CN103461471B - Application of hypsizigus marmoreus lectin and deproteinization polysaccharide in preservation of loquats - Google Patents
Application of hypsizigus marmoreus lectin and deproteinization polysaccharide in preservation of loquats Download PDFInfo
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- CN103461471B CN103461471B CN201310396752.XA CN201310396752A CN103461471B CN 103461471 B CN103461471 B CN 103461471B CN 201310396752 A CN201310396752 A CN 201310396752A CN 103461471 B CN103461471 B CN 103461471B
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Abstract
The invention belongs to the technical field of preservation of fruits, in particular to an application of hypsizigus marmoreus lectin and deproteinization polysaccharide in preservation of loquats. The invention also provides a method implementing the application. The application is characterized in that a coating preservation liquid is prepared by using one or two of hypsizigus marmoreus lectin or hypsizigus marmoreus deproteinization polysaccharide; the loquats are soaked in the coating preservation liquid for 1-4 minutes, taken out and dried in the air, and then stored at room temperature, wherein the mass percentage of the hypsizigus marmoreus lectin in the coating preservation liquid is 0.1%-0.2%, the mass percentage of the hypsizigus marmoreus deproteinization polysaccharide in the coating preservation liquid is 1%-2%. According to the application, biological natural extracts are adopted, so the prepared coating preservation liquid is applied to preservation of fruits, is safe, non-toxic and effective, and is an effective method for preservation of fruits.
Description
Technical field
The invention belongs to fruit freshness preserving technical field, be specifically related to a kind of true Ji mushroom agglutinin and the application of de-proteoglycan in loquat is fresh-keeping.
Background technology
Fruit, due to containing abundant carbohydrate, organic acid, vitamin and inorganic salts etc., thus becomes the nutrient source that the mankind are important.Fruit is not only delicious good to eat, also has the advantage such as hypotensive, delaying senility, weight-reducing, pre-cancer.But there is the perishability of stronger seasonality, regionality and fruit itself in Production of fruit, this problem making fruit storage fresh-keeping is increasingly outstanding.At present, the fresh-keeping means adopted at field of fruit freshness keeping both at home and abroad mainly contain preservation by low temperature, controlled atmosphere, fresh chemically, biological preservation etc.Although the fruit freshness preserving type of skill is various at present, because people not only require to improve to fruit freshness preserving color traditionally, also more pay close attention to the security of food, novel preservation technique mainly concentrates on biological preservation field.The application of biological way of keeping fresh in fruit will mainly concentrate on the fresh-keeping of fresh-keeping, the biological natural extract of microbial cells and metabolite thereof and utilize gene to carry out in fresh-keeping three.At present, such preservation technique is also in conceptual phase, and the mechanism of action is indefinite, and correlation technique is also immature, is not also widely used.But new bio preservation technique that is safe, nontoxic, environmental protection will become the important directions of fresh-keeping industry development from now on.Being intended to be exploitation safe, nontoxic, effective natural bacteriostatic agent, antistaling agent and providing certain theoretical foundation, is antistaling fresh exploitation effective ways, so study a kind of true Ji mushroom agglutinin and the application of de-proteoglycan in loquat is fresh-keeping has great importance.
Summary of the invention
In order to overcome above-mentioned defect, the invention provides a kind of true Ji mushroom agglutinin and the application of de-proteoglycan in loquat is fresh-keeping.
For achieving the above object, the present invention adopts following technical scheme:
The application of true Ji mushroom agglutinin in loquat is fresh-keeping.
True Ji mushroom takes off the application of proteoglycan in loquat is fresh-keeping.
Described true Ji mushroom agglutinin adopts true Ji mushroom to be raw material, true Ji mushroom agglutinin crude product is extracted to obtain in conjunction with ammonium sulfate precipitation and PBS phosphate buffer dialysis, utilizing DEAE-Sepharose Fast Flow(diethylaminethyl-Ago-Gel) ion-exchange chromatography carries out agglutinin purifying crude collecting precipitation, obtains true Ji mushroom agglutinin through desalination postlyophilization.
It is adopt true Ji mushroom to be raw material that described true Ji mushroom takes off proteoglycan, water extraction and alcohol precipitation method is utilized to carry out extracting the antibacterial polysaccharide of true Ji mushroom, after alcohol precipitation, collect polysaccharide precipitation is mycelia Thick many candies, the protein ingredient that mycelia Thick many candies application Sevag method removes wherein is carried out purifying, and the polysaccharide component after deproteination obtains true Ji mushroom through vacuum freeze drying again and takes off proteoglycan.
Described true Ji mushroom agglutinin or the application of de-proteoglycan in loquat is fresh-keeping, be true Ji mushroom agglutinin or true Ji mushroom are taken off in proteoglycan one or both, be mixed with coating anti-staling solution, loquat be placed on after soaking 1 ~ 4min in above-mentioned coating anti-staling solution and take out, dry, preserve under normal temperature condition; In described coating anti-staling solution, the mass percent of true Ji mushroom agglutinin is: 0.1%-0.2%, and in described coating anti-staling solution, true Ji mushroom takes off the mass percent of proteoglycan and is: 1%-2%.
The concrete preparation process of described true Ji mushroom agglutinin is: true Ji mushroom with mass/volume than for 1:(3 ~ 7) g/ml adds concentration for 0.05mol/L, the PBS phosphate buffer of pH=7.8, lixiviate 24h at 4 DEG C, 4 DEG C, rotating speed is refrigerated centrifuge 40 min under 8000r/min, gets supernatant; Supernatant by ammonium sulfate precipitation, collects 40% ~ 75% ammonium sulfate saturation degree sediment fraction, precipitation redissolve in concentration be 0.05mol/L, the PBS phosphate buffer of pH=7.8, load desalination in bag filter, dialysis, to outer dislysate detects without SO42-, is agglutinin crude product after freeze drying; By DEAE-Sepharose Fast Flow (diethylaminethyl-Ago-Gel) post on agglutinin crude product, with 0.05mol/L, the PBS phosphate buffer of pH=7.8 carries out wash-out and is in charge of collection, determined wavelength 280nm, the blood coagulation activity of eluting peak is detected with rabbit erythrocyte, by have blood coagulation activity by CM-Sepharose Fast Flow(carboxymethyl-Ago-Gel) chromatographic purifying, collect, namely desalination postlyophilization obtain true Ji mushroom agglutinin sterling.
