CN105875820A - Liquid compound biological preservative and preparation method and application thereof - Google Patents
Liquid compound biological preservative and preparation method and application thereof Download PDFInfo
- Publication number
- CN105875820A CN105875820A CN201610222937.2A CN201610222937A CN105875820A CN 105875820 A CN105875820 A CN 105875820A CN 201610222937 A CN201610222937 A CN 201610222937A CN 105875820 A CN105875820 A CN 105875820A
- Authority
- CN
- China
- Prior art keywords
- parts
- bacterium solution
- solution
- bacterium
- streptomyces
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
- A23B7/155—Microorganisms; Enzymes; Antibiotics
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The present invention relates to a liquid compound biological preservative. The liquid compound biological preservative is characterized by consisting of the following raw materials in parts by weight: 5-25 parts of chitosan, 5-25 parts of epsilon-polylysine, 5-25 parts of lysozyme, 5-20 parts of streptomyces hygroscopicus liquid, 5-20 parts of streptomyces microflavus liquid, 5-20 parts of streptomyces aureochromogenes liquid, 5-20 parts of lactic streptococci liquid, 5-20 parts of bacillus subtilis liquid, 5-20 parts of bacillus licheniformis liquid, 5-20 parts of beer yeast liquid, 5-20 parts of paecilomyces lilacinus liquid and 5-20 parts of trichoderma harzianum liquid. The liquid compound biological preservative is high-efficient, non-toxic, healthy and nutritious, beneficial for food safety, and especially has significant effects on preservation of nanguo pears, actinidia arguta or strawberries.
Description
Technical field
The present invention relates to technical field of food biotechnology, especially for Nanguo Pear, wild actinidia arguta, the fruit of Fructus Fragariae Ananssae
Fresh-keeping liquid complex biological preservative and preparation method thereof.
Background technology
Food is the material base of human survival and development, is the raw material maintaining human life activity indispensable, passes through
Gather, process, store, pack, a series of processes such as transport are just eaten by people.Due to physiology of respiration metabolism, pathogenic microorganism
The problem such as infect, storage method is improper causes putrid and deteriorated, not only makes national economy produce massive losses, also makes human health
Become significant threat.Therefore, to healthy, efficiently, low stain, low energy consumption, the research of food preservative technology of low cost carved and do not allowed
Slow.
Currently, cold preservation combines chemical preservative and processes the most effective technical measures being to control food spoilage, but owing to using
Health can be worked the mischief by chemical preservative, and makes phytopathogenic microorganism develop immunity to drugs it, so urgently
Need to study efficient, harmless, antiseptical bio-preservative, with the use of substituted chemistry preservative.
Bio-preservative refers to separate or utilize cell engineering, genetic engineering, fermentation engineering and enzyme work from living organism
The technology such as journey obtain, health safe to the human body, and have the material of food fresh keeping function.Biology in food fresh keeping is applied
Antistaling agent has good effect, and its preservation mechanism has the following aspects: 1. contain antibacterial substance, it is possible to suppression or
Kill the putrefactive microorganisms in food, keep the freshness of food；2. antioxidation, by preventing unsaturated fatty acids in food
The effect of Keep the quality and freshness is played in the oxidation of acid etc.；3. the activity of inhibitory enzyme, prevents food colors from changing so that it is have good
Organoleptic attribute；4. constitute protective filmy layer, be avoided that the pollution of pathogen, reduce water loss, keep food quality.
The use of bio-preservative is a kind of emerging food fresh-keeping method, and it is that one utilizes some natural materials to prepare
Become the solution of debita spissitudo, by film, soak or the method such as spray processes fresh food and suppresses or kill micro-life therein
Thing, and then reach the preservation technique of corrosion-resistanting fresh-keeping effect.Bio-preservative can suppress or kill the putrefaction bacteria in food, reduces
Air and the exposure level of food, slow down Oxidation, and the gas composition of regulation Conservation environment and relative humidity, because it has green
Color, the benefit such as efficient, healthy, expand the range of application of bio-preservative, guides following food preservative technology development and should
With.
Bio-preservative mostly extracts from organism and obtains, compared with chemical preservative, have green, safety,
The feature of health-nutrition.Use bio-preservative not only the edibility characteristic of food and nutritive value will not be produced impact, and
Also secondary pollution will not be caused.At present, by its source difference, bio-preservative is divided into crude antistaling agent and chemical preservative,
By this feature cheap, in food fresh-keeping, chemical preservative is in occupation of main status.The characteristic of bio-preservative is
Safety non-toxic, efficiency is high, and applied range must become main flow direction in following food fresh keeping.
Nanguo Pear main product, in level in Anshan city, liaoning province, so being also called " saddle fruit ", is the special product pears of the great characteristic in Liaoning Province, its
There is the plantation history of last 100 years at Anshan, liaoning Province, be referred to as " kings in pears ", be can be with the characteristic pears phase in numerous areas
The precious pears kind matched in excellence or beauty.Containing very abundant nutritional labeling in Nanguo Pear, detect through the Ministry of Agriculture, the trace that Nanguo Pear contains
Element has reached 38 kinds, has the content of many trace element to be both greater than other fruit among these.Wherein containing soluble solid
14.4%～15.5%, acid content 0.41%, and have effect of life lengthening, beauty treatment, therefore the fellow countrymen in Anshan are warm it
It is compared to pears Middle nutrition queen consort.
Actinidia arguta Sieb.et Zucc another name Fructus Actinidiae Argutae, Radix actinidiae argutae, for Actinidiaceae defoliation liana.Originate in Changbaishan area, Jilin Province
Each county, is one of famous economic wild fruit in Changbaishan area, is the king in precious wild fruit, is described as " treasure in the world is really ".Root
According to Chinese scholars, the great many of experiments of Actinidia arguta Sieb.et Zucc is shown with further investigation, Actinidia arguta Sieb.et Zucc rich in various nutritional labelings,
There is anticancer, blood pressure lowering, anti-ageing various medicinal health care function and high edible, nutrition, health care, the medical value of waiting for a long time.
Fructus Fragariae Ananssae, Rosaceae, Fragaria herbaceos perennial, the flowers and fruits of a kind of redness, have another name called raspberry, ocean the certain kind of berries, the certain kind of berries etc.,
Outward appearance is heart, and delicious red tender, sarcocarp succulence, containing special strong fruit aroma.Fructus Fragariae Ananssae rich in aminoacid, fructose, sucrose,
Glucose, citric acid, malic acid, pectin, carotene, vitamin B1, B2, nicotinic acid and mineral calcium, magnesium, phosphorus, potassium, ferrum etc., this
A little nutrients have good facilitation to growth promoter, of great advantage to old man, child.Foreign scholar studies discovery, Fructus Fragariae Ananssae
In effective ingredient, the growth of cancerous protuberance can be suppressed.Every hectogram Fructus Fragariae Ananssae 50-100 milligram Han vitamin C is higher 10 times than Fructus Mali pumilae, Fructus Vitis viniferae
Above.Scientific research, it has been demonstrated that vitamin C can eliminate intercellular lax and tense situation, makes brain cell firm in structure, skin
Skin exquisiteness is flexible, has a major impact brain and intelligent development.Eat of Fructus Fragariae Ananssae, decomposable asymmetric choice net food fat after meal, favorably digest.
