit oil tea stalk wherein the small fruit oil tea stalk refers to a solid residue obtained by removing the oil after the fruit of the small fruit oil tea is subjected to a pressing treatment or a leaching method, wherein the detoxification means removing the saponin and the small fruit Camellia oleifera is pulverized into oil tea glutinous rice powder, and evenly sprayed with acidic aqueous solution, inoculated with Rhizopus oryzae.
  • the acid is hydrochloric acid, formic acid or acetic acid.
  • the pH of the acidic aqueous solution is preferably 4-5.
  • the inoculum amount of Rhizopus oryzae is 15%-25% (mL/g), and the concentration of Rhizopus oryzae is 10 7 single spores/mL, based on the wet weight of the oil tea powder after spraying the acidic aqueous solution.
  • the heap rot temperature is 28-32 °C.
  • the rot time is 24-72 h.
  • the acidic aqueous solution is preferably an aqueous solution of formic acid having a pH of 4 to 5, and the water content of the sprayed to the camellia powder is 60-70%.
  • the inoculum amount of Rhizopus oryzae is 20% (mL/g), the concentration of Rhizopus oryzae is 10 7 single spores/mL, and the 30 ° C heap rot is 24 hours. .
  • a small fruit oil samovar prepared by the above detoxification method was prepared by the above detoxification method.
  • the above-mentioned small fruit oil tea is used as an animal feed.
  • the present inventors have found that the hydrolyzed polypeptide of Camellia oleracea has various activities, and the activity of the hydrolyzed polypeptide is affected by the proteolytic site in the Camellia oleifera: the polypeptide having a molecular weight of (5000u, 3000u) produced by trypsin hydrolysis has improved bovine freezing.
  • the anti-oxidation ability of semen can be used as a cryopreservation agent for bovine semen to reduce oxidative damage, while the polypeptide produced by alkaline protease is not obvious; a polypeptide with a molecular weight of (5000u, 3000u) produced by hydrolysis of alkaline protease can promote Lack of cows estrus and conception, while trypsin-producing peptides are not obvious;
  • the preparation method of the hydrolyzed polypeptide of the invention is simple, and only simple pulverization, enzymatic hydrolysis, ultrafiltration, lyophilization can be used, and these steps can be realized in a large production in a factory, and is easy to implement;
  • the invention realizes the waste recycling of the small fruit oil tea stalk, and can increase the economic added value of the small fruit oil samovar;
  • the Camellia oleracea polysaccharide provided by the invention can effectively antagonize the damage caused by the foot-and-mouth disease vaccine to the bull semen, and thus can be used for reducing the loss of the foot-and-mouth disease vaccine on the farm;
  • the preparation method of the camellia sinensis polysaccharide provided by the invention has few steps, and the separation and purification effect is good, and all are single components;
  • the detoxification method provided by the invention can effectively remove the saponin from the small fruit oil tea stalk, thereby removing the bitterness and spicy taste, improving the taste, and further can be used as a feed additive; in particular, when using formic acid as the acid rotant , can significantly improve the removal efficiency of saponins from Rhizopus oryzae;
  • the detoxification method provided by the invention is simple and feasible, has low cost, and is easy to be industrialized and popularized.
  • Figure 1 is a DSC curve of Camellia oleracea polypeptide 1-6;
  • Figure 2 is the effect of the Camellia oleracea polypeptide on the MDA content of the oxidation product after cryopreservation;
  • Figure 3 is the effect of camellia pupa polypeptide on estrus rate and conception rate of ill-fated cows
  • Figure 4 is an elution curve of a DEAE-52 column pure water eluate by Sephadex G-100 column chromatography
  • Figure 5 is an elution curve of a 0.3 mol/L sodium chloride eluate on a DEAE-52 column by Sephadex G-100 column chromatography;
  • Figure 6 is an elution curve of a 0.5 mol/L sodium chloride eluate on a DEAE-52 column by Sephadex G-100 column chromatography;
  • Figure 7 is a comparison of semen activity before and after the foot-and-mouth disease vaccine of each group of Holstein cattle;
  • Figure 8 is a comparison of the removal rates of total saponins in Camellia oleifera powder after the rot of the mixture of Examples 9-17 for 24 hours.
  • Camellia meiocarpa Hu. which was collected from the Camellia oleifera germplasm resources of the Tongkou State-owned Forest Farm in Minhou County, Fujian province, was used as a raw material in the spring of 1982-1983, and the oil was removed by pressing to obtain small fruit oil tea. ,spare.
  • step S1 the small fruit oil tea mash is pulverized to 80-100 mesh, soaked in water overnight, and finally a sputum powder suspension is prepared, and the mass concentration of the sputum powder suspension is 150 g/L;
  • Step S2 adding trypsin digestion (addition weight is 3.0 g / 100 g by weight of tantalum powder), the enzymatic hydrolysis conditions are: pH value, 8.0; enzymatic hydrolysis temperature, 45 ° C; enzymatic hydrolysis time, 11 hours; end of enzymatic hydrolysis After that, the solution was inactivated at a high temperature of 90 ° C for 10 minutes, cooled to room temperature, and the supernatant was allowed to stand;
  • Step S3 the supernatant is filtered 3 times with absorbent cotton, the combined filtrate is collected, and lyophilized to obtain a crude lyophilized powder;
  • step S4 the crude lyophilized powder is dissolved in ultrapure water, and sequentially separated by ultrafiltration membranes having molecular weight cutoffs of 10000 u, 5000 u, 3000 u (may be only 5000 u, 3000 u, but increasing 10000 u will increase the subsequent ultrafiltration speed).
  • the peptide having a molecular weight in the range of (5000 u, 3000 u) was collected and lyophilized.
  • step S1 the small fruit oil tea mash is pulverized to 80-100 mesh, soaked in water overnight, and finally a sputum powder suspension is prepared, and the mass concentration of the sputum powder suspension is 130 g/L;
  • Step S2 adding trypsin digestion (addition weight is 2.5 g/100 g by weight of tantalum powder), the enzymatic hydrolysis conditions are: pH value, 7.8; enzymatic hydrolysis temperature, 43 ° C; enzymatic hydrolysis time, 12 hours; end of enzymatic hydrolysis After that, the solution was inactivated at a high temperature of 90 ° C for 10 minutes, cooled to room temperature, and the supernatant was allowed to stand;
  • Step S3 the supernatant is filtered 3 times with absorbent cotton, the combined filtrate is collected, and lyophilized to obtain a crude lyophilized powder;
  • step S4 the crude lyophilized powder is dissolved in ultrapure water, and sequentially separated by ultrafiltration membranes having molecular weight cutoffs of 10000 u, 5000 u, 3000 u (may be only 5000 u, 3000 u, but increasing 10000 u will increase the subsequent ultrafiltration speed).
  • the peptide having a molecular weight in the range of (5000 u, 3000 u) was collected and lyophilized.
  • step S1 the small fruit oil tea mash is pulverized to 80-100 mesh, soaked in water overnight, and finally a sputum powder suspension is prepared, and the mass concentration of the sputum powder suspension is 170 g/L;
  • Step S2 adding trypsin digestion (addition weight is 3.5 g/100 g by weight of tantalum powder), the enzymatic hydrolysis conditions are: pH value, 8.2; enzymatic hydrolysis temperature, 47 ° C; enzymatic hydrolysis time, 10 hours; end of enzymatic hydrolysis After that, the solution was inactivated at a high temperature of 90 ° C for 10 minutes, cooled to room temperature, and the supernatant was allowed to stand;
  • Step S3 the supernatant is filtered 3 times with absorbent cotton, the combined filtrate is collected, and lyophilized to obtain a crude lyophilized powder;
  • step S4 the crude lyophilized powder is dissolved in ultrapure water, and sequentially separated by ultrafiltration membranes having molecular weight cutoffs of 10000 u, 5000 u, 3000 u (may be only 5000 u, 3000 u, but increasing 10000 u will increase the subsequent ultrafiltration speed).
  • the peptide having a molecular weight in the range of (5000 u, 3000 u) was collected and lyophilized.
  • step S1 the small fruit oil tea mash is pulverized to 80-100 mesh, soaked in water overnight, and finally a sputum powder suspension is prepared, and the mass concentration of the sputum powder suspension is 150 g/L;
  • Step S2 adding alkaline protease enzymatic hydrolysis (addition weight is 3.0 g / 100 g by weight of tantalum powder), enzymatic hydrolysis conditions are: pH value, 8.5; enzymatic hydrolysis temperature, 55 ° C; enzymatic hydrolysis time, 10 hours; enzymatic hydrolysis After completion, the alkaline protease was inactivated by treatment at 90 ° C for 10 minutes, cooled to room temperature, and the supernatant was allowed to stand;
  • Step S3 the supernatant is filtered 3 times with absorbent cotton, the combined filtrate is collected, and lyophilized to obtain a crude lyophilized powder;
  • step S4 the crude lyophilized powder is dissolved in ultrapure water, and sequentially separated by ultrafiltration membranes having molecular weight cutoffs of 10000 u, 5000 u, 3000 u (may be only 5000 u, 3000 u, but increasing 10000 u will increase the subsequent ultrafiltration speed).
  • the peptide having a molecular weight in the range of (5000 u, 3000 u) was collected and lyophilized.
  • step S1 the small fruit oil tea mash is pulverized to 80-100 mesh, soaked in water overnight, and finally a sputum powder suspension is prepared, and the mass concentration of the sputum powder suspension is 130 g/L;
  • Step S2 adding alkaline protease enzymatic hydrolysis (addition weight is 2.5 g/100 g by weight of tantalum powder), enzymatic hydrolysis conditions are: pH value, 8.3; enzymatic hydrolysis temperature, 53 ° C; enzymatic hydrolysis time, 11 hours; enzymatic hydrolysis After completion, the alkaline protease was inactivated by treatment at 90 ° C for 10 minutes, cooled to room temperature, and the supernatant was allowed to stand;
  • Step S3 the supernatant is filtered 3 times with absorbent cotton, the combined filtrate is collected, and lyophilized to obtain a crude lyophilized powder;
  • step S4 the crude lyophilized powder is dissolved in ultrapure water, and sequentially separated by ultrafiltration membranes having molecular weight cutoffs of 10000 u, 5000 u, 3000 u (may be only 5000 u, 3000 u, but increasing 10000 u will increase the subsequent ultrafiltration speed).
  • the peptide having a molecular weight in the range of (5000 u, 3000 u) was collected and lyophilized.
  • step S1 the small fruit oil tea mash is pulverized to 80-100 mesh, soaked in water overnight, and finally a sputum powder suspension is prepared, and the mass concentration of the sputum powder suspension is 170 g/L;
  • Step S2 adding alkaline protease enzymatic hydrolysis (addition weight is 3.5 g/100 g by weight of tantalum powder), enzymatic hydrolysis conditions are: pH value, 8.7; enzymatic hydrolysis temperature, 57 ° C; enzymatic hydrolysis time, 9 hours; enzymatic hydrolysis After completion, the alkaline protease was inactivated by treatment at 90 ° C for 10 minutes, cooled to room temperature, and the supernatant was allowed to stand;
  • Step S3 the supernatant is filtered 3 times with absorbent cotton, the combined filtrate is collected, and lyophilized to obtain a crude lyophilized powder;
  • step S4 the crude lyophilized powder is dissolved in ultrapure water, and sequentially separated by ultrafiltration membranes having molecular weight cutoffs of 10000 u, 5000 u, 3000 u (may be only 5000 u, 3000 u, but increasing 10000 u will increase the subsequent ultrafiltration speed).