Described true Ji mushroom takes off the concrete preparation process of proteoglycan: adopt Hot water extraction to extract true Ji mushroom polysaccharide, take the true Ji mushroom that weight in wet base is 500 g, distilled water is added than for (1 ~ 3): 1g/ml by mass/volume, with high-speed tissue mashing machine's homogenate 10 min, and be placed on 90 DEG C of water-bath lixiviate 8 h, centrifugal 5 min of 5000 rpm, get that supernatant freeze drying is concentrated into original volume 1/10, the mass concentration adding 3 times of volumes is 95% alcohol settling, centrifugal 10 min of 8000 rpm, collecting precipitation is mycelia Thick many candies; Mycelia Thick many candies is after Sevag method takes off albumen, and concentrated, adding mass concentration is 95% alcohol settling, centrifugal 10 min of 8000 rpm, and sediment freeze drying is true Ji mushroom deproteinized polysaccharide.
Described true Ji mushroom raw material can be true Ji mushroom leftover bits and pieces or fructification.
Described PBS phosphate buffer is by Na
2hPO
412H2O is dissolved in preparation in distilled water and obtains the Na that concentration is 0.2 mol/L
2hPO
4, by NaH
2pO
42H
2o to be dissolved in distilled water configuration, and to obtain concentration be 0.2 mol/L NaH
2pO
4; NaH will be prepared
2pO
4, Na
2hPO
4with water according to after the volume ratio mixing of 8.5:91.5:400, sterilizing 30min under temperature is 121 ° of C, obtains the PBS phosphate buffer that concentration is 0.05mol/L, pH=7.8, is stored in dark cold place, for subsequent use.
Containing various active material in true Ji's massee fruiting bodies, there is multiple important function simultaneously, true Ji mushroom leftover bits and pieces is the same with fructification also has various active material, and wherein containing the activated polysaccharide of a large amount of tool and activated protein, bacteriostatic activity is exactly wherein a kind of important function.In conjunction with extractive technique efficient in Food Science, carry out effectively extracting and separation and purification to antibacterial polysaccharide and agglutinin in true Ji mushroom, prepare composite coating preservative, research composite coating preservative is to the efficient fresh-keeping effect of loquat.Early stage carries out to edible mushrooms such as true Ji mushroom, Dual Mushroom mushroom, dictyophora phalloidea, mushrooms test that a large amount of active material extracts and carries out the research of bacteriostatic activity, on this basis, with true Ji mushroom for experiment material, high efficiency extraction and separation and purification are carried out to true Ji mushroom polysaccharide and agglutinin, obtain antibacterial polysaccharide and the agglutinin of higher degree, carry out the Primary Study suppressing loquat anthrax bacteria activity, prepare true Ji mushroom polysaccharide and agglutinin composite fresh-keeping film, carry out the fresh-keeping effect of composite coating preservative to loquat fresh fruit.
remarkable advantage of the present invention is:
(1) this application raw material is easy to get, and adopts leftover bits and pieces to reduce cost as raw material, improves industrial output value.
(2) adopt biological natural extract, be applied in fruit freshness preserving, safety non-toxic, decreasing pollution, useful to health.
(3) the present invention's application is obvious for loquat fresh-keeping effect, can have good economic results in society.
(4) for developing safe, nontoxic, effective natural bacteriostatic agent, antistaling agent provides certain theoretical foundation, is loquat antistaling fresh exploitation effective ways.
(5) this technology reaches the object of bacterium industry changing rejected material to useful resource recycling, the environmental protection of recycling means, and utilizing status is remarkable, realizes true Ji mushroom effective active matter first Application fresh-keeping in loquat simultaneously.
Accompanying drawing explanation
fig. 1 istrue Ji mushroom coating antistaling agent is on the impact of loquat weight-loss ratio.
fig. 2 istrue Ji mushroom coating antistaling agent is on the impact of loquat healthy fruit.
fig. 3 istrue Ji mushroom coating antistaling agent is to loquat fresh-keeping effect, and processed group 1 is coated with film formulation A, and processed group 2 is coated with film formulation B.
fig. 4 istrue Ji mushroom extract film is on the impact of loquat organic acid content.
fig. 5 istrue Ji mushroom coating antistaling agent is on the impact of loquat VC content.
fig. 6 isthe change of loquat fresh fruit storage period respiratory intensity.
fig. 7 istrue Ji mushroom coating antistaling agent is on the impact of loquat soluble solid.
Detailed description of the invention
Example is below to illustrate feature of the present invention further and unrestricted the present invention.Following examples are present pre-ferred embodiments, and all equalizations done according to the present patent application the scope of the claims change and modify, all should covering scope of the present invention.
embodiment 1
The application of true Ji mushroom agglutinin in loquat is fresh-keeping.
True Ji mushroom takes off the application of proteoglycan in loquat is fresh-keeping.
Described true Ji mushroom agglutinin adopts true Ji mushroom to be raw material, true Ji mushroom agglutinin crude product is extracted to obtain in conjunction with ammonium sulfate precipitation and PBS phosphate buffer dialysis, utilize DEAE ion-exchange chromatography to carry out agglutinin purifying crude collecting precipitation, obtain true Ji mushroom agglutinin through desalination postlyophilization.