No matter Nanguo Pear, Actinidia arguta Sieb.et Zucc or Fructus Fragariae Ananssae, pole is difficult to storage, plucks latter about 15 days and just will rot.So,
Develop a kind of novel bio-preservative the fresh-keeping of Nanguo Pear, Actinidia arguta Sieb.et Zucc or Fructus Fragariae Ananssae is significant.
Summary of the invention
It is an object of the invention to provide a kind of liquid complex biological preservative.
It is a further object to provide the preparation method of a kind of liquid complex biological preservative.
It is also another object of the present invention to provide a kind of liquid complex biological preservative in Nanguo Pear, wild actinidia arguta
Or the application on Fructus Fragariae Ananssae.
The microbial bacteria that the present invention uses is China General Microbiological culture presevation administrative center 1997,2003 and 2012
Strain disclosed in the strain catalogue of version, numbers and bacterial strain name is:
Bacterium numbering be CGMCC4.1114 streptomyces hygroscopicus (stretomyces hygroscopicus), bacterium numbering is
The streptomyces microflavus of CGMCC4.891 (Streptomyces microflavus), bacterium numbering is that the gold of CGMCC4.909 is chromogenic
Streptomycete (Streptomyces aureochromogenes), bacterium numbering is the streptococcus acidi lactici of CGMCC1.18
(Lactococcus lactis), bacterium numbering be CGMCC1.884 bacillus subtilis (Bacillus subtilis), bacterium
The Bacillus licheniformis of kind of numbered CGMCC1.1216 (Bacillus licheniformis), bacterium numbering is
The cereuisiae fermentum of CGMCC2.1528 (Saccharomyces cerevisiae), bacterium numbering is the pale purple of CGMCC3.4035
Paecilomycerol (Paecilomyces lilacinus Samson), bacterium numbering is the trichoderma harzianum of CGMCC 3.6635
(Trichoderma harzianum Rifai)。
According to liquid complex biological preservative of the present invention, it is characterised in that it is by following weight
Raw material forms:
Chitosan 5～25 parts, epsilon-polylysine 5～25 parts, lysozyme 5～25 parts, the bacterium solution 5 of streptomyces hygroscopicus～20 parts, thin
The bacterium solution 5 of yellow streptomycete～20 parts, the bacterium solution 5 of gold streptomyces chromogenes～20 parts, the bacterium solution 5 of streptococcus acidi lactici～20 parts, hay
The bacterium solution 5 of bacillus cereus～20 parts, the bacterium solution 5 of Bacillus licheniformis～20 parts, the bacterium solution 5 of cereuisiae fermentum～20 parts, pale purple
The bacterium solution 5 of paecilomycerol～20 parts, the bacterium solution 5 of trichoderma harzianum～20 parts.
According to liquid complex biological preservative of the present invention, it is characterised in that it is by following weight
Raw material forms:
Chitosan 7～20 parts, epsilon-polylysine 7～20 parts, lysozyme 7～20 parts, the bacterium solution 5 of streptomyces hygroscopicus～15 parts, thin
The bacterium solution 5 of yellow streptomycete～15 parts, the bacterium solution 5 of gold streptomyces chromogenes～15 parts, the bacterium solution 5 of streptococcus acidi lactici～15 parts, hay
The bacterium solution 5 of bacillus cereus～15 parts, the bacterium solution 5 of Bacillus licheniformis～15 parts, the bacterium solution 5 of cereuisiae fermentum～15 parts, pale purple
The bacterium solution 5 of paecilomycerol～15 parts, the bacterium solution 5 of trichoderma harzianum～15 parts.
According to liquid complex biological preservative of the present invention, it is characterised in that it is by following weight
Raw material forms:
Chitosan 10 parts, epsilon-polylysine 10 parts, lysozyme 10 parts, the bacterium solution 7.5 parts of streptomyces hygroscopicus, the bacterium of streptomyces microflavus
Liquid 7.5 parts, gold the bacterium solution 15 parts of streptomyces chromogenes, the bacterium solution 15 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of clothing bacillus cereus 10 parts, the bacterium solution 7.5 parts of cereuisiae fermentum, the bacterium solution 7.5 parts of Paecilomyces lilacinus, trichoderma harzianum
Bacterium solution 7.5 parts.
According to liquid complex biological preservative of the present invention, it is characterised in that it is by following weight
Raw material forms:
Chitosan 20 parts, epsilon-polylysine 7.5 parts, lysozyme 7.5 parts, the bacterium solution 10 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 10 parts, gold the bacterium solution 10 parts of streptomyces chromogenes, the bacterium solution 10 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 10 parts, the bacterium solution 6 parts of cereuisiae fermentum, the bacterium solution 6 parts of Paecilomyces lilacinus, trichoderma harzianum
Bacterium solution 6 parts.
According to liquid complex biological preservative of the present invention, it is characterised in that it is by following weight
Raw material forms:
Chitosan 7.5 parts, epsilon-polylysine 20 parts, lysozyme 7.5 parts, the bacterium solution 10 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 10 parts, gold the bacterium solution 10 parts of streptomyces chromogenes, the bacterium solution 10 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 10 parts, the bacterium solution 5 parts of cereuisiae fermentum, the bacterium solution 5 parts of Paecilomyces lilacinus, trichoderma harzianum
Bacterium solution 5 parts.
According to liquid complex biological preservative of the present invention, it is characterised in that it is by following weight
Raw material forms:
Chitosan 7.5 parts, epsilon-polylysine 7.5 parts, lysozyme 20 parts, the bacterium solution 10 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 10 parts, gold the bacterium solution 10 parts of streptomyces chromogenes, the bacterium solution 10 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 10 parts, the bacterium solution 7.5 parts of cereuisiae fermentum, the bacterium solution 7.5 parts of Paecilomyces lilacinus, Trichoderma harzianum
The bacterium solution of bacterium 7.5 parts.
According to liquid complex biological preservative of the present invention, it is characterised in that it is by following weight
Raw material forms:
Chitosan 7.5 parts, epsilon-polylysine 7.5 parts, lysozyme 7.5 parts, the bacterium solution 15 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 15 parts, gold the bacterium solution 15 parts of streptomyces chromogenes, the bacterium solution 15 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 10 parts, the bacterium solution 7.5 parts of cereuisiae fermentum, the bacterium solution 7.5 parts of Paecilomyces lilacinus, Trichoderma harzianum
The bacterium solution of bacterium 7.5 parts.