  • the peptide having a molecular weight in the range of (5000 u, 3000 u) was collected and lyophilized.
  • the DSC curves of the above-mentioned Camellia oleracea polypeptides 1-6 were determined, respectively, and the properties and components of Camellia oleracea polypeptide 1-3 were the same, and the properties and compositions of Camellia sinensis polypeptides 4-6 were the same, while Camellia oleracea polypeptides 1-3 and Camellia oleracea polypeptide 4 The nature and composition of -6 differ greatly.
  • the DSC curve of Camellia oleracea polypeptide 1-6 is shown in Figure 1 (NETZSCH STA 449C synchronous thermal analyzer, sample mass 5mg, measuring temperature range 30-900 ° C, argon flow rate 30mL / min, heating rate 10 ° C / min) .
  • the bull breed is Holstein cattle; the bull semen dilution uses TCM-199.
  • the malondialdehyde (MDA) test kit was purchased from Nanjing Institute of Bioengineering.
  • polypeptides 1-6 prepared in Examples 1-6 were accurately weighed and dissolved in sterile ultrapure water to prepare a stock solution having a concentration of 5 mg/mL.
  • the instruments used should be washed and disinfected beforehand; the glassware is washed and sent to the drying oven for drying, the fake vagina and other instruments are wiped and disinfected with 75% alcohol cotton balls, the fake vagina is installed with a protective cover, and the fake vagina after disinfection Can only be used once.
  • the temperature of the intravaginal water injection is about 40 °C
  • the water injection amount is about 300mL
  • the temperature in the incubator is controlled at 40°C-42°C
  • the inner tube temperature during the semen collection is kept between 38°C and 40°C.
  • the inner tube temperature can be different according to the The cow should be properly adjusted to a maximum of 43 °C.
  • the lubricant is evenly applied to the first 2/3 of the vaginal inner tube, and the lubricant is prepared in a 1:1 ratio with white petrolatum and liquid paraffin oil.
  • the inner tube pressure of the fake vagina (inflated from the fake vaginal piston hole before collecting the sperm) is preferably a triangle with a false vaginal opening.
  • Taiwanese cattle should be strong, gentle, and free of disease, and the vulva and buttocks should be cleaned. When the bulls are collecting sperm, they use incentives, climbs, and repeated creeps to stimulate the bulls. It is also possible to change the location of the collection and replace the Taiwanese cattle to increase the excitement of the bulls.
  • the site should be kept quiet, the floor cleaned and the mat should be laid to prevent the bull from slipping when collecting.
  • the sperm is collected by the right hand.
  • the excavator is holding the fake vagina in the right hand.
  • Standing on the right rear of the bull when the bull jumps, the forelimb climbs onto the Taiwanese cow and then quickly carries the bull's foreskin.
  • the right hand holds the fake vagina and the Taiwanese cow into a 40-degree clip.
  • the angle, the false vaginal opening is obliquely downward, and the left and right hands cooperate to introduce the bull penis into the fake vagina naturally.
  • the bull rushes forward and completes the ejaculation action.
  • the TCM-199 dilution was preheated in a 37 ° C water bath, and the polypeptide stock solution was added to prepare a bovine semen frozen dilution at a concentration of 10 ⁇ g/mL, and then the bovine semen was mixed with the dilution at a volume ratio of 1:4. A diluted semen is obtained.
  • control group was diluted with pure TCM-199 dilution.
  • Semen freezing The diluted semen was firstly pre-cooled into a 0.25 mL thin tube, and then placed in a -5 ° C freezer for 2.5 h. After the end of the balance, the thin tube was placed in a non-rigid foam box, liquid nitrogen was added to the foam box, and the thin tube was fumigated for 30 min at a temperature of about -120 °C. Finally, the straw is placed in liquid nitrogen for cryopreservation.
  • the MDA content in the semen after thawing in the control group and each polypeptide group was determined.
  • the index can reflect the degree of oxidation in the semen freezing process and the antioxidant effect of each peptide.
  • the MDA assay was performed according to the instructions of the malondialdehyde test kit.
  • Malondialdehyde is the end product of lipid oxidation, and MDA production can exacerbate membrane damage and is cytotoxic.
  • MDA Malondialdehyde
  • the MDA content of the Camellia oleracea polypeptide group 1-3 was significantly decreased, and the MDA content of the Camellia oleracea polypeptide group 4-6 was not significantly decreased, indicating that the Camellia oleracea polypeptide 1-3 can significantly inhibit the oxidative damage of bovine semen.
  • Table 1 and Figure 2 The results are shown in Table 1 and Figure 2.
  • the singular Simmental cows with the same monthly age (between 3.5 and 4.0 months) were randomly divided into the control group and the oil tea ⁇ polypeptide 1-6 groups, 20 in each group.
  • the control group was fed with conventional feed, and the Camellia oleracea polypeptide group 1-6 was added with 5 g/kg of Camellia oleracea polypeptide 1-6 in a conventional feed.
  • Each group of cows was fed for 75 consecutive days, and the performance of estrus was observed within 75 days and the breeding and conception were accepted.
  • the hydrolyzed polypeptide of Camellia oleracea L. of the present invention has various activities, and the activity of the hydrolyzed polypeptide is affected by the proteolytic site in the Camellia oleifera: the polypeptide having a molecular weight of (5000 u, 3000 u) produced by trypsin hydrolysis has an improved bovine jelly.
  • the anti-oxidation ability of semen can be used as a cryopreservation agent for bovine semen to reduce oxidative damage, while the polypeptide produced by alkaline protease is not obvious; a polypeptide with a molecular weight of (5000u, 3000u) produced by hydrolysis of alkaline protease can promote The estrus cow is estrus and fertilized, while the polypeptide produced by trypsin is not obvious.
  • the preparation method of the hydrolyzed polypeptide of the present invention is simple, and only simple pulverization, enzymatic hydrolysis, ultrafiltration, lyophilization can be used, and these steps are all It is possible to realize large-scale production in the factory and is easy to implement.
  • the invention realizes the waste recycling of the small fruit oil tea stalk, and can improve the economic added value of the small fruit oil samovar.
  • Example 7 Preparation and average molecular weight determination of Camellia oleracea polysaccharide 1-3
  • step S1 the small fruit oil tea mash is pulverized to 80-100 mesh, and 1 kg plus 15 L water boiling water is refluxed and extracted three times, each time for 2 hours, each extract is filtered, the filtrate is combined, and concentrated under reduced pressure to 150 mL, Sevag method Deproteinization
  • Step S2 adding 4 volumes of absolute ethanol to the concentrated solution after deproteinization, stirring uniformly, standing at 4 ° C for 24 hours, centrifuging at 5 ⁇ 10 3 rpm for 5 minutes, collecting the precipitate;
  • step S3 the precipitate is dissolved in deionized water, and applied to a DEAE-52 column, and successively eluted with a gradient of pure water, 0.3 mol/L sodium chloride solution, and 0.5 mol/L sodium chloride solution.
  • step S4 the pure water eluate was concentrated to 5 mL, and applied to a Sephadex G-100 column (60 cm ⁇ 2.6 cm) at a flow rate of 0.3 mL/min, eluted with deionized water, and collected in steps (each tube). 10min).
  • the content of polysaccharides in each tube (A490) was determined by sulfuric acid-phenol method.
  • the number of tubes collected was plotted on the abscissa and A490 was plotted on the ordinate.
  • the chromatographic elution curve of Sephadex G-100 (shown in Figure 4) was plotted and the main elution was combined. The corresponding eluate of the peak is freeze-dried to obtain the oil tea ⁇ polysaccharide 1;
  • step S5 the 0.3 mol/L sodium chloride eluate was concentrated to 5 mL, and applied to a Sephadex G-100 column (60 cm ⁇ 2.6 cm) at a flow rate of 0.3 mL/min, and eluted with deionized water.
  • Step collection (10 min per tube).
  • the content of polysaccharides in each tube was determined by sulfuric acid-phenol method (A490).
  • the number of tubes collected was plotted on the abscissa and A490 was plotted on the ordinate.
  • the chromatographic elution curve of Sephadex G-100 was plotted (as shown in Figure 5). The corresponding eluate of the peak is freeze-dried to obtain the oil tea ⁇ polysaccharide 2;
  • step S6 the 0.5 mol/L sodium chloride eluate was concentrated to 5 mL, and applied to a Sephadex G-100 column (60 cm ⁇ 2.6 cm) at a flow rate of 0.3 mL/min, and eluted with deionized water. Step collection (10 min per tube). The content of polysaccharides in each tube (A490) was determined by sulfuric acid-phenol method. The number of tubes collected was plotted on the abscissa and A490 was plotted on the ordinate. The chromatographic elution curve of Sephadex G-100 (shown in Figure 6) was plotted and the main elution was combined. The eluate corresponding to the peak was freeze-dried to obtain the Camellia oleifera polysaccharide 3.
  • a polysaccharide control of a known molecular weight is formulated into a 1.0 mg/mL standard solution using a mobile phase, and the literature analysis of a hydrophilic isolated from the aqueous extract of the edible mushroom, Pleurotus sajor-caju, cultivar Black Japan. Carbohyd Res, 2008), determined by molecular exclusion HPLC method. Taking the logarithmic value of the molecular weight of the polysaccharide reference as the ordinate and the retention time as the abscissa, the standard curve equation of the molecular weight of the polysaccharide was obtained.
  • Camellia sinensis polysaccharides 1-3 are single symmetrical peaks and are single components.
  • Example 8 Camellia oleracea polysaccharide 1-3 Antagonizes foot-and-mouth disease vaccine against bull semen damage
  • the bull breed is Holstein cattle; the bull semen dilution uses TCM-199.
  • the foot-and-mouth disease vaccine is a foot-and-mouth disease virus type O Asian type I bivalent inactivated vaccine, and the inoculation dose is 2.5 mL/head.
  • the oil tea ⁇ polysaccharide 1-3 was accurately weighed and dissolved in physiological saline to prepare an injection solution having a concentration of 5 mg/mL.
  • the instruments used should be washed and disinfected beforehand; the glassware is washed and sent to the drying oven for drying, the fake vagina and other instruments are wiped and disinfected with 75% alcohol cotton balls, the fake vagina is installed with a protective cover, and the fake vagina after disinfection Can only be used once.
  • the temperature of the intravaginal water injection is about 40 °C
  • the water injection amount is about 300mL
  • the temperature in the incubator is controlled at 40°C-42°C
  • the inner tube temperature during the semen collection is kept between 38°C and 40°C.
  • the inner tube temperature can be different according to the The cow should be properly adjusted to a maximum of 43 °C.
  • the lubricant is evenly applied to the first 2/3 of the vaginal inner tube, and the lubricant is prepared in a 1:1 ratio with white petrolatum and liquid paraffin oil.
  • the inner tube pressure of the fake vagina (inflated from the fake vaginal piston hole before collecting the sperm) is preferably a triangle with a false vaginal opening.