It is adopt true Ji mushroom to be raw material that described true Ji mushroom takes off proteoglycan, water extraction and alcohol precipitation method is utilized to carry out extracting the antibacterial polysaccharide of true Ji mushroom, after alcohol precipitation, collect polysaccharide precipitation is mycelia Thick many candies, the protein ingredient that mycelia Thick many candies application Sevag method removes wherein is carried out purifying, and the polysaccharide component after deproteination obtains true Ji mushroom through vacuum freeze drying again and takes off proteoglycan.
Described true Ji mushroom agglutinin or the application of de-proteoglycan in loquat is fresh-keeping, be true Ji mushroom agglutinin or true Ji mushroom are taken off in proteoglycan one or both, be mixed with coating anti-staling solution, loquat be placed on after soaking 1min in above-mentioned coating anti-staling solution and take out, dry, preserve under normal temperature condition; In described coating anti-staling solution, the mass percent of true Ji mushroom agglutinin is: 0.1%, and in described coating anti-staling solution, true Ji mushroom takes off the mass percent of proteoglycan and is: 2%.
The concrete preparation process of described true Ji mushroom agglutinin is: true Ji mushroom adds concentration for 0.05mol/L with mass/volume than for 1:3g/ml, the PBS phosphate buffer of pH=7.8, lixiviate 24h at 4 DEG C, 4 DEG C, rotating speed is refrigerated centrifuge 40 min under 8000r/min, gets supernatant; Supernatant by ammonium sulfate precipitation, collects 40% ammonium sulfate saturation degree sediment fraction, precipitation redissolve in concentration be 0.05mol/L, the PBS phosphate buffer of pH=7.8, load desalination in bag filter, dialysis, to outer dislysate detects without SO42-, is agglutinin crude product after freeze drying; By DEAE-Sepharose Fast Flow post on agglutinin crude product, with 0.05mol/L, the PBS phosphate buffer of pH=7.8 carries out wash-out and is in charge of collection, determined wavelength 280nm, the blood coagulation activity of eluting peak is detected with rabbit erythrocyte, by have blood coagulation activity by CM-Sepharose Fast Flow chromatographic purifying, collect, namely desalination postlyophilization obtain true Ji mushroom agglutinin sterling.
Described true Ji mushroom takes off the concrete preparation process of proteoglycan: adopt Hot water extraction to extract true Ji mushroom polysaccharide, take the true Ji mushroom that weight in wet base is 500 g, distilled water is added than for 1:1g/ml by mass/volume, with high-speed tissue mashing machine's homogenate 10 min, and be placed on 90 DEG C of water-bath lixiviate 8 h, centrifugal 5 min of 5000 rpm, get that supernatant freeze drying is concentrated into original volume 1/10, the mass concentration adding 3 times of volumes is 95% alcohol settling, centrifugal 10 min of 8000 rpm, collecting precipitation is mycelia Thick many candies; Mycelia Thick many candies is after Sevag method takes off albumen, and concentrated, adding mass concentration is 95% alcohol settling, centrifugal 10 min of 8000 rpm, and sediment freeze drying is true Ji mushroom deproteinized polysaccharide.
Described true Ji mushroom raw material is true Ji mushroom leftover bits and pieces.
Described PBS phosphate buffer is by Na
2hPO
412H
2o is dissolved in preparation in distilled water and obtains the Na that concentration is 0.2 mol/L
2hPO
4, by NaH
2pO
42H
2o to be dissolved in distilled water configuration, and to obtain concentration be 0.2 mol/L NaH
2pO
4; NaH will be prepared
2pO
4, Na
2hPO
4with water according to after the volume ratio mixing of 8.5:91.5:400, sterilizing 30min under temperature is 121 ° of C, obtains the PBS phosphate buffer that concentration is 0.05mol/L, pH=7.8, is stored in dark cold place, for subsequent use.
embodiment 2
The application of true Ji mushroom agglutinin in loquat is fresh-keeping.
True Ji mushroom takes off the application of proteoglycan in loquat is fresh-keeping.
Described true Ji mushroom agglutinin adopts true Ji mushroom to be raw material, true Ji mushroom agglutinin crude product is extracted to obtain in conjunction with ammonium sulfate precipitation and PBS phosphate buffer dialysis, utilize DEAE ion-exchange chromatography to carry out agglutinin purifying crude collecting precipitation, obtain true Ji mushroom agglutinin through desalination postlyophilization.
It is adopt true Ji mushroom to be raw material that described true Ji mushroom takes off proteoglycan, water extraction and alcohol precipitation method is utilized to carry out extracting the antibacterial polysaccharide of true Ji mushroom, after alcohol precipitation, collect polysaccharide precipitation is mycelia Thick many candies, the protein ingredient that mycelia Thick many candies application Sevag method removes wherein is carried out purifying, and the polysaccharide component after deproteination obtains true Ji mushroom through vacuum freeze drying again and takes off proteoglycan.
Described true Ji mushroom agglutinin or the application of de-proteoglycan in loquat is fresh-keeping, be true Ji mushroom agglutinin or true Ji mushroom are taken off in proteoglycan one or both, be mixed with coating anti-staling solution, loquat be placed on after soaking 4min in above-mentioned coating anti-staling solution and take out, dry, preserve under normal temperature condition; In described coating anti-staling solution, the mass percent of true Ji mushroom agglutinin is: 0.2%, and in described coating anti-staling solution, true Ji mushroom takes off the mass percent of proteoglycan and is: 1%.