According to liquid complex biological preservative of the present invention, it is characterised in that it is by following weight
Raw material forms:
Chitosan 7.5 parts, epsilon-polylysine 7.5 parts, lysozyme 7.5 parts, the bacterium solution 10 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 10 parts, gold the bacterium solution 10 parts of streptomyces chromogenes, the bacterium solution 10 parts of streptococcus acidi lactici, the bacterium solution 15 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 15 parts, the bacterium solution 7.5 parts of cereuisiae fermentum, the bacterium solution 7.5 parts of Paecilomyces lilacinus, Trichoderma harzianum
The bacterium solution of bacterium 7.5 parts.
A kind of method preparing liquid complex biological preservative as above, it is characterised in that following steps:
A, chitosan, epsilon-polylysine, the preparation method of lysozyme:
A, the preparation method of chitosan:
Weigh Crusta Penaeus seu Panulirus 25 grams, add the hydrochloric acid solution 250 milliliters of 1 mol/L, stirring, stand 12 hours, filter, discard filtrate；Add
5% sodium hydroxide solution, to solid-to-liquid ratio 1:8, stirring, water proof boils 1 hour, filters, obtains chitin, dries, and pulverizes, standby；
Take 5 grams of chitin powder in there-necked flask, add 45% sodium hydroxide solution 100 milliliters, little in 110～120 DEG C of stirring reactions 1
Time, cooling, centrifugal, remove supernatant, water washing and precipitating, then be centrifuged, then remove supernatant, water washing and precipitating, then remove supernatant, with
95% washing with alcohol precipitation, pours in bottle,suction, and sucking filtration stays filter cake, prepares chitosan, dry, standby heavy；
B, the preparation method of epsilon-polylysine:
The streptomyces albus (Streptomyces albulus) that bacterium numbering is 4.121 is inoculated in containing 5% glycerol, 1% sulfur
Acid ammonium, 0.5% yeast extract, 0.05% Magnesium sulfate heptahydrate, 0.003% ferrous sulfate heptahydrate, 0.004% zinc sulphate heptahydrate, pH is 6.8
Fluid medium in, at a temperature of 30 DEG C, cultivate 48 hours continuously, obtain the epsilon-polylysine that content is 0.38 grams per liter, standby
With；
C, the preparation method of lysozyme:
The preparation of Ovum Gallus domesticus album: the egg two ends that 4～5 fresh are respectively struck a duck eye, makes Ovum Gallus domesticus album flow out, tune Ovum Gallus domesticus album pH value is
8, it is gently mixed 5 minutes so that it is homogenous consistency, uses two-layer filtered through gauze, standby；
Ovum Gallus domesticus album crude product separates: the Ovum Gallus domesticus album of above-mentioned filtration measures 100 milliliters and is slowly stirred while add isopyknic deionization
Water, uniformly after adjust pH value to 7 at the hydrochloric acid solution being stirred continuously lower 1 mol/L, filter with absorbent cotton, collect filtrate, prepared
Egg white solution, standby；
D152 Macroporous weak acid cation exchange resin chromatographs:
1) D152 resin treatment: first with distilled water, D152 resin is washed away foreign material, leaches, with 1 mol/L sodium hydroxide solution
Soak, and stir 4～8 hours, sucking filtration, be washed till pH7.5, sucking filtration with distilled water, then press upper with the hydrochloric acid solution of 1 mol/L
Stating the method that 1 mol/L sodium hydroxide solution processes resin, until being all transformed into Hydrogen, sucking filtration, being washed till with distilled water
PH5.5, keeps overnight, sucking filtration, with 2 mol/L sodium hydroxide solutions by the method for above-mentioned process resin, is allowed to be changed into sodium
Type, pH value to 6.5, blots solution, adds the phosphate buffer balance resin of 0.02 mol/L of pH6.5, prepare resin and suspend
Liquid；
2) dress post: 1.6 centimetres of cut-off footpath, the chromatographic column of a length of 30 centimetres, injects treated resin suspension, closes from top
Close chromatographic column outlet, after resin settles, release the solution of excess, add resin suspension, to pitch deposition to 15～20
Cm height, continuously adds the 0.02 mol/L phosphate buffer balance resin of pH6.5, makes effluent in chromatographic column top
Till pH is 6.5, close chromatography column outlet, keep liquid level to exceed resin surface 1 centimetre；
3) upper prop absorption: above-mentioned egg white solution is added to resin top, opens outlet and make it flow slowly in post, flow velocity is 1 milli
Liter/min；
4) eluting: with the phosphate buffer eluting foreign protein of 0.02 mol/L of pH6.5, during collecting eluent,
By the elution profile of effective ultraviolet spectrophotometer inspection foreign protein, after baseline starts to walk and puts down, use instead containing 1.0 mol/L
The concentration of the pH value 6.5 of sodium chloride is 0.02 mol/L sodium phosphate buffer eluting, collects eluent；
5) Polyethylene Glycol concentrates: is merged by above-mentioned eluent and loads in bag filter, puts in container, and outside, coated with Polyethylene Glycol, holds
Device is added a cover, and the Polyethylene Glycol that the water in enzyme liquid is dialyzed outside film is absorbed, and when being concentrated to 5 milliliters, washes away dialysis with distilled water
Polyethylene Glycol outside film, takes out concentrated solution；
6) dialysis desalination: distilled water dialysis desalination 24 hours, prepares Ovum Gallus domesticus album crude product；
Sephadex G50 molecular sieve column chromatography:
1) dress post: first the Sephadex G50 chromatographic column sucking filtration preserved with 20% ethanol is removed ethanol, with 6 grams per liter sodium chloride
Solution stirring Sephadex G50 several minutes, then sucking filtration, repeated multiple times till without alcohol taste, add colloid amass 6 grams of 1/4/
Rise sodium chloride solution, be sufficiently stirred for, ultrasonic removing bubble, load the glass chromatography column of 1.6 × 50 centimetres, post bed 45 centimetres；
2) loading: the Ovum Gallus domesticus album crude product of above-mentioned preparation is added chromatographic column；
3) eluting: after sample stream is complete, adds the sodium chloride solution 50 milliliters of 6 grams per liters, the sample on eluting post jamb the most by several times, connects
Connecing constant flow pump, making flow velocity is 0.5 ml/min, collects with fraction collector, every 10 minutes one pipes；
4) Polyethylene Glycol concentrates: merges Peak Activity solution, when being concentrated into 5 milliliters with Polyethylene Glycol, washes away dialyzer with distilled water
Outer Polyethylene Glycol, takes out concentrated solution；
5) dialysis desalination: distilled water dialysis desalination 24 hours, collects dialysis solution, measures volume, prepare lysozyme, standby；
B, the preparation of composite bacteria agent capable:
Prepare the bacterium solution of described streptomyces hygroscopicus, the bacterium solution of streptomyces microflavus, the fermentation process of bacterium solution of gold streptomyces chromogenes:
A, slant strains activation culture: take Gause I synthetic fluid culture medium, access the slant culture of a kind of mentioned microorganism
Thing, under the conditions of 27～28 DEG C on shaking table shaken cultivation 96～120 hours, speed of agitator is 60～120 revs/min, then transfers