  • Taiwanese cattle should be strong, gentle, and free of disease, and the vulva and buttocks should be cleaned. When the bulls are collecting sperm, they use incentives, climbs, and repeated creeps to stimulate the bulls. It is also possible to change the location of the collection and replace the Taiwanese cattle to increase the excitement of the bulls.
  • the site should be kept quiet, the floor cleaned and the mat should be laid to prevent the bull from slipping when collecting.
  • the sperm is collected by the right hand.
  • the excavator is holding the fake vagina in the right hand.
  • Standing on the right rear of the bull when the bull jumps, the forelimb climbs onto the Taiwanese cow and then quickly carries the bull's foreskin.
  • the right hand holds the fake vagina and the Taiwanese cow into a 40-degree clip.
  • the angle, the false vaginal opening is obliquely downward, and the left and right hands cooperate to introduce the bull penis into the fake vagina naturally.
  • the bull rushes forward and completes the ejaculation action.
  • the group of Camellia oleracea polysaccharides, the group of Camellia oleracea polysaccharides, and the group of Camellia oleracea polysaccharides were administered intramuscularly at a dose of 5 mg/100 kg every three days. -3.
  • each group of Holstein cattle was injected with foot-and-mouth disease virus O-type Asian type I bivalent inactivated vaccine, the dose was 2.5 mL/head, and the observation period was 30 days. All Holstein cattle were successfully immunized after observation and testing. Ten days after the end of the observation period, each group of Holstein cattle was re-seeded according to the above method, and the semen activity was measured.
  • the oleophylla polysaccharide provided by the invention can effectively antagonize the damage caused by the foot-and-mouth disease vaccine to the bull semen, and can be used to reduce the loss of the vaccination of the foot-and-mouth disease vaccine on the farm;
  • the preparation method of the succulent polysaccharide of the present invention has few steps and separation The purification effect is good and all are single components.
  • Rhizopus oryzae AS3.866 purchased from the Guangdong Provincial Collection of Microorganisms.
  • Incline medium PDA medium.
  • the access oryzae slant medium cultured at 28 °C 72h, until inclined surface covered with spores, spores were added to 10mL of sterile water and washed under the broken with glass beads, made of a single concentration of 10 7 spores / mL, Spore suspension.
  • step S1 the small fruit oil tea smash is pulverized into oil tea glutinous rice powder, and the particle size is 40-60 mesh;
  • Step S2 uniformly spraying the oil tea powder into a hydrochloric acid aqueous solution having a pH of 4.5, and the water content of the oil tea powder is 65%;
  • Step S3 inoculating a suspension of Rhizopus oryzae spores having a concentration of 10 7 single spores/mL, the inoculum amount is 20% (mL/g);
  • step S4 the rot is prepared at 30 ° C for 24-72 h, and the saponin content is determined by taking an appropriate amount of bismuth powder at 24, 48, and 72 h, respectively.
  • Example 10 Hydrochloric acid rot + Rhizopus oryzae transformation
  • Rhizopus oryzae AS3.866 purchased from the Guangdong Provincial Collection of Microorganisms.
  • Incline medium PDA medium.
  • the access oryzae slant medium cultured at 28 °C 72h, until inclined surface covered with spores, spores were added to 10mL of sterile water and washed under the broken with glass beads, made of a single concentration of 10 7 spores / mL, Spore suspension.
  • step S1 the small fruit oil tea smash is pulverized into oil tea glutinous rice powder, and the particle size is 40-60 mesh;
  • Step S2 uniformly spraying the oil tea powder with a hydrochloric acid aqueous solution having a pH of 4, and the water content of the oil tea powder is 60%;
  • Step S3 inoculating a suspension of Rhizopus oryzae spores having a concentration of 10 7 single spores/mL, the inoculum amount is 15% (mL/g);
  • step S4 the mixture is rotted at 32 ° C for 24-72 h, and the appropriate amount of glutinous rice powder is taken at 24, 48, 72 h to determine the saponin content.
  • Example 11 Hydrochloric acid rot + Rhizopus oryzae transformation
  • Rhizopus oryzae AS3.866 purchased from the Guangdong Provincial Collection of Microorganisms.
  • Incline medium PDA medium.
  • the access oryzae slant medium cultured at 28 °C 72h, until inclined surface covered with spores, spores were added to 10mL of sterile water and washed under the broken with glass beads, made of a single concentration of 10 7 spores / mL, Spore suspension.
  • step S1 the small fruit oil tea smash is pulverized into oil tea glutinous rice powder, and the particle size is 40-60 mesh;
  • Step S2 uniformly spraying the oil tea powder into a hydrochloric acid aqueous solution having a pH of 5, and the water content of the oil tea powder is 70%;
  • Step S3 inoculating a suspension of Rhizopus oryzae spores having a concentration of 10 7 single spores/mL, the inoculum amount is 25% (mL/g);
  • step S4 the rot is prepared at 28 ° C for 24-72 h, and the saponin content is determined by taking an appropriate amount of bismuth powder at 24, 48, and 72 h, respectively.
  • Example 12 Formic acid rot + Rhizopus oryzae transformation
  • Rhizopus oryzae AS3.866 purchased from the Guangdong Provincial Collection of Microorganisms.
  • Incline medium PDA medium.
  • the access oryzae slant medium cultured at 28 °C 72h, until inclined surface covered with spores, spores were added to 10mL of sterile water and washed under the broken with glass beads, made of a single concentration of 10 7 spores / mL, Spore suspension.
  • step S1 the small fruit oil tea smash is pulverized into oil tea glutinous rice powder, and the particle size is 40-60 mesh;
  • Step S2 uniformly spraying the oil tea powder into a formic acid aqueous solution having a pH of 4.5, and the water content of the oil tea powder is 65%;
  • Step S3 inoculating a suspension of Rhizopus oryzae spores having a concentration of 10 7 single spores/mL, the inoculum amount is 20% (mL/g);
  • step S4 the rot is prepared at 30 ° C for 24-72 h, and the saponin content is determined by taking an appropriate amount of bismuth powder at 24, 48, and 72 h, respectively.
  • Example 13 Formic acid rot + Rhizopus oryzae transformation
  • Rhizopus oryzae AS3.866 purchased from the Guangdong Provincial Collection of Microorganisms.
  • Incline medium PDA medium.
  • the access oryzae slant medium cultured at 28 °C 72h, until inclined surface covered with spores, spores were added to 10mL of sterile water and washed under the broken with glass beads, made of a single concentration of 10 7 spores / mL, Spore suspension.
  • step S1 the small fruit oil tea smash is pulverized into oil tea glutinous rice powder, and the particle size is 40-60 mesh;
  • step S2 the oil tea powder is uniformly sprayed with a formic acid aqueous solution having a pH of 4, and the water content of the oil tea powder is 60%;
  • Step S3 inoculating a suspension of Rhizopus oryzae spores having a concentration of 10 7 single spores/mL, the inoculum amount is 15% (mL/g);
  • step S4 the mixture is rotted at 32 ° C for 24-72 h, and the appropriate amount of glutinous rice powder is taken at 24, 48, 72 h to determine the saponin content.
  • Example 14 Formic acid rot + Rhizopus oryzae transformation
  • Rhizopus oryzae AS3.866 purchased from the Guangdong Provincial Collection of Microorganisms.
  • Incline medium PDA medium.
  • the access oryzae slant medium cultured at 28 °C 72h, until inclined surface covered with spores, spores were added to 10mL of sterile water and washed under the broken with glass beads, made of a single concentration of 10 7 spores / mL, Spore suspension.
  • step S1 the small fruit oil tea smash is pulverized into oil tea glutinous rice powder, and the particle size is 40-60 mesh;
  • Step S2 uniformly spraying the oil tea powder with a formic acid aqueous solution having a pH of 5, and the water content of the oil tea powder is 70%;
  • Step S3 inoculating a suspension of Rhizopus oryzae spores having a concentration of 10 7 single spores/mL, the inoculum amount is 25% (mL/g);
  • step S4 the rot is prepared at 28 ° C for 24-72 h, and the saponin content is determined by taking an appropriate amount of bismuth powder at 24, 48, and 72 h, respectively.
  • Rhizopus oryzae AS3.866 purchased from the Guangdong Provincial Collection of Microorganisms.
  • Incline medium PDA medium.
  • the access oryzae slant medium cultured at 28 °C 72h, until inclined surface covered with spores, spores were added to 10mL of sterile water and washed under the broken with glass beads, made of a single concentration of 10 7 spores / mL, Spore suspension.
  • Step S2 uniformly spraying the oil tea powder into the acetic acid aqueous solution having a pH of 4.5, and the water content of the oil tea powder is 65%;
  • Rhizopus oryzae AS3.866 purchased from the Guangdong Provincial Collection of Microorganisms.
  • Incline medium PDA medium.
  • the access oryzae slant medium cultured at 28 °C 72h, until inclined surface covered with spores, spores were added to 10mL of sterile water and washed under the broken with glass beads, made of a single concentration of 10 7 spores / mL, Spore suspension.
  • step S1 the small fruit oil tea smash is pulverized into oil tea glutinous rice powder, and the particle size is 40-60 mesh;
  • Step S2 uniformly spraying the oil tea powder into the acetic acid aqueous solution having a pH of 4, and the water content of the oil tea powder is 60%;
  • Step S3 inoculating a suspension of Rhizopus oryzae spores having a concentration of 10 7 single spores/mL, the inoculum amount is 15% (mL/g);
  • step S4 the mixture is rotted at 32 ° C for 24-72 h, and the appropriate amount of glutinous rice powder is taken at 24, 48, 72 h to determine the saponin content.
  • Rhizopus oryzae AS3.866 purchased from the Guangdong Provincial Collection of Microorganisms.
  • Incline medium PDA medium.
  • the access oryzae slant medium cultured at 28 °C 72h, until inclined surface covered with spores, spores were added to 10mL of sterile water and washed under the broken with glass beads, made of a single concentration of 10 7 spores / mL, Spore suspension.
  • step S1 the small fruit oil tea smash is pulverized into oil tea glutinous rice powder, and the particle size is 40-60 mesh;
  • Step S2 uniformly spraying the oil tea powder into the acetic acid aqueous solution having a pH of 5, and the water content of the oil tea powder is 70%;
  • Step S3 inoculating a suspension of Rhizopus oryzae spores having a concentration of 10 7 single spores/mL, the inoculum amount is 25% (mL/g);
  • step S4 the rot is prepared at 28 ° C for 24-72 h, and the saponin content is determined by taking an appropriate amount of bismuth powder at 24, 48, and 72 h, respectively.
  • Total saponin removal rate (%) (absorption value per unit mass of oil tea ⁇ powder acidification - absorption value per unit mass of oil tea ⁇ powder pile rot) / unit mass oil tea ⁇ powder acidification before absorption value ⁇ 100%.
  • the detoxification method provided by the invention can effectively remove the saponin from the small fruit oil tea stalk, thereby removing the bitterness and improving the mouthfeel, and further can be used as a feed additive; in particular, when formic acid is used as the acid rotant, The removal efficiency of the saponin of Rhizopus oryzae can be significantly improved; and the detoxification method provided by the invention is simple and feasible, low in cost, and easy to industrialize and popularize.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Water Supply & Treatment (AREA)
  • Environmental Sciences (AREA)
  • Dentistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