The concrete preparation process of described true Ji mushroom agglutinin is: true Ji mushroom adds concentration for 0.05mol/L with mass/volume than for 1:7g/ml, the PBS phosphate buffer of pH=7.8, lixiviate 24h at 4 DEG C, 4 DEG C, rotating speed is refrigerated centrifuge 40 min under 8000r/min, gets supernatant; Supernatant by ammonium sulfate precipitation, collects 75% ammonium sulfate saturation degree sediment fraction, precipitation redissolve in concentration be 0.05mol/L, the PBS phosphate buffer of pH=7.8, load desalination in bag filter, dialysis, to outer dislysate detects without SO42-, is agglutinin crude product after freeze drying; By DEAE-Sepharose Fast Flow post on agglutinin crude product, with 0.05mol/L, the PBS phosphate buffer of pH=7.8 carries out wash-out and is in charge of collection, determined wavelength 280nm, the blood coagulation activity of eluting peak is detected with rabbit erythrocyte, by have blood coagulation activity by CM-Sepharose Fast Flow chromatographic purifying, collect, namely desalination postlyophilization obtain true Ji mushroom agglutinin sterling.
Described true Ji mushroom takes off the concrete preparation process of proteoglycan: adopt Hot water extraction to extract true Ji mushroom polysaccharide, take the true Ji mushroom that weight in wet base is 500 g, distilled water is added than for 3:1g/ml by mass/volume, with high-speed tissue mashing machine's homogenate 10 min, and be placed on 90 DEG C of water-bath lixiviate 8 h, centrifugal 5 min of 5000 rpm, get that supernatant freeze drying is concentrated into original volume 1/10, the mass concentration adding 3 times of volumes is 95% alcohol settling, centrifugal 10 min of 8000 rpm, collecting precipitation is mycelia Thick many candies; Mycelia Thick many candies is after Sevag method takes off albumen, and concentrated, adding mass concentration is 95% alcohol settling, centrifugal 10 min of 8000 rpm, and sediment freeze drying is true Ji mushroom deproteinized polysaccharide.
Described true Ji mushroom raw material is true Ji mushroom leftover bits and pieces.
Described PBS phosphate buffer is by Na
2hPO
412H2O is dissolved in preparation in distilled water and obtains the Na that concentration is 0.2 mol/L
2hPO
4, by NaH
2pO
42H
2o to be dissolved in distilled water configuration, and to obtain concentration be 0.2 mol/L NaH
2pO
4; NaH will be prepared
2pO
4, Na
2hPO
4with water according to after the volume ratio mixing of 8.5:91.5:400, sterilizing 30min under temperature is 121 ° of C, obtains the PBS phosphate buffer that concentration is 0.05mol/L, pH=7.8, is stored in dark cold place, for subsequent use.
embodiment 3
The application of true Ji mushroom agglutinin in loquat is fresh-keeping.
True Ji mushroom takes off the application of proteoglycan in loquat is fresh-keeping.
Described true Ji mushroom agglutinin adopts true Ji mushroom to be raw material, true Ji mushroom agglutinin crude product is extracted to obtain in conjunction with ammonium sulfate precipitation and PBS phosphate buffer dialysis, utilize DEAE ion-exchange chromatography to carry out agglutinin purifying crude collecting precipitation, obtain true Ji mushroom agglutinin through desalination postlyophilization.
It is adopt true Ji mushroom to be raw material that described true Ji mushroom takes off proteoglycan, water extraction and alcohol precipitation method is utilized to carry out extracting the antibacterial polysaccharide of true Ji mushroom, after alcohol precipitation, collect polysaccharide precipitation is mycelia Thick many candies, the protein ingredient that mycelia Thick many candies application Sevag method removes wherein is carried out purifying, and the polysaccharide component after deproteination obtains true Ji mushroom through vacuum freeze drying again and takes off proteoglycan.
Described true Ji mushroom agglutinin or the application of de-proteoglycan in loquat is fresh-keeping, be true Ji mushroom agglutinin or true Ji mushroom are taken off in proteoglycan one or both, be mixed with coating anti-staling solution, loquat be placed on after soaking 2.5min in above-mentioned coating anti-staling solution and take out, dry, preserve under normal temperature condition; In described coating anti-staling solution, the mass percent of true Ji mushroom agglutinin is: 0.15%, and in described coating anti-staling solution, true Ji mushroom takes off the mass percent of proteoglycan and is: 1.5%.
The concrete preparation process of described true Ji mushroom agglutinin is: true Ji mushroom adds concentration for 0.05mol/L with mass/volume than for 1:5g/ml, the PBS phosphate buffer of pH=7.8, lixiviate 24h at 4 DEG C, 4 DEG C, rotating speed is refrigerated centrifuge 40 min under 8000r/min, gets supernatant; Supernatant by ammonium sulfate precipitation, collects 57.5% ammonium sulfate saturation degree sediment fraction, precipitation redissolve in concentration be 0.05mol/L, the PBS phosphate buffer of pH=7.8, load desalination in bag filter, dialysis, to outer dislysate detects without SO42-, is agglutinin crude product after freeze drying; By DEAE-Sepharose Fast Flow post on agglutinin crude product, with 0.05mol/L, the PBS phosphate buffer of pH=7.8 carries out wash-out and is in charge of collection, determined wavelength 280nm, the blood coagulation activity of eluting peak is detected with rabbit erythrocyte, by have blood coagulation activity by CM-Sepharose Fast Flow chromatographic purifying, collect, namely desalination postlyophilization obtain true Ji mushroom agglutinin sterling.
Described true Ji mushroom takes off the concrete preparation process of proteoglycan: adopt Hot water extraction to extract true Ji mushroom polysaccharide, take the true Ji mushroom that weight in wet base is 500 g, distilled water is added than for 2:1g/ml by mass/volume, with high-speed tissue mashing machine's homogenate 10 min, and be placed on 90 DEG C of water-bath lixiviate 8 h, centrifugal 5 min of 5000 rpm, get that supernatant freeze drying is concentrated into original volume 1/10, the mass concentration adding 3 times of volumes is 95% alcohol settling, centrifugal 10 min of 8000 rpm, collecting precipitation is mycelia Thick many candies; Mycelia Thick many candies is after Sevag method takes off albumen, and concentrated, adding mass concentration is 95% alcohol settling, centrifugal 10 min of 8000 rpm, and sediment freeze drying is true Ji mushroom deproteinized polysaccharide.