Cultivating in identical culture medium, method is transferred 3～5 times continuously like this, is applied to flat board solid after being diluted by the culture fluid of acquisition
In body culture medium, cultivating 4～5 days under the conditions of 27～28 DEG C, after growing bacterium colony, picking macrocolony is at same solid plate
Ruling in culture medium, picking individual colonies is transferred to solid slant culture base, cultivates and obtains naturalized strain, saves backup；B, two grades
Shaking flask spawn culture: take Gause I synthetic fluid culture medium, accesses primary inclined plane strain, cultivate at 27～28 DEG C 96～
120 hours, speed of agitator was 60～120 revs/min, obtained second-level shake flask seed liquor；According to production scale, use seed tank further
Carry out the amplification culture of seed；
C, the fermenting and producing of microbial inoculum: take Gause I synthetic fluid culture medium, access the fermentation seed liquid of above-mentioned preparation,
In pH7.2～7.4, temperature 27～28 DEG C, ferment 4～5 days, to obtain final product, can be standby as producing bacterium solution；
Prepare the fermentation process of the bacterium solution of the bacterium solution of described cereuisiae fermentum, the bacterium solution of Paecilomyces lilacinus, trichoderma harzianum:
A, slant strains activation culture: take PDA fluid medium, access the slant culture of a kind of mentioned microorganism, 25～28
Under the conditions of DEG C on shaking table shaken cultivation 48～72 hours, speed of agitator is 60～120 revs/min, is then transferred to identical training
Supporting in base and cultivate, method is transferred 3～5 times continuously like this, is applied on Solid media for plates after being diluted by the culture fluid of acquisition,
Cultivating 3～5 days under the conditions of 25～28 DEG C, rotating speed is 60～120 revs/min, and after growing sturdy mycelia, the sturdy mycelia of picking is same
Rule in the solid plate culture medium of sample, select sturdy mycelia and be transferred to solid slant culture base, cultivate and obtain naturalized strain, protect
Deposit standby；
B, second-level shake flask spawn culture: take PDA fluid medium, access primary inclined plane strain, cultivate at 25～28 DEG C 48～
72 hours, speed of agitator was 60～120 revs/min, obtained second-level shake flask seed liquor；According to production scale, use seed tank further
Carry out the amplification culture of seed；
C, the fermenting and producing of microbial inoculum: take PDA fluid medium, access the fermentation seed liquid of above-mentioned preparation, in pH7.0～
7.2, temperature 25～28 DEG C ferment 3～5 days, to obtain final product, and as producing, bacterium solution is standby；
Prepare the fermentation process of the bacterium solution of the bacterium solution of described streptococcus acidi lactici, the bacterium solution of bacillus subtilis, Bacillus licheniformis:
A, slant strains activation culture: take beef extract-peptone fluid medium, access the slant culture of a kind of mentioned microorganism
Thing, under the conditions of 30～40 DEG C on shaking table shaken cultivation 20～48 hours, rotating speed is 60～120 revs/min, is then transferred to phase
Cultivating in same culture medium, method is transferred 3～5 times continuously like this, is applied to flat-plate solid training after being diluted by the culture fluid of acquisition
Supporting on base, cultivate 1～2 day under the conditions of 30～40 DEG C, rotating speed is 60～120 revs/min, and after growing bacterium colony, picking macrocolony exists
Ruling in same solid plate culture medium, picking individual colonies is transferred to solid slant culture base, cultivates and obtains naturalized strain, protects
Deposit standby；
B, second-level shake flask spawn culture: take beef extract-peptone fluid medium, access primary inclined plane strain, at 30～40 DEG C
Cultivating 20～48 hours, rotating speed is 60～120 revs/min, obtains second-level shake flask seed liquor；According to production scale, use kind further
Sub-tank carries out the amplification culture of seed；
C, the fermenting and producing of microbial inoculum: take beef extract-peptone fluid medium, access the fermentation seed liquid of above-mentioned preparation,
In pH7.2～8.5, temperature 30～40 DEG C, fermenting 1～2 day, to obtain final product, as producing, bacterium solution is standby；
C, the preparation of liquid complex biological preservative:
According to the parts by weight of raw materials proportioning of above-mentioned composite bacteria agent capable, the various bacterium solution above-mentioned step B prepared mix, make compound bacteria
Agent, through inspection, make to reach 200,000,000/milliliter containing total living bacteria count, the quantity of each effective bacterium must not less than 20,000,000/in the least
More than Shenging, meet standard GB/T-20287-2006, according still further to the weight of raw material components as above, by above-mentioned
Chitosan prepared by step, epsilon-polylysine, lysozyme are mixed homogeneously with composite bacteria agent capable, to obtain final product.
The purposes of a kind of liquid complex biological preservative as above, it is characterised in that it should as fruit antistaling agent
For Nanguo Pear, wild actinidia arguta or Fructus Fragariae Ananssae.
The raw material components feature that the present invention uses is as follows:
Bacillus subtilis can improve in pathogenic bacteria and adverse circumstance are endangered primosome by crop the Scavenging activity producing oxygen, and regulation cell is micro-
Biotic environment, maintains the normal physiological metabolism of cell and biochemical reaction, improves the resistance of crop, and the health care increasing crop is made
With, plant growth can be promoted, improve yield.
Bacillus licheniformis cell form and arrangement, in shaft-like, Dan Sheng, can promote body to produce antibacterial substance, kill
Pathogenic bacterium.It can produce resistant activity material, and the biology with uniqueness takes oxygen mechanism of action by force, and the growth that can suppress pathogenic bacterium is numerous
Grow.
Streptomyces lactis can produce nisin, typically will not by the microbial hydrolysis in food, but can be by pepsin
Enzyme hydrolysis, so the meeting aminoacid being broken down into little molecule quickly after people is edible, harmless, meanwhile will not change
Normal clump count in human body, the resistance also will not intersected with commonly used other antibiotic generation, therefore, it is a kind of
Crude antistaling agent to human non-toxic.It is to gram positive bacterias such as Listerella, staphylococcus, sheet coccus, enterococcus, and
The sporiferous antibacterial such as bacillus, clostruidium all has preferable inhibition, is that the food of a kind of efficient green is protected
Fresh dose, extend fruit storage period 4～6 times.
Streptomyces microflavus produces T-1384, spiramycin, to Gram-positive and negative bacteria, yeast, thread very
Bacterium has inhibitory action.The upper diseases prevention that is often used in agriculture is kept a full stand of seedings, and can make bacterial manure simultaneously, can convert Nitrogen In Soils P elements and improve soil
Fertility, and have the effect stimulating plant growth.