一种油茶粕多肽及其制备方法、应用。油茶粕多肽是以小果油茶粕为原料，经胰蛋白酶水解后，收集分子量在(5000u,3000u]的多肽并冷冻干燥，具有提高牛冻存精液抗氧化能力的作用。

Description

一种油茶粕多肽、多糖及其制备方法、应用和油茶粕脱毒方法 技术领域
本发明涉及一种油茶粕多肽、多糖及其制备方法、应用和油茶粕脱毒方法。
背景技术
小果油茶(Camellia meiocarpa Hu.)是我国山茶属中栽培面积和年产量仅次于普通油茶(Camellia oleifera Abel.)的主栽物种，主要分布于我国淮河、长江以南的福建、江西、广西、湖南、贵州等地。小果油茶又名江西子、小茶、鸡心子、小叶油茶、羊屎子等。果小、叶小、芽小，芽、苞片没有毛是其显著特征。
小果油茶粕是小果油茶提取油脂后得到的副产品。虽然，目前已有茶场将这些油茶粕脱毒后用作动物饲料，但是，由于工艺不成熟，绝大部分油茶粕仍然是当废料扔掉，或者堆腐后用作茶场的肥料，利用度低，附加值低。为了提高小谷油茶粕的利用度，提高其经济附加值，申请人对茶粕进行了深度研究与开发。
发明内容
本发明的目的在于克服现有技术的不足，提供一种油茶粕多肽、多糖及其制备方法、应用和油茶粕脱毒方法。
本发明的上述目的是通过下面的技术方案得以实现的：
胰蛋白酶水解制备牛精液冷冻保存添加剂：
一种油茶粕多肽，以小果油茶粕为原料，经胰蛋白酶水解后，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥即得。
优选地，所述小果油茶粕指小果油茶的果实经过压榨处理或浸出法处理去除油脂后得到的固体残渣。
上述油茶粕多肽的制备方法，包括如下步骤：
步骤S1，将小果油茶粕粉碎至80-100目，加水浸泡，最后制成粕粉悬浮液；
步骤S2，加入胰蛋白酶酶解，酶解条件为：pH值，7.8-8.2；酶解温度，43-47℃；酶解时间，10-12小时；酶解结束后，加热使胰蛋白酶灭活，冷却至常温，静置取上清；
步骤S3，将上清液用脱脂棉过滤，收集滤液，冻干得粗品冻干粉；
步骤S4，粗品冻干粉用超纯水溶解，用不同截留分子量的超滤膜进行分离，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥即得。
优选地，所述不同截留分子量的超滤膜包括截留分子量分别为5000u、3000u的超滤膜。
优选地，还包括截留分子量10000u的超滤膜。
优选地，加热使胰蛋白酶灭活的方法为：90℃高温处理10分钟。
优选地，粕粉悬浮液优选130-170g/L。
优选地，所述胰蛋白酶的添加重量以粕粉重量计为2.5-3.5g/100g。
优选地，所述胰蛋白酶的酶解条件优选pH值8.0、温度45℃、时间11小时。
上述油茶粕多肽在牛精液冷冻保存方面的应用。
碱性蛋白酶水解制备促进乏情母牛发情和受胎的多肽：
一种油茶粕多肽，以小果油茶粕为原料，经碱性蛋白酶水解后，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥即得。
优选地，所述小果油茶粕指小果油茶的果实经过压榨处理或浸出法处理去除油脂后得到的固体残渣。
上述油茶粕多肽的制备方法，包括如下步骤：
步骤S1，将小果油茶粕粉碎至80-100目，加水浸泡，最后制成粕粉悬浮液；
步骤S2，加入碱性蛋白酶酶解，酶解条件为：pH值，8.3-8.7；酶解温度，53-57℃；酶解时间，9-11小时；酶解结束后，加热使碱性蛋白酶灭活，冷却至常温，静置取上清；
步骤S3，将上清液用脱脂棉过滤，收集滤液，冻干得粗品冻干粉；
步骤S4，粗品冻干粉用超纯水溶解，用不同截留分子量的超滤膜进行分离，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥即得。
优选地，所述不同截留分子量的超滤膜包括截留分子量分别为5000u、3000u的超滤膜。
优选地，还包括截留分子量10000u的超滤膜。
优选地，加热使碱性蛋白酶灭活的方法为：90℃高温处理10分钟。
优选地，粕粉悬浮液优选130-170g/L。
优选地，所述碱性蛋白酶的添加重量以粕粉重量计为2.5-3.5g/100g。
优选地，所述碱性蛋白酶的酶解条件优选pH值8.5、温度55℃、时间10小时。
上述油茶粕多肽在制备促进乏情母牛发情和受胎的饲料或药物方面的应用。
油茶粕多糖及应用：
一种油茶粕多糖，从小果油茶粕中分离得到，平均分子量为75-95kDa。
优选地，以小果油茶粕为原料，经水提醇沉、DEAE-52纤维素柱层析、Sephadex G-l00柱层析纯化得到。
优选地，平均分子量为75.3、85.6或94.7kDa。
优选地，所述小果油茶粕指小果油茶的果实经过压榨处理或浸出法处理去除油脂后得到的固体残渣。
上述油茶粕多糖的制备方法，包括如下步骤：
步骤S1，将小果油茶粕粉碎至80-100目，沸水回流提取，过滤，合并滤液，浓缩；
步骤S2，向浓缩液中添加加入4-5倍体积的无水乙醇，低温沉淀，离心收集沉淀；
步骤S3，将沉淀溶于去离子水中，加样于DEAE-52层析柱，依次用纯水和不同浓度氯化钠溶液梯度洗脱，分别收集纯水洗脱液、0.3mol/L氯化钠洗脱液和0.5mol/L氯化钠洗脱液；
步骤S4，分别将纯水洗脱液、0.3mol/L氯化钠洗脱液和0.5mol/L氯化钠洗脱液浓缩后的浓缩液上样于Sephadex G-l00层析柱，用去离子水洗脱，分别收集洗脱峰、冷冻干燥。
优选地，步骤S2沉淀条件优选4℃静置24小时；离心条件优选5×10 3rpm离心5分钟。
优选地，步骤S4用硫酸-苯酚法检测各管多糖的含量，以收集的管数为横坐标、紫外吸光度值为纵坐标，绘制Sephadex G-100色谱洗脱曲线，各浓缩液经Sephadex G-100层析柱分离后各得1个主要洗脱峰，收集洗脱峰、冷冻干燥。
优选地，紫外检测波长为490nm。
优选地，步骤S2醇沉前先用Sevag法脱蛋白。
上述油茶粕多糖拮抗口蹄疫疫苗对公牛精液损伤的用途。
油茶粕脱毒方法：
一种小果油茶粕的脱毒方法，所述小果油茶粕指小果油茶的果实经过压榨处理或浸出法处理去除油脂后得到的固体残渣，所述脱毒指脱除皂苷，将小果油茶粕粉碎成油茶粕粉，均匀喷洒酸性水溶液后接种米根霉堆腐。
优选地，所述酸为盐酸、甲酸或乙酸。
优选地，酸性水溶液pH值优选4-5。
优选地，以喷洒酸性水溶液后的油茶粕粉湿重计，米根霉的接种量为15％-25％(mL/g)，米根霉菌液浓度为10 7个单孢子/mL。
优选地，堆腐温度为28-32℃。
优选地，堆腐时间为24-72h。
优选地，酸性水溶液优选pH值为4-5的甲酸水溶液，喷洒至油茶粕粉的含水量为60-70％。
优选地，以喷洒酸性水溶液后的油茶粕粉湿重计，米根霉的接种量为20％(mL/g)，米根霉菌液浓度为10 7个单孢子/mL，30℃堆腐24h。
一种由上述脱毒方法制备的小果油茶粕。
上述小果油茶粕用作动物饲料的用途。
本发明的优点：
1、本发明发现，油茶粕的水解多肽具有多种活性，并且水解多肽的活性受油茶粕中蛋白 水解位点影响：经胰蛋白酶水解产生的分子量为(5000u,3000u]的多肽具有提高牛冻存精液抗氧化能力的作用，可以作为牛精液冻存保护剂降低氧化损伤，而经碱性蛋白酶产生的多肽则不明显；经碱性蛋白酶水解产生的分子量为(5000u,3000u]的多肽可以促进乏情母牛发情和受胎，而经胰蛋白酶产生的多肽则不明显；
2、本发明水解多肽的制备方法简单，仅使用简单的粉碎、酶解、超滤、冻干即可，这些步骤都是可以在工厂中实现大生产化，易于实施；
3、本发明实现了小果油茶粕的废物再利用，可以提高小果油茶粕的经济附加值；
4、本发明提供的油茶粕多糖可以有效拮抗口蹄疫疫苗对公牛精液造成的损伤，从而可以用于降低接种口蹄疫疫苗对农场的损失；
5、本发明提供的油茶粕多糖的制备方法步骤少，分离纯化效果好，均为单一组分；
6、本发明提供的脱毒方法可以有效脱除小果油茶粕中的皂苷，从而脱除其苦味和辛辣味，改善口感，进而可以用作饲料添加剂；尤其是，使用甲酸作为酸腐剂时，可以显著提高米根霉对小果油茶粕中皂苷的脱除效率；
7、本发明提供的脱毒方法简易可行，成本低，易于工业化生产和推广。
附图说明
图1为油茶粕多肽1-6的DSC曲线；
图2为油茶粕多肽对精液冻存后氧化产物MDA含量的影响；
图3为油茶粕多肽对乏情母牛发情率和受胎率的影响；
图4为DEAE-52层析柱纯水洗脱液经Sephadex G-100柱层析的洗脱曲线；
图5为DEAE-52层析柱0.3mol/L氯化钠洗脱液经Sephadex G-100柱层析的洗脱曲线；
图6为DEAE-52层析柱0.5mol/L氯化钠洗脱液经Sephadex G-100柱层析的洗脱曲线；
图7为各组荷斯坦牛接种口蹄疫疫苗前后精液活率比较；
图8为实施例9-17堆腐24h后油茶粕粉中总皂苷脱除率比较。
具体实施方式
下面结合附图和实施例具体介绍本发明实质性内容，但并不以此限定本发明的保护范围。
下述实施例中，以1982-1983年春定植在福建省闽侯桐口国有林场油茶种质资源收集圃的小果油茶(Camellia meiocarpa Hu.)为原材料，经压榨工艺去除油脂得小果油茶粕，备用。
实施例1：胰蛋白酶制备油茶粕多肽1
包括如下步骤：
步骤S1，将小果油茶粕粉碎至80-100目，加水浸泡过夜，最后制成粕粉悬浮液，粕粉悬浮液的质量浓度为150g/L；
步骤S2，加入胰蛋白酶酶解(添加重量以粕粉重量计为3.0g/100g)，酶解条件为：pH值，8.0；酶解温度，45℃；酶解时间，11小时；酶解结束后，90℃高温处理10分钟使胰蛋白酶灭活，冷却至常温，静置取上清；
步骤S3，将上清液用脱脂棉过滤3次，收集合并滤液，冻干得粗品冻干粉；
步骤S4，粗品冻干粉用超纯水溶解，依次用截留分子量分别为10000u、5000u、3000u(也可以仅使用5000u、3000u，但增加10000u会利于提高后续超滤速度)的超滤膜进行分离，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥。
实施例2：胰蛋白酶制备油茶粕多肽2
包括如下步骤：
步骤S1，将小果油茶粕粉碎至80-100目，加水浸泡过夜，最后制成粕粉悬浮液，粕粉悬浮液的质量浓度为130g/L；
步骤S2，加入胰蛋白酶酶解(添加重量以粕粉重量计为2.5g/100g)，酶解条件为：pH值，7.8；酶解温度，43℃；酶解时间，12小时；酶解结束后，90℃高温处理10分钟使胰蛋白酶灭活，冷却至常温，静置取上清；
步骤S3，将上清液用脱脂棉过滤3次，收集合并滤液，冻干得粗品冻干粉；
步骤S4，粗品冻干粉用超纯水溶解，依次用截留分子量分别为10000u、5000u、3000u(也可以仅使用5000u、3000u，但增加10000u会利于提高后续超滤速度)的超滤膜进行分离，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥。
实施例3：胰蛋白酶制备油茶粕多肽3
包括如下步骤：
步骤S1，将小果油茶粕粉碎至80-100目，加水浸泡过夜，最后制成粕粉悬浮液，粕粉悬浮液的质量浓度为170g/L；
步骤S2，加入胰蛋白酶酶解(添加重量以粕粉重量计为3.5g/100g)，酶解条件为：pH值，8.2；酶解温度，47℃；酶解时间，10小时；酶解结束后，90℃高温处理10分钟使胰蛋白酶灭活，冷却至常温，静置取上清；
步骤S3，将上清液用脱脂棉过滤3次，收集合并滤液，冻干得粗品冻干粉；
步骤S4，粗品冻干粉用超纯水溶解，依次用截留分子量分别为10000u、5000u、3000u(也可以仅使用5000u、3000u，但增加10000u会利于提高后续超滤速度)的超滤膜进行分离，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥。
实施例4：碱性蛋白酶制备油茶粕多肽4
包括如下步骤：
步骤S1，将小果油茶粕粉碎至80-100目，加水浸泡过夜，最后制成粕粉悬浮液，粕粉悬浮液的质量浓度为150g/L；