Described true Ji mushroom raw material is true Ji mushroom leftover bits and pieces.
Described PBS phosphate buffer is by Na
2hPO
412H
2o is dissolved in preparation in distilled water and obtains the Na that concentration is 0.2 mol/L
2hPO
4, by NaH
2pO
42H
2o to be dissolved in distilled water configuration, and to obtain concentration be 0.2 mol/L NaH
2pO
4; NaH will be prepared
2pO
4, Na
2hPO
4with water according to after the volume ratio mixing of 8.5:91.5:400, sterilizing 30min under temperature is 121 ° of C, obtains the PBS phosphate buffer that concentration is 0.05mol/L, pH=7.8, is stored in dark cold place, for subsequent use.
embodiment 4
The present embodiment sets forth a kind of specific implementation method of the present invention further for true Ji mushroom mushroom pin sample, the present invention includes but be not limited to the present embodiment.
Test strain: true Ji mushroom (Hypsizygus marmoreus (Peck) H.E.Bigelow) mushroom pin, by Shunchang, Fujian Province, legendary god of farming Gu Ye Co., Ltd provides; Loquat anthrax bacteria (Colletotrichum gloeosporioides), is provided by Fujian Normal University's school of life and health sciences.
Test fruit: loquat is bought in fruit market, Fuzhou City, rejects time fruit after transporting laboratory back, selects size, solid colour, do not have the fruit of mechanical damage to refrigerate.
Medicine and reagent: glucose, sucrose, peptone, beef extract, yeast extract, agar, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, vitamin B1, ascorbic acid, 2,6-dichloropheno-lindophenol, oxalic acid, sodium acid carbonate, benzinum 60-90, chloroform, ethyl acetate, acetone, n-butanol, absolute ethyl alcohol, formaldehyde, glutaraldehyde, isoamyl acetate, osmic acid, uranium acetate, lead citrate, it is pure that above reagent is common domestic analysis.CM-cellulose sodium salt 300-800(Shanghai Run Jie chemical reagent Co., Ltd), tlc silica gel GF254 chemical pure (Qingdao Marine Chemical Co., Ltd.), chromatographic silica gel (100-200 order, traditional Chinese medicines reagent), Sephadex LH-20 gel (Sweden GE Healthcare), Epon812 epoxy resin.
Laboratory apparatus: electro-heating standing-temperature cultivator; Rotary Evaporators RE52-1; The multiplex vavuum pump of SHB-IIIS circulating water type; Ao Haosi electronic balance CP114; LMQ-R3260 automatic high pressure autoclave; UD-650 type table above formula clean bench; Constant temperature culture oscillator ZHWY-200B; ZF-
type Quartz ultraviolet detector (Shanghai Gu Cun electric light instrument plant); Chromatographic column (Φ 1.6cm × 60cm, Φ 2.6cm × 40cm); HL-2N constant flow pump (Shanghai Hu Xi analytical instrument factory); Freeze drier FD-1A-50B; S-100N automatic fraction collector (Shanghai Hu Xi analytical instrument factory); Leica EM UC6 ultramicrotome; JEOL/EO SEM; JEOL perspective electron microscope.Modern instrument factory of HWF-1 type infrared carbon dioxide analyzer Jintan City; Acid base titration pipe.
Culture medium:
(1) PDA culture medium: potato 200g, sucrose 20g, agar 20g, water 1000mL, pH nature.Peeling potatoes, is cut into block and boils half an hour, then use filtered through gauze, then sugaring and agar, supplies water to 1000 mL after dissolving.
(2) rose bengal medium: rose bengal medium is that Huankai Microbes Tech Co., Ltd., Guangdong produces, rose bengal medium 31.6g, water 1000mL.
Coating-film fresh-keeping agent prescription: formula A:1% true Ji mushroom takes off proteoglycan+0.1% true Ji mushroom agglutinin; The true Ji mushroom of formula B:1% takes off proteoglycan; C K: control group.
Test method
(1) preparation of true Ji mushroom mushroom pin deproteinized polysaccharide
Hot water extraction is adopted to extract true Ji mushroom mushroom pin polysaccharide, take 500 g(weight in wet bases) true Ji mushroom mushroom pin, by mass/volume than being 2:1(and w:v=2:1) add distilled water, with high-speed tissue mashing machine's homogenate 10 min, and be placed on 90 DEG C of water-bath lixiviate 8 h, centrifugal 5 min of 5000 rpm, get supernatant freeze drying and be concentrated into 1/10,3 times of 95% alcohol settling, centrifugal 10 min of 8000 rpm, collecting precipitation is mycelia Thick many candies.Thick many candies is after Sevag method takes off albumen, and concentrated, 95% alcohol settling, centrifugal 10 min of 8000 rpm, sediment freeze drying is true Ji mushroom mushroom pin deproteinized polysaccharide.
(2) preparation of true Ji mushroom agglutinin
Various true Ji mushroom adds PBS phosphate buffer (0.05mol/L, pH=7.8) lixiviate 24h at 4 DEG C, refrigerated centrifuge (8000r/min, 40 min) at 4 DEG C with mass/volume than for 1:5, gets supernatant.Supernatant, by ammonium sulfate precipitation, collects 40% ~ 75% ammonium sulfate saturation degree sediment fraction.Precipitation is redissolved in a small amount of PBS phosphate buffer (0.05mol/L, pH=7.8) and is loaded desalination in bag filter, and dialysis, to outer dislysate detects without SO42-, is agglutinin crude product after freeze drying.By DEAE-Sepharose Fast Flow post on agglutinin crude product, with PBS phosphate buffer (0.05mol/L, pH=7.8) carry out wash-out and be in charge of collection, determined wavelength 280nm, the blood coagulation activity at each peak is detected with rabbit erythrocyte, collect Peak Activity, again Peak Activity is passed through CM-Sepharose Fast Flow chromatographic purifying, namely collection, desalination postlyophilization obtain true Ji mushroom agglutinin sterling, by gained agglutinin through SDS-PAGE(SDS-PAGE) detect, be shown as the single band of about 35kDa size.