Streptomyces hygroscopicus produces suppression gram-positive bacterium, mycobacteria, the hygromycin (hygromycin) of protozoon,
Suppression mycobacteria, virus, tumor, filamentous fungi, the hygroscopin (hygroscopin) of yeast, suppression Gram-positive is thin
Bacterium, filamentous fungi, the Multiple Classes of Antibiotics such as hygrostatin (hygrostatin) of yeast.
Gold streptomyces chromogenes suppression gram-positive bacterium, pan penicillium sp.Produce antifungal antibiotic；To trichosporon also
Having effect, Antagonism is good, suppresses gram-positive bacterium, root nodule bacteria；Weak suppression gram negative bacteria, high inhibition is multiple thread
Fungus and some yeast and some streptomycetes.
The secondary metabolite that trichoderma harzianum produces has fungistatic effect to various agricultural pathogenic bacterium.
Cereuisiae fermentum is to Pb2+Adsorbance increase with the increase of pH value, reach absorbance maximum as pH=6.
Paecilomyces lilacinus belongs to endoparasitism fungus, is the Important Natural Enemy of some plant nematodes, it is possible to parasitize
In worm's ovum, also can infect larva and female worm, can substantially alleviate the plant lines such as various crop root-knot nematode, Cyst nematode, Ditylenchus dipsaci
The harm of parasitosis.
Chitosan can not be adsorbed to bacterium surface in the case of cell wall contains abundant teichoic acid, therefore,
For this bacterioid, chitosan can upset its metabolism with the anion generation flocculation in Cytoplasm intracellular
Process, makes the macromolecular components such as intracellular protein and glucose leak, thus reaches the purpose of sterilization.Chitosan leads to
Cross and form thin film on fresh food top layer, it is possible to slow down the loss of food water and nutritional labeling, with the oxygen in air
Isolate with causing rotten microorganism, thus food is difficult to putrid and deteriorated.
Epsilon-polylysine, when pH ＜ 7, can suppress major part antibacterial and the normal growth of fungus, to its better resistance as
The gram negative bacteria rejection ability such as escherichia coli, Salmonella is the most pretty good, and can kill thermostability bacillus cereus
With some virus.
Lysozyme (Lysozyme) is alkaline globulin, the most more stable.It can act on the thin of cell
After birth, it is possible to the glycoprotein on hydrolysis film, thus cause the cell can not normal growth.To gram positive bacteria, lysozyme can
There is extraordinary inhibition.
The liquid complex biological preservative of the present invention is various bacterium solution to be prepared by described material rate mixture, can be well
Keep each Microflora ratio in composite bacteria agent capable the most stable.Composite bacteria agent capable carries out, with relying on natural environment, the microorganism decomposed
Flora and compared with the flora of the mixture without single culture, after using single culture, then the flora that mixture is together, due to can
To select rational proportioning according to actual needs, the biological nature of each flora self in microbial inoculum can be played fully collaborative with mutual
Effect, so, carry out the scope of application carried out a biological disposal upon widely with it.
The liquid complex biological preservative of the present invention is a kind of efficient, nontoxic, health-nutrition, fruit to food safety is protected
Fresh dose, especially notable to Nanguo Pear, Actinidia arguta Sieb.et Zucc or strawberry preservation effect.
Detailed description of the invention
Following embodiment is only used for explaining the present invention, is not intended that the restriction to right,
Those skilled in the art according to description it is contemplated that other alternative means, all should be in the protection of the claims in the present invention
Within the scope of.
The microorganism fungus kind that the embodiment of the present invention uses, select China General Microbiological culture presevation administrative center 1997,
2003, the strain disclosed in the strain catalogue of version in 2012, numbers and bacterial strain name is:
Bacterium numbering be CGMCC4.1114 streptomyces hygroscopicus (stretomyces hygroscopicus), bacterium numbering is
The streptomyces microflavus of CGMCC4.891 (Streptomyces microflavus), bacterium numbering is that the gold of CGMCC4.909 is chromogenic
Streptomycete (Streptomyces aureochromogenes), bacterium numbering is the streptococcus acidi lactici of CGMCC1.18
(Lactococcus lactis), bacterium numbering be CGMCC1.884 bacillus subtilis (Bacillus subtilis), bacterium
The Bacillus licheniformis of kind of numbered CGMCC1.1216 (Bacillus licheniformis), bacterium numbering is
The cereuisiae fermentum of CGMCC2.1528 (Saccharomyces cerevisiae), bacterium numbering is the pale purple of CGMCC3.4035
Paecilomycerol (Paecilomyces lilacinus Samson), bacterium numbering is the trichoderma harzianum of CGMCC 3.6635
(Trichoderma harzianum Rifai)。
The liquid complex biological preservative preparation method of embodiment is following steps:
A, chitosan, epsilon-polylysine, the preparation method of lysozyme:
A, the preparation method of chitosan:
Weigh Crusta Penaeus seu Panulirus 25 grams, add the hydrochloric acid solution 250 milliliters of 1 mol/L, stirring, stand 12 hours, filter, discard filtrate；Add
Entering 5% sodium hydroxide solution, to solid-to-liquid ratio 1:8, stirring, water proof boils 1 hour, filters, obtains chitin, dries, and pulverizes, stand-by；
Take 5 grams of chitin powder in there-necked flask, add 45% sodium hydroxide solution 100 milliliters, in 115 DEG C of stirring reactions 1 hour, cold
But, centrifugal, remove supernatant, water washing and precipitating, then be centrifuged, then remove supernatant, water washing and precipitating, then remove supernatant, with 95% second
Alcohol washing precipitation, pours in bottle,suction, and sucking filtration stays filter cake, prepares chitosan, dry, standby；
B, the preparation method of epsilon-polylysine:
The streptomyces albus (Streptomyces albulus) that bacterium numbering is 4.121 is inoculated in containing 5% glycerol, 1% sulfur
Acid ammonium, 0.5% yeast extract, 0.05% Magnesium sulfate heptahydrate, 0.003% ferrous sulfate heptahydrate, 0.004% zinc sulphate heptahydrate, pH is 6.8
Fluid medium in, at a temperature of 30 DEG C, cultivate 48 hours continuously, prepared content is the epsilon-polylysine of 0.38 grams per liter, standby
With；
C, the preparation method of lysozyme:
The preparation of Ovum Gallus domesticus album: the egg two ends that 4～5 fresh are respectively struck a duck eye, makes Ovum Gallus domesticus album flow out, tune Ovum Gallus domesticus album pH value is
8, it is gently mixed 5 minutes so that it is homogenous consistency, uses two-layer filtered through gauze, standby；
Ovum Gallus domesticus album crude product separates: the Ovum Gallus domesticus album of above-mentioned filtration measures 100 milliliters and is slowly stirred while add isopyknic deionization
Water, uniformly after adjust pH value to 7 at the hydrochloric acid solution being stirred continuously lower 1 mol/L, filter with absorbent cotton, collect filtrate, prepared
Egg white solution, standby；
D152 Macroporous weak acid cation exchange resin chromatographs:
1) D152 resin treatment: first with distilled water, D152 resin is washed away foreign material, leaches, with 1 mol/L sodium hydroxide solution
Soak, and stir 6 hours, sucking filtration, be washed till pH7.5, sucking filtration, then the hydrochloric acid solution by 1 mol/L with distilled water, by above-mentioned
1 mol/L sodium hydroxide solution processes the method for resin, until being all transformed into Hydrogen, sucking filtration, is washed till with distilled water
PH5.5, keeps overnight, sucking filtration, then with 2 mol/L sodium hydroxide solutions by the method for above-mentioned process resin, is allowed to be changed into sodium
Type, pH value to 6.5, blots solution, adds the phosphate buffer balance resin of 0.02 mol/L of pH6.5, prepare resin and suspend
Liquid；
2) dress post: 1.6 centimetres of cut-off footpath, the chromatographic column of a length of 30 centimetres, injects treated resin suspension, closes from top
Close chromatographic column outlet, after resin settles, release the solution of excess, add resin suspension, to pitch deposition to 20 centimetres