步骤S2，加入碱性蛋白酶酶解(添加重量以粕粉重量计为3.0g/100g)，酶解条件为：pH值，8.5；酶解温度，55℃；酶解时间，10小时；酶解结束后，90℃高温处理10分钟使碱性蛋白酶灭活，冷却至常温，静置取上清；
步骤S3，将上清液用脱脂棉过滤3次，收集合并滤液，冻干得粗品冻干粉；
步骤S4，粗品冻干粉用超纯水溶解，依次用截留分子量分别为10000u、5000u、3000u(也可以仅使用5000u、3000u，但增加10000u会利于提高后续超滤速度)的超滤膜进行分离，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥。
实施例5：碱性蛋白酶制备油茶粕多肽5
包括如下步骤：
步骤S1，将小果油茶粕粉碎至80-100目，加水浸泡过夜，最后制成粕粉悬浮液，粕粉悬浮液的质量浓度为130g/L；
步骤S2，加入碱性蛋白酶酶解(添加重量以粕粉重量计为2.5g/100g)，酶解条件为：pH值，8.3；酶解温度，53℃；酶解时间，11小时；酶解结束后，90℃高温处理10分钟使碱性蛋白酶灭活，冷却至常温，静置取上清；
步骤S3，将上清液用脱脂棉过滤3次，收集合并滤液，冻干得粗品冻干粉；
步骤S4，粗品冻干粉用超纯水溶解，依次用截留分子量分别为10000u、5000u、3000u(也可以仅使用5000u、3000u，但增加10000u会利于提高后续超滤速度)的超滤膜进行分离，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥。
实施例6：碱性蛋白酶制备油茶粕多肽6
包括如下步骤：
步骤S1，将小果油茶粕粉碎至80-100目，加水浸泡过夜，最后制成粕粉悬浮液，粕粉悬浮液的质量浓度为170g/L；
步骤S2，加入碱性蛋白酶酶解(添加重量以粕粉重量计为3.5g/100g)，酶解条件为：pH值，8.7；酶解温度，57℃；酶解时间，9小时；酶解结束后，90℃高温处理10分钟使碱性蛋白酶灭活，冷却至常温，静置取上清；
步骤S3，将上清液用脱脂棉过滤3次，收集合并滤液，冻干得粗品冻干粉；
步骤S4，粗品冻干粉用超纯水溶解，依次用截留分子量分别为10000u、5000u、3000u(也可以仅使用5000u、3000u，但增加10000u会利于提高后续超滤速度)的超滤膜进行分离，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥。
分别测定上述油茶粕多肽1-6的DSC曲线，可见油茶粕多肽1-3的性质和成分一样，油茶粕多肽4-6的性质和成分一样，而油茶粕多肽1-3与油茶粕多肽4-6的性质和成分区别较大。油茶粕多肽1-6的DSC曲线如图1所示(NETZSCH STA 449C型同步热分析仪，样品质量5mg，测定温度范围30～900℃，氩气流速30mL/min，升温速度10℃/min)。
效果实施例1：油茶粕多肽对牛精液冷冻保存的抗氧化作用
一、实验材料
公牛品种为荷斯坦牛；公牛精液稀释液采用TCM-199。
丙二醛(MDA)测试盒购于南京建成生物工程研究所。
分别精确称取实施例1-6制备的多肽1-6用无菌超纯水溶解，制成浓度5mg/mL的储备液。
二、实验方法
1、精液的采集和检查(采集和检查方法参考文献：注射口蹄疫疫苗对种公牛精液品质的影响研究，中国牛业科学，2014)
所用的器械事先要进行冲洗，消毒；玻璃器皿洗净后送入干燥箱干燥，假阴道和其他器械等用75％酒精棉球擦拭消毒，假阴道安装好套上保护套，消毒后的假阴道只能使用一次。假阴道内注水温度在40℃左右，注水量约在300mL，恒温箱内温度控制在40℃-42℃，采精时的内胎温度保持在38℃-40℃之间，内胎温度可根据不同的牛做适当的调整，最高不可超过43℃。润滑剂均匀涂抹在假阴道内胎的前2/3，润滑剂用白凡士林与液态石蜡油按1：1比例调制。假阴道的内胎压力(采精前从假阴道活塞孔打气)以假阴道口呈三角形为宜。台牛要健壮、性情温顺、无疫病，外阴、臀部清洗干净。公牛采精时采取引诱、被爬跨、反复空爬等方法刺激公牛兴奋。还可更换采精地点，更换台牛等方法增加种公牛的兴奋度。采精场地应保持安静，地面清洁并铺设防滑垫，以免公牛采精时滑倒。当公牛性欲旺盛时采精，采精员右手持假阴道，站在公牛右后方，当公牛起跳，前肢爬上台牛后迅速向前托着公牛包皮，右手持假阴道与台牛成40度夹角，假阴道口斜向下方，左右手配合将公牛阴茎自然地引入假阴道内，公牛向前一冲即完成射精动作，待公牛前肢落地，缓慢地把假阴道脱出。打开活塞放气，小心取下集精管，迅速转移至精液检测室进行检测。选取新鲜精液(应呈乳白稍带黄色或奶油色不透明液体)，取一滴原精液加盖玻片后在400倍的恒温(37℃)可视显微镜下观察呈直线运动精子的百分数，活率不低于80％的精液的精液用于精液冷冻。
按照上述方法，随机采集10头健康荷斯坦牛的精液。
2、精液稀释
将TCM-199稀释液放入37℃水浴锅中预热，加入多肽储备液混合制成浓度为10μg/mL的牛精液冷冻稀释液，然后将牛精液与该稀释液按照体积比1：4混合得到稀释精液。
对照组使用纯TCM-199稀释液对牛精液进行稀释。
3、精液冷冻与解冻
(1)精液冷冻。先将稀释精液装入0.25mL的细管中预冷，然后放入-5℃的冷柜中平衡2.5h。平衡结束后，将细管放入不严密的泡沫箱中，向泡沫箱中添加液氮，熏蒸细管30min，温度-120℃左右。最后将细管投入液氮中冷冻保存。
(2)解冻。将保存35d后的冻精细管取出，立即放入37℃水浴锅中解冻20s。
4、抗氧化能力测定
分别测定对照组和各多肽组解冻后精液中MDA含量，该指标可以体现各组精液冷冻过程中的氧化程度以及各多肽的抗氧化效果。MDA测定按照丙二醛测试盒说明书进行。
5、统计学方法
采用SPSS 19.0软件统计分析，数据以均值±偏差表示，组间比较采用单因素方差分析。
三、实验结果
丙二醛(MDA)是脂质氧化的终产物，MDA的产生会加剧膜的损伤并具有细胞毒性。与对照组相比，油茶粕多肽1-3组MDA含量显著降低，油茶粕多肽4-6组MDA含量降低不明显，表明油茶粕多肽1-3可以显著抑制冻存对牛精液的氧化损伤。结果如表1和图2所示。
表1 各组精液冻存后MDA含量比较(n＝10)
Figure PCTCN2018082479-appb-000001
效果实施例2：油茶粕多肽对乏情母牛发情、受胎作用影响
一、实验材料
母牛品种为西门塔尔牛。判断母牛乏情的标准：无生殖系统炎症及畸形，但产后3个月后仍然不出现发情、影响配种受胎的母牛为乏情母牛。
二、实验方法
1、实验分组和饲养
将月龄基本一致(3.5-4.0月之间)的乏情西门塔尔母牛随机分为对照组、油茶粕多肽1-6组，每组20头。对照组给予常规饲料喂养，油茶粕多肽1-6组在常规饲料中分别添加5g/kg的油茶粕多肽1-6。各组母牛连续喂养75天，观察75天内表现发情并接受配种及受胎情况。
2、统计学方法
采用SPSS 19.0软件统计分析，数据以均值±偏差表示，组间比较采用单因素方差分析。
三、实验结果
1、油茶粕多肽对乏情母牛发情率的影响
与对照组相比，油茶粕多肽4-6组乏情母牛发情率显著提高，而油茶粕多肽1-3组不明显，表明油茶粕多肽4-6具有促进乏情母牛发情的作用。具体结果见表2和图3。
2、油茶粕多肽对乏情母牛受胎率的影响
与对照组相比，油茶粕多肽4-6组乏情母牛受胎率显著提高，而油茶粕多肽1-3组不明显，表明油茶粕多肽4-6具有促进乏情母牛受胎的作用。具体结果见表2和图3。
表2 油茶粕多肽对乏情母牛发情率和受胎率的影响
Figure PCTCN2018082479-appb-000002
综上可见，本发明油茶粕的水解多肽具有多种活性，并且水解多肽的活性受油茶粕中蛋白水解位点影响：经胰蛋白酶水解产生的分子量为(5000u,3000u]的多肽具有提高牛冻存精液抗氧化能力的作用，可以作为牛精液冻存保护剂降低氧化损伤，而经碱性蛋白酶产生的多肽则不明显；经碱性蛋白酶水解产生的分子量为(5000u,3000u]的多肽可以促进乏情母牛发情和受胎，而经胰蛋白酶产生的多肽则不明显。而且，本发明水解多肽的制备方法简单，仅使用简单的粉碎、酶解、超滤、冻干即可，这些步骤都是可以在工厂中实现大生产化，易于实施。本发明实现了小果油茶粕的废物再利用，可以提高小果油茶粕的经济附加值。
实施例7：油茶粕多糖1-3的制备和平均分子量测定
包括如下步骤：
步骤S1，将小果油茶粕粉碎至80-100目，取1kg加15L水沸水回流提取3次，每次提取2小时，将每次提取液过滤，合并滤液，减压浓缩至150mL，Sevag法脱蛋白；
步骤S2，向脱蛋白后的浓缩液中添加加入4倍体积的无水乙醇，搅拌均匀后置于4℃低温静置24小时，5×10 3rpm离心5分钟，收集沉淀；
步骤S3，将沉淀溶于去离子水中，加样于DEAE-52层析柱，依次用纯水、0.3mol/L氯化钠溶液、0.5mol/L氯化钠溶液梯度洗脱各洗脱15个柱体积，流速1.0mL/min，分别收集纯 水洗脱液、0.3mol/L氯化钠洗脱液和0.5mol/L氯化钠洗脱液；
步骤S4，将纯水洗脱液浓缩至5mL，上样于Sephadex G-100层析柱(60cm×2.6cm)中，流速0.3mL/min，用去离子水洗脱，分步收集(每管10min)。用硫酸-苯酚法检测各管多糖的含量(A490)，以收集的管数为横坐标、A490为纵坐标，绘制Sephadex G-100色谱洗脱曲线(如图4所示)，合并主要洗脱峰对应的洗脱液，冷冻干燥得油茶粕多糖1；
步骤S5，将0.3mol/L氯化钠洗脱液浓缩至5mL，上样于Sephadex G-100层析柱(60cm×2.6cm)中，流速0.3mL/min，用去离子水洗脱，分步收集(每管10min)。用硫酸-苯酚法检测各管多糖的含量(A490)，以收集的管数为横坐标、A490为纵坐标，绘制Sephadex G-100色谱洗脱曲线(如图5所示)，合并主要洗脱峰对应的洗脱液，冷冻干燥得油茶粕多糖2；
步骤S6，将0.5mol/L氯化钠洗脱液浓缩至5mL，上样于Sephadex G-100层析柱(60cm×2.6cm)中，流速0.3mL/min，用去离子水洗脱，分步收集(每管10min)。用硫酸-苯酚法检测各管多糖的含量(A490)，以收集的管数为横坐标、A490为纵坐标，绘制Sephadex G-100色谱洗脱曲线(如图6所示)，合并主要洗脱峰对应的洗脱液，冷冻干燥得油茶粕多糖3。
分子排阻HPLC法测定油茶粕多糖1-3的平均分子量：
将已知分子量的多糖对照品用流动相配成1.0mg/mL标准溶液，参考文献条件(Structural analysis of a polysaccharide isolated from the aqueous extract of an edible mushroom,Pleurotus sajor-caju,cultivar Black Japan.Carbohyd Res,2008)，用分子排阻HPLC法测定。以多糖对照品分子量的对数值为纵坐标、保留时间为横坐标作图，得多糖分子量的标准曲线方程。称取适量油茶粕多糖1-3样品，配制1.0mg/mL溶液，用0.45μm滤膜过滤，在相同条件下进行HPLC分析。将各样品保留时间代入标准曲线方程，计算得油茶粕多糖1、2、3的平均分子量分别为75.3、85.6、94.7kDa。油茶粕多糖1-3均为单一对称峰，为单一组分。
实施例8：油茶粕多糖1-3拮抗口蹄疫疫苗对公牛精液损伤
一、实验材料
公牛品种为荷斯坦牛；公牛精液稀释液采用TCM-199。
口蹄疫疫苗为口蹄疫病毒O型亚洲Ⅰ型二价灭活疫苗，接种剂量2.5mL/头。
分别精确称取油茶粕多糖1-3用生理盐水溶解，制成浓度5mg/mL的注射液。
二、实验方法
1、精液的采集、检查、分组(采集和检查方法参考文献：注射口蹄疫疫苗对种公牛精液品质的影响研究，中国牛业科学，2014)