Described PBS phosphate buffer is the preparation first carrying out mother liquor: 0.2M Na
2hPO
4: take 71.6g Na
2hPO
412H
2o, is dissolved in 1000ml water; 0.2M NaH
2pO
4: take 31.2g NaH
2pO
42H
2o, is dissolved in 1000ml water.Carry out the preparation of PBS phosphate buffer (pH=7.8 0.05mol/L) again: first join 0.2M PBS phosphate buffer (pH=7.8,100ml): the NaH getting 8.5ml 0.2mol/L
2pO
4, the Na of 91.5ml 0.2mol/L
2hPO
4; Then the 0.2M PBS phosphate buffer (pH=7.8) of joining is diluted four times, sterilizing 30min under temperature is 121 ° of C, obtain 0.05M PBS phosphate buffer (pH=7.8), be stored in dark cold place, for subsequent use.
(3) loquat anthrax bacteria Activity determination is pressed down
Employing mycelial growth rate method (Lin Chenqiang, Lin Rongbin, Cai Haisong, etc. Pleurotus tuber-regium antibacterial substance compares [J] with food preservative bacteriostatic activity. edible mushroom journal, 2007,14 (3): 62-66.):
1. bacterium cake preparation: the loquat anthrax bacteria getting activation, is connected on PDA culture medium, cultivates 5 ~ 6d in 28 DEG C ± 1, with conidium under aseptic washing, makes spore suspension (concentration is about 106/mL).Get 5mL spore suspension, pour into and melt and be cooled in the rose bengal medium of 45 DEG C ~ 50 DEG C, after shaking up, in rapid packing culture dish, pour 10mL in each culture dish, be to be solidifiedly placed in constant incubator, 28 DEG C ± 1 is inverted cultivation 2 ~ 3d covers with full ware to mycelia.Some bacterium cakes are made for subsequent use with the card punch that diameter is 0.9cm.
2. pastille culture medium flat plate preparation: accurately make pastille culture medium flat plate by 0.1mL liquid coating rose bengal medium, contrast is then coated with the solvent of same volume.
3. suppress mycelial growth experiment: put down gently in pastille culture medium central authorities (every ware one ferfas cake, mycelia faces down) with transfer needle picking bacterium cake, each process establish 3 parallel, and establish contrast.Survey colony diameter by right-angled intersection method after 28 DEG C ± 1 constant temperature culture a period of time, calculate for examination Fungal hyphal growth relative inhibition, compare inhibition.Computing formula: relative inhibition (%)=(contrast bacterium colony average diameter-process bacterium colony average diameter) ÷ (contrast bacterium colony average diameter-bacterium cake diameter) × 100%.
According to the logarithm value of drug concentration (x) with probability value (y) of mycelial growth inhibition rate, with Origin8.0 software fitting a straight line virulence regression equation, and obtain half-inhibition concentration (IC50), than serious Ji mushroom polysaccharide and true Ji mushroom agglutinin to the inhibitory action of loquat anthrax bacteria.
(4) coating method and loquat physiological index determining
Coating method: often organize sample and select loquat of uniform size 10, take out after soaking 1min respectively in different fresh-keeping liquids, airing, put into enamel tray, preserve under normal temperature condition, often organize repetition 4 times, and carry out grab sample analysis every 2d.
(5) physiological index determining
1) weight-loss ratio [10]=(weightless grams/former grams) × 100%
2) healthy fruit=(intact fruit number/inspection total fruit number) × 100%
When speck diameter is greater than 10mm, then assert that this fruit is for fruit of rotting;
3) mensuration of solubility solid sugar: measure with WYA abbe's refractometer;
4) VC measures: 2,6-dichloroindophenol titration;
5) organic acid measures: acid-base titration;
6) respiratory intensity measures: measure with infrared carbon dioxide analyzer.
Test results and analysis
(1) true Ji mushroom deproteinized polysaccharide suppresses loquat anthracnose active
Adopt mycelial growth rate method to determine 0.3125,0.6250,1.250,2.500, the true Ji mushroom deproteinized polysaccharide of 5.000mgmL-1 5 gradient concentrations is to the inhibit activities (table 1) of loquat anthrax bacteria mycelial growth.Through Origin8.0 software fitting a straight line virulence regression equation (this step is completed by computer software by experimenter's guidance, lower same), after known 72h, the ρ IC50 of true Ji mushroom deproteinized polysaccharide to loquat anthrax bacteria mycelial growth is 0.56 mgmL-1.
(2) true Ji mushroom agglutinin suppresses loquat anthracnose active
Measure through mycelial growth rate method, the true Ji mushroom agglutinin of 0.3125,0.6250,1.250,2.500,5.000 mgmL-1,5 gradient concentrations has certain inhibitory action (table 2) to loquat anthrax bacteria mycelial growth.As calculated, after drawing 72h, the ρ IC50 of true Ji mushroom agglutinin to loquat anthrax bacteria mycelial growth is 3.14 mgmL-1.
Obtained by half-inhibition concentration results contrast in table 1 and table 2, the inhibitory action of true Ji mushroom polysaccharide to loquat anthrax bacteria mycelia is better than true Ji mushroom agglutinin.