Highly, continuously add the 0.02 mol/L phosphate buffer balance resin of pH6.5 in chromatographic column top, make the effluent pH be
Till 6.5, close chromatography column outlet, keep liquid level to exceed resin surface 1 centimetre；
3) upper prop absorption: above-mentioned egg white solution is added to resin top, opens outlet and make it flow slowly in post, flow velocity is 1 milli
Liter/min；
4) eluting: with the phosphate buffer eluting foreign protein of 0.02 mol/L of pH6.5, during collecting eluent,
By the elution profile of effective ultraviolet spectrophotometer inspection foreign protein, after baseline starts to walk and puts down, use instead containing 1.0 mol/L
The concentration of the pH value 6.5 of sodium chloride is 0.02 mol/L sodium phosphate buffer eluting, collects eluent；
5) Polyethylene Glycol concentrates: is merged by above-mentioned eluent and loads in bag filter, puts in container, and outside, coated with Polyethylene Glycol, holds
Device is added a cover, and the Polyethylene Glycol that the water in enzyme liquid is dialyzed outside film is absorbed, and when being concentrated to 5 milliliters, washes away dialysis with distilled water
Polyethylene Glycol outside film, takes out concentrated solution；
6) dialysis desalination: distilled water dialysis desalination 24 hours, prepares Ovum Gallus domesticus album crude product；
Sephadex G50 molecular sieve column chromatography:
1) dress post: first the Sephadex G50 chromatographic column sucking filtration preserved with 20% ethanol is removed ethanol, with 6 grams per liter sodium chloride
Solution stirring Sephadex G50 several minutes, then sucking filtration, repeated multiple times till without alcohol taste, add colloid amass 6 grams of 1/4/
Rise sodium chloride solution, be sufficiently stirred for, ultrasonic removing bubble, load the glass chromatography column of 1.6 × 50 centimetres, post bed 45 centimetres；
2) loading: the Ovum Gallus domesticus album crude product of above-mentioned preparation is added chromatographic column；
3) eluting: after sample stream is complete, adds the sodium chloride solution 50 milliliters of 6 grams per liters, the sample on eluting post jamb the most by several times, connects
Connecing constant flow pump, making flow velocity is 0.5 ml/min, collects with fraction collector, every 10 minutes one pipes；
4) Polyethylene Glycol concentrates: merges Peak Activity solution, when being concentrated into 5 milliliters with Polyethylene Glycol, washes away dialyzer with distilled water
Outer Polyethylene Glycol, takes out concentrated solution；
5) dialysis desalination: distilled water dialysis desalination 24 hours, collects dialysis solution, measures volume, prepare lysozyme, standby；
B, the preparation of composite bacteria agent capable:
Prepare the bacterium solution of described streptomyces hygroscopicus, the bacterium solution of streptomyces microflavus, the fermentation process of bacterium solution of gold streptomyces chromogenes:
A, slant strains activation culture: take Gause I synthetic fluid culture medium, access the slant culture of a kind of mentioned microorganism
Thing, under the conditions of 27～28 DEG C on shaking table shaken cultivation 100 hours, speed of agitator is 100 revs/min, is then transferred to identical
Cultivating in culture medium, method is transferred 3～5 times continuously like this, is applied to Solid media for plates after being diluted by the culture fluid of acquisition
On, cultivate 4～5 days under the conditions of 27～28 DEG C, after growing bacterium colony, picking macrocolony is in same solid plate culture medium
Line, picking individual colonies is transferred to solid slant culture base, cultivates and obtains naturalized strain, saves backup；B, second-level shake flask strain
Cultivating: take Gause I synthetic fluid culture medium, access primary inclined plane strain, cultivate 100 hours at 27～28 DEG C, stirring turns
Speed is 100 revs/min, obtains second-level shake flask seed liquor；According to production scale, seed tank is used to carry out the amplification training of seed further
Support；
C, the fermenting and producing of microbial inoculum: take Gause I synthetic fluid culture medium, access the fermentation seed liquid of above-mentioned preparation,
In pH7.2～7.4, temperature 27～28 DEG C, ferment 4～5 days, to obtain final product, can be standby as producing bacterium solution；
Prepare the fermentation process of the bacterium solution of the bacterium solution of described cereuisiae fermentum, the bacterium solution of Paecilomyces lilacinus, trichoderma harzianum:
A, slant strains activation culture: take PDA fluid medium, access the slant culture of a kind of mentioned microorganism, 26～27
Under the conditions of DEG C on shaking table shaken cultivation 60 hours, speed of agitator is 100 revs/min, be then transferred in identical culture medium training
Supporting, method is transferred 3～5 times continuously like this, is applied on Solid media for plates, 26～27 DEG C after being diluted by the culture fluid of acquisition
Under the conditions of cultivate 3～5 days, rotating speed is 100 revs/min, and after growing sturdy mycelia, the sturdy mycelia of picking is at same solid plate
Rule in culture medium, select sturdy mycelia and be transferred to solid slant culture base, cultivate and obtain naturalized strain, save backup；
B, second-level shake flask spawn culture: take PDA fluid medium, access primary inclined plane strain, cultivates 60 little at 26～27 DEG C
Time, speed of agitator is 100 revs/min, obtains second-level shake flask seed liquor；According to production scale, seed tank is used to carry out seed further
Amplification culture；
C, the fermenting and producing of microbial inoculum: take PDA fluid medium, access the fermentation seed liquid of above-mentioned preparation, in pH7.0～
7.2, temperature 26～27 DEG C ferment 3～5 days, to obtain final product, and as producing, bacterium solution is standby；
Prepare the fermentation process of the bacterium solution of the bacterium solution of described streptococcus acidi lactici, the bacterium solution of bacillus subtilis, Bacillus licheniformis:
A, slant strains activation culture: take beef extract-peptone fluid medium, access the slant culture of a kind of mentioned microorganism
Thing, under the conditions of 35 DEG C on shaking table shaken cultivation 35 hours, rotating speed is 100 revs/min, is then transferred in identical culture medium
Cultivating, method is transferred 3～5 times continuously like this, is applied on Solid media for plates, 35 DEG C after being diluted by the culture fluid of acquisition
Under the conditions of cultivate 2 days, rotating speed is 100 revs/min, and after growing bacterium colony, picking macrocolony is in same solid plate culture medium
Line, picking individual colonies is transferred to solid slant culture base, cultivates and obtains naturalized strain, saves backup；
B, second-level shake flask spawn culture: take beef extract-peptone fluid medium, access primary inclined plane strain, cultivates at 35 DEG C
35 hours, rotating speed was 100 revs/min, obtained second-level shake flask seed liquor；According to production scale, seed tank is used to carry out seed further
Amplification culture；
C, the fermenting and producing of microbial inoculum: take beef extract-peptone fluid medium, access the fermentation seed liquid of above-mentioned preparation,
In pH7.2～8.5, temperature 35 DEG C, fermenting 2 days, to obtain final product, as producing, bacterium solution is standby；
C, the preparation of liquid complex biological preservative:
According to the parts by weight of raw materials proportioning of composite bacteria agent capable described in examples below, the various bacterium solution above-mentioned step B prepared are mixed
Closing, make composite bacteria agent capable, through inspection, make to reach 200,000,000/milliliter containing total living bacteria count, the quantity of each effective bacterium must not be lacked
More than 20,000,000/milliliter, meet standard GB/T-20287-2006, according still further to the raw material group described in examples below
The weight divided, chitosan above-mentioned steps prepared, epsilon-polylysine, lysozyme are mixed homogeneously with composite bacteria agent capable, i.e.