所用的器械事先要进行冲洗，消毒；玻璃器皿洗净后送入干燥箱干燥，假阴道和其他器械等用75％酒精棉球擦拭消毒，假阴道安装好套上保护套，消毒后的假阴道只能使用一次。 假阴道内注水温度在40℃左右，注水量约在300mL，恒温箱内温度控制在40℃-42℃，采精时的内胎温度保持在38℃-40℃之间，内胎温度可根据不同的牛做适当的调整，最高不可超过43℃。润滑剂均匀涂抹在假阴道内胎的前2/3，润滑剂用白凡士林与液态石蜡油按1：1比例调制。假阴道的内胎压力(采精前从假阴道活塞孔打气)以假阴道口呈三角形为宜。台牛要健壮、性情温顺、无疫病，外阴、臀部清洗干净。公牛采精时采取引诱、被爬跨、反复空爬等方法刺激公牛兴奋。还可更换采精地点，更换台牛等方法增加种公牛的兴奋度。采精场地应保持安静，地面清洁并铺设防滑垫，以免公牛采精时滑倒。当公牛性欲旺盛时采精，采精员右手持假阴道，站在公牛右后方，当公牛起跳，前肢爬上台牛后迅速向前托着公牛包皮，右手持假阴道与台牛成40度夹角，假阴道口斜向下方，左右手配合将公牛阴茎自然地引入假阴道内，公牛向前一冲即完成射精动作，待公牛前肢落地，缓慢地把假阴道脱出。打开活塞放气，小心取下集精管，迅速转移至精液检测室进行检测。选取新鲜精液(应呈乳白稍带黄色或奶油色不透明液体)，取一滴原精液加盖玻片后在400倍的恒温(37℃)可视显微镜下观察呈直线运动精子的百分数，选择精液活率约为80％的荷斯坦牛随机分为对照组、油茶粕多糖1组、油茶粕多糖2组、油茶粕多糖3组，每组5只。各组荷斯坦牛正常饲养30天，其中，油茶粕多糖1组、油茶粕多糖2组、油茶粕多糖3组荷斯坦牛每三天分别按照5mg/100kg的剂量肌肉注射给药油茶粕多糖1-3。正常饲养30天后各组荷斯坦牛注射口蹄疫病毒O型亚洲Ⅰ型二价灭活疫苗，接种剂量2.5mL/头，观察期30天。经观察和检测，所有荷斯坦牛均免疫成功。观察期结束10天后，按照上述方法对各组荷斯坦牛再次采精，测定精液活率。
2、统计学方法
采用SPSS 19.0软件统计分析，数据以均值±偏差表示，组间比较采用单因素方差分析。
三、实验结果
实验结果显示，对照组荷斯坦牛经口蹄疫疫苗免疫后，精液活率显著降低(P＜0.05)；与对照组相比，油茶粕多糖1组、油茶粕多糖2组、油茶粕多糖3组荷斯坦牛精液活率显著升高(P＜0.05)。结果如表3和图7所示。
表3 各组荷斯坦牛接种口蹄疫疫苗前后精液活率比较(n＝5)
Figure PCTCN2018082479-appb-000003
综上可见，本发明提供的油茶粕多糖可以有效拮抗口蹄疫疫苗对公牛精液造成的损伤，从而可以用于降低接种口蹄疫疫苗对农场的损失；本发明提供的油茶粕多糖的制备方法步骤少，分离纯化效果好，均为单一组分。
实施例9：盐酸酸腐+米根霉转化
1、实验材料
菌种：米根霉(Rhizopus oryzae)AS3.866，购于广东省微生物菌种保藏中心。
斜面培养基：PDA培养基。
2、孢子液的制备
将米根霉接入斜面培养基，28℃下培养72h，待斜面长满孢子后，加入10mL无菌水将孢子洗下并用玻璃珠打散，制成浓度为10 7个单孢子/mL的孢子悬浮液。
3、脱毒方法
包括如下步骤：
步骤S1，将小果油茶粕粉碎成油茶粕粉，粒径为40-60目；
步骤S2，将油茶粕粉均匀喷洒pH值为4.5的盐酸水溶液，至油茶粕粉的含水量为65％；
步骤S3，接种浓度为10 7个单孢子/mL的米根霉孢子悬浮液，接种量为20％(mL/g)；
步骤S4，于30℃堆腐24-72h，分别于24、48、72h取适量粕粉提取、测定皂苷含量。
实施例10：盐酸酸腐+米根霉转化
1、实验材料
菌种：米根霉(Rhizopus oryzae)AS3.866，购于广东省微生物菌种保藏中心。
斜面培养基：PDA培养基。
2、孢子液的制备
将米根霉接入斜面培养基，28℃下培养72h，待斜面长满孢子后，加入10mL无菌水将孢子洗下并用玻璃珠打散，制成浓度为10 7个单孢子/mL的孢子悬浮液。
3、脱毒方法
包括如下步骤：
步骤S1，将小果油茶粕粉碎成油茶粕粉，粒径为40-60目；
步骤S2，将油茶粕粉均匀喷洒pH值为4的盐酸水溶液，至油茶粕粉的含水量为60％；
步骤S3，接种浓度为10 7个单孢子/mL的米根霉孢子悬浮液，接种量为15％(mL/g)；
步骤S4，于32℃堆腐24-72h，分别于24、48、72h取适量粕粉提取、测定皂苷含量。
实施例11：盐酸酸腐+米根霉转化
1、实验材料
菌种：米根霉(Rhizopus oryzae)AS3.866，购于广东省微生物菌种保藏中心。
斜面培养基：PDA培养基。
2、孢子液的制备
将米根霉接入斜面培养基，28℃下培养72h，待斜面长满孢子后，加入10mL无菌水将孢子洗下并用玻璃珠打散，制成浓度为10 7个单孢子/mL的孢子悬浮液。
3、脱毒方法
包括如下步骤：
步骤S1，将小果油茶粕粉碎成油茶粕粉，粒径为40-60目；
步骤S2，将油茶粕粉均匀喷洒pH值为5的盐酸水溶液，至油茶粕粉的含水量为70％；
步骤S3，接种浓度为10 7个单孢子/mL的米根霉孢子悬浮液，接种量为25％(mL/g)；
步骤S4，于28℃堆腐24-72h，分别于24、48、72h取适量粕粉提取、测定皂苷含量。
实施例12：甲酸酸腐+米根霉转化
1、实验材料
菌种：米根霉(Rhizopus oryzae)AS3.866，购于广东省微生物菌种保藏中心。
斜面培养基：PDA培养基。
2、孢子液的制备
将米根霉接入斜面培养基，28℃下培养72h，待斜面长满孢子后，加入10mL无菌水将孢子洗下并用玻璃珠打散，制成浓度为10 7个单孢子/mL的孢子悬浮液。
3、脱毒方法
包括如下步骤：
步骤S1，将小果油茶粕粉碎成油茶粕粉，粒径为40-60目；
步骤S2，将油茶粕粉均匀喷洒pH值为4.5的甲酸水溶液，至油茶粕粉的含水量为65％；
步骤S3，接种浓度为10 7个单孢子/mL的米根霉孢子悬浮液，接种量为20％(mL/g)；
步骤S4，于30℃堆腐24-72h，分别于24、48、72h取适量粕粉提取、测定皂苷含量。
实施例13：甲酸酸腐+米根霉转化
1、实验材料
菌种：米根霉(Rhizopus oryzae)AS3.866，购于广东省微生物菌种保藏中心。
斜面培养基：PDA培养基。
2、孢子液的制备
将米根霉接入斜面培养基，28℃下培养72h，待斜面长满孢子后，加入10mL无菌水将孢子洗下并用玻璃珠打散，制成浓度为10 7个单孢子/mL的孢子悬浮液。
3、脱毒方法
包括如下步骤：
步骤S1，将小果油茶粕粉碎成油茶粕粉，粒径为40-60目；
步骤S2，将油茶粕粉均匀喷洒pH值为4的甲酸水溶液，至油茶粕粉的含水量为60％；
步骤S3，接种浓度为10 7个单孢子/mL的米根霉孢子悬浮液，接种量为15％(mL/g)；
步骤S4，于32℃堆腐24-72h，分别于24、48、72h取适量粕粉提取、测定皂苷含量。
实施例14：甲酸酸腐+米根霉转化
1、实验材料
菌种：米根霉(Rhizopus oryzae)AS3.866，购于广东省微生物菌种保藏中心。
斜面培养基：PDA培养基。
2、孢子液的制备
将米根霉接入斜面培养基，28℃下培养72h，待斜面长满孢子后，加入10mL无菌水将孢子洗下并用玻璃珠打散，制成浓度为10 7个单孢子/mL的孢子悬浮液。
3、脱毒方法
包括如下步骤：
步骤S1，将小果油茶粕粉碎成油茶粕粉，粒径为40-60目；
步骤S2，将油茶粕粉均匀喷洒pH值为5的甲酸水溶液，至油茶粕粉的含水量为70％；
步骤S3，接种浓度为10 7个单孢子/mL的米根霉孢子悬浮液，接种量为25％(mL/g)；
步骤S4，于28℃堆腐24-72h，分别于24、48、72h取适量粕粉提取、测定皂苷含量。
实施例15：乙酸酸腐+米根霉转化
1、实验材料
菌种：米根霉(Rhizopus oryzae)AS3.866，购于广东省微生物菌种保藏中心。
斜面培养基：PDA培养基。
2、孢子液的制备
将米根霉接入斜面培养基，28℃下培养72h，待斜面长满孢子后，加入10mL无菌水将孢子洗下并用玻璃珠打散，制成浓度为10 7个单孢子/mL的孢子悬浮液。
3、脱毒方法
包括如下步骤：
步骤S1，将小果油茶粕粉碎成油茶粕粉，粒径为40-60目；
步骤S2，将油茶粕粉均匀喷洒pH值为4.5的乙酸水溶液，至油茶粕粉的含水量为65％；
步骤S3，接种浓度为10 7个单孢子/mL的米根霉孢子悬浮液，接种量为20％(mL/g)；
步骤S4，于30℃堆腐24-72h，分别于24、48、72h取适量粕粉提取、测定皂苷含量。
实施例16：乙酸酸腐+米根霉转化
1、实验材料
菌种：米根霉(Rhizopus oryzae)AS3.866，购于广东省微生物菌种保藏中心。
斜面培养基：PDA培养基。
2、孢子液的制备
将米根霉接入斜面培养基，28℃下培养72h，待斜面长满孢子后，加入10mL无菌水将孢子洗下并用玻璃珠打散，制成浓度为10 7个单孢子/mL的孢子悬浮液。
3、脱毒方法
包括如下步骤：
步骤S1，将小果油茶粕粉碎成油茶粕粉，粒径为40-60目；
步骤S2，将油茶粕粉均匀喷洒pH值为4的乙酸水溶液，至油茶粕粉的含水量为60％；
步骤S3，接种浓度为10 7个单孢子/mL的米根霉孢子悬浮液，接种量为15％(mL/g)；
步骤S4，于32℃堆腐24-72h，分别于24、48、72h取适量粕粉提取、测定皂苷含量。
实施例17：乙酸酸腐+米根霉转化
1、实验材料
菌种：米根霉(Rhizopus oryzae)AS3.866，购于广东省微生物菌种保藏中心。
斜面培养基：PDA培养基。
2、孢子液的制备
将米根霉接入斜面培养基，28℃下培养72h，待斜面长满孢子后，加入10mL无菌水将孢子洗下并用玻璃珠打散，制成浓度为10 7个单孢子/mL的孢子悬浮液。
3、脱毒方法
包括如下步骤：
步骤S1，将小果油茶粕粉碎成油茶粕粉，粒径为40-60目；
步骤S2，将油茶粕粉均匀喷洒pH值为5的乙酸水溶液，至油茶粕粉的含水量为70％；
步骤S3，接种浓度为10 7个单孢子/mL的米根霉孢子悬浮液，接种量为25％(mL/g)；
步骤S4，于28℃堆腐24-72h，分别于24、48、72h取适量粕粉提取、测定皂苷含量。
粕粉中总皂苷提取及含量测定方法
分别称取堆腐不同时间后的粕粉各3份(平行操作)，每份1g(以酸化前的油茶粕粉质量计)，置于100mL锥形瓶中，加入60％乙醇溶液30mL，超声提取30min，4000r/min离心5min，取上清液，残渣重复提取1次，合并上清液回收乙醇至无醇味，用水饱和的正丁醇萃 取4次，每次10mL，合并萃取液，挥干溶剂，残渣用甲醇溶解，转移至50mL量瓶中，加甲醇稀释至刻度，作为供试品溶液。精密量取供试品溶液400μL，置于干燥的具塞试管中，60℃蒸干，取出放冷。依次加入5％香草醛冰醋酸溶液0.2mL和高氯酸0.8mL，摇匀，于60℃水浴中保温15min，取出，用冰水浴冷却10min，加入4mL冰醋酸，摇匀，测定480nm处的吸收值。同法测定酸化前的油茶粕粉提取液于480nm处的吸收值，计算总皂苷脱除率(％)：
总皂苷脱除率(％)＝(单位质量油茶粕粉酸化前吸收值-单位质量油茶粕粉堆腐后吸收值)/单位质量油茶粕粉酸化前吸收值×100％。
测定、计算结果如表4和图8所示。
表4 实施例9-17堆腐24、48、72h后油茶粕粉中总皂苷脱除率比较
  堆腐24h脱除率(％) 堆腐48h脱除率(％) 堆腐72h脱除率(％)
实施例1 59.5±3.4 83.7±4.1 96.5±4.7
实施例2 60.1±3.2 80.9±3.8 96.2±4.3
实施例3 58.7±3.7 81.8±3.9 96.8±4.5
实施例4 97.3±4.6 98.5±4.3 99.2±4.8
实施例5 96.5±4.5 97.2±4.6 97.9±4.5
实施例6 97.0±4.9 98.1±4.7 98.8±4.2
实施例7 66.8±3.1 86.1±4.5 95.9±5.0
实施例8 64.2±3.3 84.8±4.2 96.4±4.6
实施例9 65.7±3.0 84.6±4.4 95.6±4.3
由上可见，本发明提供的脱毒方法可以有效脱除小果油茶粕中的皂苷，从而脱除其苦味，改善口感，进而可以用作饲料添加剂；尤其是，使用甲酸作为酸腐剂时，可以显著提高米根霉对小果油茶粕中皂苷的脱除效率；而且，本发明提供的脱毒方法简易可行，成本低，易于工业化生产和推广。
上述实施例的作用在于具体介绍本发明的实质性内容，但本领域技术人员应当知道，不应将本发明的保护范围局限于该具体实施例。