(3) true Ji mushroom extract coating antistaling agent is on the impact of loquat
A coating antistaling agent is on the impact of loquat weight-loss ratio
Loquat adopt after respiration and transpiration its dehydration can be caused weightless, along with the prolongation of storage time, the weight-loss ratio of loquat increases.Soak 1 min with formula A and formula B to loquat respectively, and establish control group, carry out the fresh-keeping test of loquat, regularly weigh for often organizing loquat, result as shown in Figure 1.
Formula A and formula B effectively can reduce the weight-loss ratio of loquat to loquat coating problems, the effect difference of wherein fill a prescription A and formula B is not obvious.When room temperature storage 12d, the weight-loss ratio of formula A and formula B is used to be respectively 4.65% and 4.91%, comparatively control group (8.17%) respectively low 3.52% and 3.25%, this may be that true Ji mushroom polysaccharide forms the sealing of film to fruit surface breathing duct at fruit surface, reduce fruit water evaporation, reduce respiratory intensity, thus reduce the weight-loss ratio of fruit.
B coating antistaling agent is on the impact of loquat healthy fruit
Different formulations preservative film on the impact of healthy fruit in loquat fresh fruit shelf time as Fig. 2, shown in 3.
Soft and the succulence of loquat, is vulnerable to mechanical damage and germ infringement, very easily rots and lose commodity value after adopting.Painting film formulation A and formula B antistaling agent can extend the freshness date of loquat, when room temperature storage 12d, control group loquat all rots, and be coated with film formulation A and formula B fresh-keeping group of healthy fruit is respectively 70% and 50%, wherein fill a prescription A fresh-keeping group of healthy fruit higher than formula B, this may be more effective containing antibacterial components in formula A, suppresses germ to encroach on, thus improve healthy fruit in preservation process.
C coating antistaling agent is on the impact of loquat organic acid content
Containing multiple organic acid in loquat fresh fruit, such as malic acid, tartaric acid, oxalic acid, citric acid, lactic acid and fumaric acid etc., wherein account for major part with malic acid.In loquat fresh fruit storage, fresh fruit organic acid content can continue to increase aging rate along with the process of fruit maturation aging, and due to respiration consumption effect, organic acid content can constantly reduce.With organic acid changes of contents in determination of acid-basetitration loquat coating-film fresh-keeping process in this test, result as shown in the figure.
As can be seen from Figure 4, compared with blank group, the true Ji mushroom extract film of formula A and B can delay the minimizing of loquat fresh fruit organic acid content in loquat storage.The effect of formula A is comparatively better than formula B.
D coating antistaling agent is on the impact of loquat VC content
VC is a kind of reducing substances, shields to fruits and vegetables, when content is reduced to a certain degree, along with the accumulation gradually of fruit interior free yl, will constantly accelerate fruit senescence speed.VC is nutriment important in fruits and vegetables simultaneously, is also the important indicator weighing fresh-keeping effect.Different formulations is as shown below to loquat antistaling fresh effect.
As can be seen from Figure 5, along with the prolongation of time, in three groups of test loquats, VC content all has downward trend, downward trend control group ﹥ formula B group ﹥ formula A group, illustrate that the loquat of coating-film fresh-keeping film has good preservation, its reason may be that the film forming of true Ji mushroom extract enters pericarp to oxygen and serves inhibitory action, reduces the oxidational losses of VC.It may be have inhibitory action containing antibacterial components to some enzyme in loquat in formula A that the decline of formula A group VC content becomes to being less than formula B group, reduces the activity of enzyme, strengthens the oxidation resistance of fruit, thus improve fruit quality.
E true Ji mushroom extract film is on the impact of loquat respiratory rate
Loquat is in storage, and accretion rate can aggravate gradually along with the increasing of aging rate, the CO discharged in body
2speed also continue to increase.By measuring the CO in loquat storage ambient
2concentration, just can measure the respiratory rate of loquat, and then learns the fresh-keeping effect of coating antistaling agent to loquat.To CO in film different preservative film loquat environment in closed environment
2concentration carries out results of regular determination, and detect different preservative film to loquat antistaling fresh effect, result as shown in Figure 6.
In this experiment, two kinds of preservative films all have certain fresh-keeping effect, and along with the prolongation of fresh fruit storage time, the loquat fresh fruit CO2 rate of release being coated with film formulation A and B preservative film presents to be increased slowly, and the speed of increase is slow compared with blank group.Illustrate that formula A and B preservative film has certain preservation, the effect of the A that wherein fills a prescription is better than the effect of formula B.
F coating antistaling agent is on the impact of loquat soluble solid
Soluble solid mainly refers to molten capacitive glucide or other soluble substances, as can be seen from Figure 7, along with the prolongation of time, the loquat soluble solid of 3 test group all has downward trend, downward trend control group ﹥ formula B group ﹥ formula A group, but the loquat downward trend of coating-film fresh-keeping is starkly lower than control group, this may be that true Ji mushroom polysaccharide forms at fruit surface the respiratory intensity that film can reduce fruit, thus reduces the consumption of molten capacitive glucide or other soluble substances.
In addition, have also been made test the same manner as in Example 4 to embodiment 1,2,3, the result of result of the test and embodiment 4 without significant difference, therefore is not repeated.
In sum, in antibacterial silk growth experiment, true Ji mushroom leftover bits and pieces polysaccharide and agglutinin have certain inhibitory action to loquat anthrax bacteria mycelial growth, along with incubation time extends, the inhibitory action of mycelial growth is weakened to some extent, the argy wormwood ethanol extract that this and Jiang Maosheng etc. are reported exists with processing time lengthening to phytopathogen inhibiting rate that downward trend is similar, analyzes that its reason may there are differences at the biomass of different growing stage to thalline, thalline is relevant to factors such as the metabolism of active material.