?.
Embodiment 1: be made up of the raw material of following weight:
Chitosan 10 parts, epsilon-polylysine 10 parts, lysozyme 10 parts, the bacterium solution 7.5 parts of streptomyces hygroscopicus, the bacterium of streptomyces microflavus
Liquid 7.5 parts, gold the bacterium solution 15 parts of streptomyces chromogenes, the bacterium solution 15 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of clothing bacillus cereus 10 parts, the bacterium solution 7.5 parts of cereuisiae fermentum, the bacterium solution 7.5 parts of Paecilomyces lilacinus, trichoderma harzianum
Bacterium solution 7.5 parts.
Embodiment 2: be made up of the raw material of following weight:
Chitosan 20 parts, epsilon-polylysine 7.5 parts, lysozyme 7.5 parts, the bacterium solution 10 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 10 parts, gold the bacterium solution 10 parts of streptomyces chromogenes, the bacterium solution 10 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 10 parts, the bacterium solution 6 parts of cereuisiae fermentum, the bacterium solution 6 parts of Paecilomyces lilacinus, trichoderma harzianum
Bacterium solution 6 parts.
Embodiment 3: be made up of the raw material of following weight:
Chitosan 7.5 parts, epsilon-polylysine 20 parts, lysozyme 7.5 parts, the bacterium solution 10 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 10 parts, gold the bacterium solution 10 parts of streptomyces chromogenes, the bacterium solution 10 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 10 parts, the bacterium solution 5 parts of cereuisiae fermentum, the bacterium solution 5 parts of Paecilomyces lilacinus, trichoderma harzianum
Bacterium solution 5 parts.
Embodiment 4: be made up of the raw material of following weight:
Chitosan 7.5 parts, epsilon-polylysine 7.5 parts, lysozyme 20 parts, the bacterium solution 10 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 10 parts, gold the bacterium solution 10 parts of streptomyces chromogenes, the bacterium solution 10 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 10 parts, the bacterium solution 7.5 parts of cereuisiae fermentum, the bacterium solution 7.5 parts of Paecilomyces lilacinus, Trichoderma harzianum
The bacterium solution of bacterium 7.5 parts.
Embodiment 5: be made up of the raw material of following weight:
Chitosan 7.5 parts, epsilon-polylysine 7.5 parts, lysozyme 7.5 parts, the bacterium solution 15 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 15 parts, gold the bacterium solution 15 parts of streptomyces chromogenes, the bacterium solution 15 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 10 parts, the bacterium solution 7.5 parts of cereuisiae fermentum, the bacterium solution 7.5 parts of Paecilomyces lilacinus, Trichoderma harzianum
The bacterium solution of bacterium 7.5 parts.
Embodiment 6: be made up of the raw material of following weight:
Chitosan 7.5 parts, epsilon-polylysine 7.5 parts, lysozyme 7.5 parts, the bacterium solution 10 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 10 parts, gold the bacterium solution 10 parts of streptomyces chromogenes, the bacterium solution 10 parts of streptococcus acidi lactici, the bacterium solution 15 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 15 parts, the bacterium solution 7.5 parts of cereuisiae fermentum, the bacterium solution 7.5 parts of Paecilomyces lilacinus, Trichoderma harzianum
The bacterium solution of bacterium 7.5 parts.
The using method of the liquid complex biological preservative of the embodiment of the present invention and comparative test result:
Embodiment of the present invention liquid complex biological preservative is used to protect for the fruit of Nanguo Pear, Actinidia arguta Sieb.et Zucc or Fructus Fragariae Ananssae
Fresh, its using method is: after bio-preservative 12～15 times of dilutions, and dip-coating fruit 5 minutes is natural at a temperature of 20～25 DEG C
Draining, storage at normal temperature, result of the test is as follows by contrast:
The weight-loss ratio of Nanguo Pear, Actinidia arguta Sieb.et Zucc or Fructus Fragariae Ananssae does not uses group to be 3～4 times of bio-preservative use group, use group
Can improve Nanguo Pear, Actinidia arguta Sieb.et Zucc or the Fructus Fragariae Ananssae shelf life of nearly 15～18 days, explanation process group fresh-keeping effect is clearly.
Embodiment of the present invention liquid complex biological preservative 12～15 times are used to be diluted under variable concentrations, sample is whole
Immersing, take out, naturally drain at a temperature of 20～25 DEG C after 10 seconds, comparative test result is as follows:
The liquid complex biological preservative of the embodiment of the present invention is obvious to aspergillus niger fungistatic effect, antibacterial circle diameter 22mm with
On；Putrefying bacteria escherichia coli, staphylococcus aureus and Salmonella are all had obvious inhibitory action, and inhibition zone all reaches
More than 25mm.Through the lowest more treated than not having of polyphenol oxidase activity peak value that the antistaling agent of embodiment processes
20.6%～23.8%, thus stop the degree of fruit storage later stage fruit browning.
When being stored to 25 days, through embodiment liquid complex biological preservative process fruit mda content be 1.05 μ
Mol/g, and undressed mda content is 1.44 μm ol/g, contrast has marked difference, illustrates that antistaling agent process has
It is beneficial to keep the integrity of film, and then maintains the freshness of fruit.