Claims (40)

  1. 一种油茶粕多肽，其特征在于：以小果油茶粕为原料，经胰蛋白酶水解后，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥即得。
  2. 根据权利要求1所述的油茶粕多肽，其特征在于：所述小果油茶粕指小果油茶的果实经过压榨处理或浸出法处理去除油脂后得到的固体残渣。
  3. 权利要求1或2所述油茶粕多肽的制备方法，其特征在于，包括如下步骤：
    步骤S1，将小果油茶粕粉碎至80-100目，加水浸泡，最后制成粕粉悬浮液；
    步骤S2，加入胰蛋白酶酶解，酶解条件为：pH值，7.8-8.2；酶解温度，43-47℃；酶解时间，10-12小时；酶解结束后，加热使胰蛋白酶灭活，冷却至常温，静置取上清；
    步骤S3，将上清液用脱脂棉过滤，收集滤液，冻干得粗品冻干粉；
    步骤S4，粗品冻干粉用超纯水溶解，用不同截留分子量的超滤膜进行分离，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥即得。
  4. 根据权利要求3所述的制备方法，其特征在于：所述不同截留分子量的超滤膜包括截留分子量分别为5000u、3000u的超滤膜。
  5. 根据权利要求4所述的制备方法，其特征在于：还包括截留分子量10000u的超滤膜。
  6. 根据权利要求3所述的制备方法，其特征在于，加热使胰蛋白酶灭活的方法为：90℃高温处理10分钟。
  7. 根据权利要求3所述的制备方法，其特征在于：粕粉悬浮液优选130-170g/L。
  8. 根据权利要求3所述的制备方法，其特征在于，所述胰蛋白酶的添加重量以粕粉重量计为2.5-3.5g/100g。
  9. 根据权利要求8所述的制备方法，其特征在于，所述胰蛋白酶的酶解条件优选pH值8.0、温度45℃、时间11小时。
  10. 权利要求1或2所述的油茶粕多肽在牛精液冷冻保存方面的应用。
  11. 一种油茶粕多肽，其特征在于：以小果油茶粕为原料，经碱性蛋白酶水解后，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥即得。
  12. 根据权利要求11所述的油茶粕多肽，其特征在于：所述小果油茶粕指小果油茶的果实经过压榨处理或浸出法处理去除油脂后得到的固体残渣。
  13. 权利要求11或12所述油茶粕多肽的制备方法，其特征在于，包括如下步骤：
    步骤S1，将小果油茶粕粉碎至80-100目，加水浸泡，最后制成粕粉悬浮液；
    步骤S2，加入碱性蛋白酶酶解，酶解条件为：pH值，8.3-8.7；酶解温度，53-57℃；酶解时间，9-11小时；酶解结束后，加热使碱性蛋白酶灭活，冷却至常温，静置取上清；
    步骤S3，将上清液用脱脂棉过滤，收集滤液，冻干得粗品冻干粉；
    步骤S4，粗品冻干粉用超纯水溶解，用不同截留分子量的超滤膜进行分离，收集分子量在(5000u,3000u]范围内的肽段，冷冻干燥即得。
  14. 根据权利要求13所述的制备方法，其特征在于：所述不同截留分子量的超滤膜包括截留分子量分别为5000u、3000u的超滤膜。
  15. 根据权利要求14所述的制备方法，其特征在于：还包括截留分子量10000u的超滤膜。
  16. 根据权利要求13所述的制备方法，其特征在于，加热使碱性蛋白酶灭活的方法为：90℃高温处理10分钟。
  17. 根据权利要求13所述的制备方法，其特征在于：粕粉悬浮液优选130-170g/L。
  18. 根据权利要求13所述的制备方法，其特征在于，所述碱性蛋白酶的添加重量以粕粉重量计为2.5-3.5g/100g。
  19. 根据权利要求18所述的制备方法，其特征在于，所述碱性蛋白酶的酶解条件优选pH值8.5、温度55℃、时间10小时。
  20. 权利要求11或12所述的油茶粕多肽在制备促进乏情母牛发情和受胎的饲料或药物方面的应用。
  21. 一种油茶粕多糖，其特征在于：从小果油茶粕中分离得到，平均分子量为75-95kDa。
  22. 根据权利要求21所述的油茶粕多糖，其特征在于：以小果油茶粕为原料，经水提醇沉、DEAE-52纤维素柱层析、Sephadex G-l00柱层析纯化得到。
  23. 根据权利要求22所述的油茶粕多糖，其特征在于：平均分子量为75.3、85.6或94.7kDa。
  24. 根据权利要求21所述的油茶粕多糖，其特征在于：所述小果油茶粕指小果油茶的果实经过压榨处理或浸出法处理去除油脂后得到的固体残渣。
  25. 权利要求21-24任一所述油茶粕多糖的制备方法，其特征在于，包括如下步骤：
    步骤S1，将小果油茶粕粉碎至80-100目，沸水回流提取，过滤，合并滤液，浓缩；
    步骤S2，向浓缩液中添加加入4-5倍体积的无水乙醇，低温沉淀，离心收集沉淀；
    步骤S3，将沉淀溶于去离子水中，加样于DEAE-52层析柱，依次用纯水和不同浓度氯化钠溶液梯度洗脱，分别收集纯水洗脱液、0.3mol/L氯化钠洗脱液和0.5mol/L氯化钠洗脱液；
    步骤S4，分别将纯水洗脱液、0.3mol/L氯化钠洗脱液和0.5mol/L氯化钠洗脱液浓缩后的浓缩液上样于Sephadex G-l00层析柱，用去离子水洗脱，分别收集洗脱峰、冷冻干燥。
  26. 根据权利要求25所述的制备方法，其特征在于：步骤S2沉淀条件优选4℃静置24小时；离心条件优选5×10 3rpm离心5分钟。
  27. 根据权利要求25所述的制备方法，其特征在于：步骤S4用硫酸-苯酚法检测各管多糖的含量，以收集的管数为横坐标、紫外吸光度值为纵坐标，绘制Sephadex G-100色谱洗脱曲线，各浓缩液经Sephadex G-100层析柱分离后各得1个主要洗脱峰，收集洗脱峰、冷冻干燥。
  28. 根据权利要求27所述的制备方法，其特征在于：紫外检测波长为490nm。
  29. 根据权利要求25所述的制备方法，其特征在于：步骤S2醇沉前先用Sevag法脱蛋白。
  30. 权利要求21-24任一所述油茶粕多糖拮抗口蹄疫疫苗对公牛精液损伤的用途。
  31. 一种小果油茶粕的脱毒方法，所述小果油茶粕指小果油茶的果实经过压榨处理或浸出法处理去除油脂后得到的固体残渣，所述脱毒指脱除皂苷，其特征在于：将小果油茶粕粉碎成油茶粕粉，均匀喷洒酸性水溶液后接种米根霉堆腐。
  32. 根据权利要求31所述的脱毒方法，其特征在于：所述酸为盐酸、甲酸或乙酸。
  33. 根据权利要求32所述的脱毒方法，其特征在于：酸性水溶液pH值优选4-5。
  34. 根据权利要求31所述的脱毒方法，其特征在于：以喷洒酸性水溶液后的油茶粕粉湿重计，米根霉的接种量为15％-25％(mL/g)，米根霉菌液浓度为10 7个单孢子/mL。
  35. 根据权利要求31所述的脱毒方法，其特征在于：堆腐温度为28-32℃。
  36. 根据权利要求31所述的脱毒方法，其特征在于：堆腐时间为24-72h。
  37. 根据权利要求31所述的脱毒方法，其特征在于：所述酸性水溶液优选pH值为4-5的甲酸水溶液，喷洒至油茶粕粉的含水量为60-70％。
  38. 根据权利要求37所述的脱毒方法，其特征在于：以喷洒酸性水溶液后的油茶粕粉湿重计，米根霉的接种量为20％(mL/g)，米根霉菌液浓度为10 7个单孢子/mL，30℃堆腐24h。
  39. 一种由权利要求31-38任一所述脱毒方法制备的小果油茶粕。
  40. 权利要求39所述的小果油茶粕用作动物饲料的用途。
PCT/CN2018/082479 2017-12-11 2018-04-10 一种油茶粕多肽、多糖及其制备方法、应用和油茶粕脱毒方法 WO2019114159A1 (zh)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
CN201711304360.0A CN108060196B (zh) 2017-12-11 2017-12-11 一种油茶粕多肽、制备方法及促进乏情母牛发情和受胎的作用
CN201711304360.0 2017-12-11
CN201711304376.1A CN107964033B (zh) 2017-12-11 2017-12-11 一种油茶粕多肽、制备方法及在牛精液冷冻保存方面的应用
CN201711308451.1 2017-12-11
CN201711308451.1A CN108003253B (zh) 2017-12-11 2017-12-11 一种油茶粕多糖、制备方法及拮抗口蹄疫疫苗对公牛精液损伤的作用
CN201711304358.3A CN108041282B (zh) 2017-12-11 2017-12-11 一种油茶粕的脱毒方法及脱毒油茶粕用作动物饲料的用途
CN201711304358.3 2017-12-11
CN201711304376.1 2017-12-11