True Ji mushroom leftover bits and pieces polysaccharide has significant linear medicine efficacy relation to loquat anthrax bacteria.Choose 5 gradient concentration true Ji mushroom polysaccharide be respectively 0.3125,0.6250,1.250,2.500,5.000mgmL-1, carry out the test suppressing loquat anthrax bacteria mycelial growth, obtain anthracnose mycelial growth inhibition rate and be respectively 4.55%, 9.55%, 21.32%, 40.91% and 68.19%, by Origin8.0 analytical test data, the virulence regression equation of the rate that is inhibited and antibacterial polysaccharide concentration, equation is y=86.373x+71.773, and coefficient correlation reaches 0.965.Show that true Ji mushroom active polysaccharide has the functional activity of good suppression loquat anthrax bacteria growth, and present linear medicine efficacy relation in certain data area.In the test that the suppression loquat anthrax-bacilus of true Ji mushroom leftover bits and pieces agglutinin grows, with the same function also with good suppression loquat anthrax bacteria growth of active polysaccharide, show that in true Ji mushroom leftover bits and pieces, active polysaccharide and agglutinin have effective inhibitory action to loquat anthrax bacteria, there are the potentiality that exploitation becomes the agent of loquat active fresh-keeping.
Utilize true Ji mushroom leftover bits and pieces polysaccharide and agglutinin to develop formula A and the B of two kinds of loquat antistaling fresh agent, A:1% true Ji mushroom of wherein filling a prescription takes off proteoglycan+0.1% true Ji mushroom agglutinin; The true Ji mushroom of formula B:1% takes off proteoglycan, arranges blank group CK simultaneously.Carry out composite coating preservative to test loquat antistaling fresh.With healthy fruit, weight-loss ratio, solubility solid sugar content, VC content, organic acid and respiratory intensity for index, measure the antibacterial polysaccharide of true Ji mushroom and agglutinin composite coating preservative to the fresh-keeping effect of loquat.Experimental result shows, the true Ji mushroom extract coating antistaling agent of two kinds of formulas all serves certain fresh-keeping effect to loquat, inhibit the respiratory intensity of fruit to some extent, decrease the consumption of nutriment, slow down organic acid accretion rate in fresh fruit body, effectively can keep higher fresh fruit healthy fruit, reduce the rotten loss caused because of pathogen contamination, delay the aging of loquat significantly.Wherein through the loquat of formula A film, at room temperature storage 12d, healthy fruit reaches 70%, and comparatively control group rotten level is lower, and weight-loss ratio reduces, and VC and soluble solid nutritional labeling can be preserved preferably, and fresh-keeping effect is slightly better than formula B.
Claims (4)
1. the application of true Ji mushroom agglutinin in loquat is fresh-keeping, is characterized in that: true Ji mushroom agglutinin is mixed with coating anti-staling solution, is placed on by loquat after soaking 1 ~ 4min in above-mentioned coating anti-staling solution and takes out, dry, preserve under normal temperature condition; The concrete preparation process of described true Ji mushroom agglutinin is: true Ji mushroom adds concentration for 0.05mol/L with mass/volume than for 1:3 ~ 7g/ml, the PBS phosphate buffer of pH=7.8, lixiviate 24h at 4 DEG C, 4 DEG C, rotating speed is refrigerated centrifuge 40 min under 8000r/min, gets supernatant; Supernatant by ammonium sulfate precipitation, collects 40% ~ 75% ammonium sulfate saturation degree sediment fraction, and it is the PBS phosphate buffer of 0.05mol/L, pH=7.8 that precipitation is redissolved in concentration, loads desalination in bag filter, dialysis to outer dislysate without SO
4 2-till detecting, after freeze drying, be agglutinin crude product; By DEAE-Sepharose Fast Flow post on agglutinin crude product, with 0.05mol/L, the PBS phosphate buffer of pH=7.8 carries out wash-out and is in charge of collection, determined wavelength 280nm, the blood coagulation activity of eluting peak is detected with rabbit erythrocyte, by have blood coagulation activity by CM-Sepharose Fast Flow chromatographic purifying, collect, namely desalination postlyophilization obtain true Ji mushroom agglutinin sterling.
2. the application of true Ji mushroom agglutinin according to claim 1 in loquat is fresh-keeping, is characterized in that: in described coating anti-staling solution, the mass percent of true Ji mushroom agglutinin is: 0.1%-0.2%.
3. the application of true Ji mushroom agglutinin according to claim 1 in loquat is fresh-keeping, is characterized in that: described true Ji mushroom raw material is true Ji mushroom leftover bits and pieces.
4. the application of true Ji mushroom agglutinin according to claim 1 in loquat is fresh-keeping, is characterized in that: described PBS phosphate buffer is by Na
2hPO
412H
2o is dissolved in preparation in distilled water and obtains the Na that concentration is 0.2 mol/L
2hPO
4, by NaH
2pO
42H
2o to be dissolved in distilled water configuration, and to obtain concentration be 0.2 mol/L NaH
2pO
4; NaH will be prepared
2pO
4, Na
2hPO
4with water according to after the volume ratio mixing of 8.5:91.5:400, sterilizing 30min under temperature is 121 ° of C, obtains the PBS phosphate buffer that concentration is 0.05mol/L, pH=7.8, is stored in dark cold place, for subsequent use.
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| S.K Lam et al.Hypsin,a Novel Thermostable Ribosome-Inactivating Protein with Antifungal and Antiproliferative Activities from Fruiting Bodies of the Edible Mushroom Hypsizigus marmoreus.《Biochemical and Biophysical Research Communications》.2001,第285卷(第4期),1071-1075. * |
| 斑玉蕈Hypsizygus marmoreus凝集素的部分性质和细胞凝集活性分析;林玉满等;《菌物学报》;20061231;第25卷(第2期);第284-291页 * |
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