Publications (1)

Publication Number Publication Date
WO2019114159A1 true WO2019114159A1 (zh) 2019-06-20

Family

ID=66819913

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/082479 WO2019114159A1 (zh) 2017-12-11 2018-04-10 一种油茶粕多肽、多糖及其制备方法、应用和油茶粕脱毒方法

Country Status (1)

Country Link
WO (1) WO2019114159A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112870103A (zh) * 2021-03-30 2021-06-01 安徽省金天柱农业科技有限公司 一种油茶籽多肽的护肤精华液及其制备工艺

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101220345A (zh) * 2008-01-23 2008-07-16 西北农林科技大学 一种牛冷冻精液稀释液及其制备方法
CN101513220A (zh) * 2009-03-24 2009-08-26 中国农业科学院油料作物研究所 一种油料饼粕蛋白饲料的制备方法
CN102048026A (zh) * 2010-12-06 2011-05-11 中南林业科技大学 一种功能性饲料用添加剂油茶粕蛋白多肽的制备方法
CN102993328A (zh) * 2012-12-31 2013-03-27 广西师范大学 一种从油茶枯中综合提取多糖、多酚和皂素的方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101220345A (zh) * 2008-01-23 2008-07-16 西北农林科技大学 一种牛冷冻精液稀释液及其制备方法
CN101513220A (zh) * 2009-03-24 2009-08-26 中国农业科学院油料作物研究所 一种油料饼粕蛋白饲料的制备方法
CN102048026A (zh) * 2010-12-06 2011-05-11 中南林业科技大学 一种功能性饲料用添加剂油茶粕蛋白多肽的制备方法
CN102993328A (zh) * 2012-12-31 2013-03-27 广西师范大学 一种从油茶枯中综合提取多糖、多酚和皂素的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GONG JIJUN: "The Research on the Preparation and Bioactivities of Peptide Derived from Camelia Oleifera Abel Seed Meal", CHINA DOCTORAL DISSERTATIONS- AGRICULTURE SCIENCE AND TECHNOLOGY, no. 5, 15 May 2012 (2012-05-15), pages 2 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112870103A (zh) * 2021-03-30 2021-06-01 安徽省金天柱农业科技有限公司 一种油茶籽多肽的护肤精华液及其制备工艺

Similar Documents

Publication Publication Date Title
CN104844723A (zh) 一种铁皮石斛提取物的制备方法及应用
CN111787931B (zh) 分离的赤红球菌细胞壁骨架在制备治疗人乳头瘤病毒感染的药物中的用途
CN101607990A (zh) 一种蚯蚓活性蛋白及其提取方法与应用
CN106822890B (zh) 一种用于防治断奶仔猪腹泻的药物组合物及其制备方法和应用
CN105505887A (zh) 一种猪繁殖与呼吸综合征病毒稀释液及其制备方法
CN112316128B (zh) 一种犬瘟热病毒犬细小病毒犬传染性肝炎病毒三联活疫苗
WO2004060381A1 (fr) Peau de lapin comprenant une substance bioactive et son utilisation
WO2019114159A1 (zh) 一种油茶粕多肽、多糖及其制备方法、应用和油茶粕脱毒方法
CN101934068B (zh) 胸腺肽注射液的制备方法
CN116570640B (zh) 葵花盘生物碱及衍生物在降尿酸溶解痛风石产品中的应用
CN113402626A (zh) 一种香水莲花多糖提取物及其制备方法和应用
CN109512851B (zh) 一种医用抑菌助产凝胶的制备方法
JP2022531109A (ja) 熱損傷の治療におけるロドコッカスルーバー製品の使用
CN116694509A (zh) 一株代谢l-色氨酸产吲哚衍生物且具有增强肠道屏障功能的植物乳杆菌及其应用
CN106177900B (zh) 一种抗人乳头瘤病毒的凝胶及其制备方法
CN1995062A (zh) 一种小麦麸皮抗冻蛋白的制备方法
CN108113001B (zh) 一种缓解胃溃疡的益生菌冻干粉
CN103495180B (zh) 一种猪繁殖与呼吸综合征复合活疫苗用冻干保护剂
CN106798920B (zh) 一种复合免疫佐剂及其制备方法和应用
CN1329041C (zh) 猪胎素的生产方法
CN102816226A (zh) 一种猪小肠抗菌肽pr39的制备方法
CN113249298A (zh) 一种草鱼动脉球组织细胞系及其应用
CN101686992B (zh) 卡介菌多糖核酸提取物在制备治疗变态反应性皮肤病的药物中的应用及其注射剂和制备方法
CN101623304A (zh) 一种用于鼻腔给药的药物组合物
CN114099602B (zh) 一种抗加德纳菌的妇科外用物及其制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18889028

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18889028

Country of ref document: EP

Kind code of ref document: A1