The peroxidase from fruit processed through the liquid complex biological preservative of embodiment is 0.6U/mg, and unprocessed
Peroxidase be 0.5U/mg, antistaling agent improves the catalytic capability of enzyme, stops Fruit anatomy and aging.
The fruit catalase processed through the liquid complex biological preservative of embodiment is 16.5U/mg, and without place
The peroxidase of reason is 10.5U/mg, and antistaling agent improves the catalytic capability of peroxidase, stops the aging of fruit.
Claims (10)
1. a liquid complex biological preservative, it is characterised in that it is made up of the raw material of following weight:
Chitosan 5～25 parts, epsilon-polylysine 5～25 parts, lysozyme 5～25 parts, the bacterium solution 5 of streptomyces hygroscopicus～20 parts, thin
The bacterium solution 5 of yellow streptomycete～20 parts, the bacterium solution 5 of gold streptomyces chromogenes～20 parts, the bacterium solution 5 of streptococcus acidi lactici～20 parts, hay
The bacterium solution 5 of bacillus cereus～20 parts, the bacterium solution 5 of Bacillus licheniformis～20 parts, the bacterium solution 5 of cereuisiae fermentum～20 parts, pale purple
The bacterium solution 5 of paecilomycerol～20 parts, the bacterium solution 5 of trichoderma harzianum～20 parts.
Liquid complex biological preservative the most according to claim 1, it is characterised in that it is by following weight
Raw material forms:
Chitosan 7～20 parts, epsilon-polylysine 7～20 parts, lysozyme 7～20 parts, the bacterium solution 5 of streptomyces hygroscopicus～15 parts, thin
The bacterium solution 5 of yellow streptomycete～15 parts, the bacterium solution 5 of gold streptomyces chromogenes～15 parts, the bacterium solution 5 of streptococcus acidi lactici～15 parts, hay
The bacterium solution 5 of bacillus cereus～15 parts, the bacterium solution 5 of Bacillus licheniformis～15 parts, the bacterium solution 5 of cereuisiae fermentum～15 parts, pale purple
The bacterium solution 5 of paecilomycerol～15 parts, the bacterium solution 5 of trichoderma harzianum～15 parts.
Liquid complex biological preservative the most according to claim 1 and 2, it is characterised in that it is to be joined by following weight portion
The raw material composition of ratio:
Chitosan 10 parts, epsilon-polylysine 10 parts, lysozyme 10 parts, the bacterium solution 7.5 parts of streptomyces hygroscopicus, the bacterium of streptomyces microflavus
Liquid 7.5 parts, gold the bacterium solution 15 parts of streptomyces chromogenes, the bacterium solution 15 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of clothing bacillus cereus 10 parts, the bacterium solution 7.5 parts of cereuisiae fermentum, the bacterium solution 7.5 parts of Paecilomyces lilacinus, trichoderma harzianum
Bacterium solution 7.5 parts.
Liquid complex biological preservative the most according to claim 1 and 2, it is characterised in that it is to be joined by following weight portion
The raw material composition of ratio:
Chitosan 20 parts, epsilon-polylysine 7.5 parts, lysozyme 7.5 parts, the bacterium solution 10 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 10 parts, gold the bacterium solution 10 parts of streptomyces chromogenes, the bacterium solution 10 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 10 parts, the bacterium solution 6 parts of cereuisiae fermentum, the bacterium solution 6 parts of Paecilomyces lilacinus, trichoderma harzianum
Bacterium solution 6 parts.
Liquid complex biological preservative the most according to claim 1 and 2, it is characterised in that it is to be joined by following weight portion
The raw material composition of ratio:
Chitosan 7.5 parts, epsilon-polylysine 20 parts, lysozyme 7.5 parts, the bacterium solution 10 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 10 parts, gold the bacterium solution 10 parts of streptomyces chromogenes, the bacterium solution 10 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 10 parts, the bacterium solution 5 parts of cereuisiae fermentum, the bacterium solution 5 parts of Paecilomyces lilacinus, trichoderma harzianum
Bacterium solution 5 parts.
Liquid complex biological preservative the most according to claim 1 and 2, it is characterised in that it is to be joined by following weight portion
The raw material composition of ratio:
Chitosan 7.5 parts, epsilon-polylysine 7.5 parts, lysozyme 20 parts, the bacterium solution 10 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 10 parts, gold the bacterium solution 10 parts of streptomyces chromogenes, the bacterium solution 10 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 10 parts, the bacterium solution 7.5 parts of cereuisiae fermentum, the bacterium solution 7.5 parts of Paecilomyces lilacinus, Trichoderma harzianum
The bacterium solution of bacterium 7.5 parts.
Liquid complex biological preservative the most according to claim 1 and 2, it is characterised in that it is to be joined by following weight portion
The raw material composition of ratio:
Chitosan 7.5 parts, epsilon-polylysine 7.5 parts, lysozyme 7.5 parts, the bacterium solution 15 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 15 parts, gold the bacterium solution 15 parts of streptomyces chromogenes, the bacterium solution 15 parts of streptococcus acidi lactici, the bacterium solution 10 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 10 parts, the bacterium solution 7.5 parts of cereuisiae fermentum, the bacterium solution 7.5 parts of Paecilomyces lilacinus, Trichoderma harzianum
The bacterium solution of bacterium 7.5 parts.
Liquid complex biological preservative the most according to claim 1 and 2, it is characterised in that it is to be joined by following weight portion
The raw material composition of ratio:
Chitosan 7.5 parts, epsilon-polylysine 7.5 parts, lysozyme 7.5 parts, the bacterium solution 10 parts of streptomyces hygroscopicus, streptomyces microflavus
Bacterium solution 10 parts, gold the bacterium solution 10 parts of streptomyces chromogenes, the bacterium solution 10 parts of streptococcus acidi lactici, the bacterium solution 15 parts of bacillus subtilis,
The bacterium solution of Bacillus licheniformis 15 parts, the bacterium solution 7.5 parts of cereuisiae fermentum, the bacterium solution 7.5 parts of Paecilomyces lilacinus, Trichoderma harzianum
The bacterium solution of bacterium 7.5 parts.
9. the preparation method of the liquid complex biological preservative described in a claim 1～8, it is characterised in that following steps:
A, chitosan, epsilon-polylysine, the preparation method of lysozyme:
A, the preparation method of chitosan:
Weigh Crusta Penaeus seu Panulirus 25 grams, add the hydrochloric acid solution 250 milliliters of 1 mol/L, stirring, stand 12 hours, filter, discard filtrate；Add
5% sodium hydroxide solution, to solid-to-liquid ratio 1:8, stirring, water proof boils 1 hour, filters, obtains chitin, dries, and pulverizes, standby；
Take 5 grams of chitin powder in there-necked flask, add 45% sodium hydroxide solution 100 milliliters, little in 110～120 DEG C of stirring reactions 1
Time, cooling, centrifugal, remove supernatant, water washing and precipitating, then be centrifuged, then remove supernatant, water washing and precipitating, then remove supernatant, with
95% washing with alcohol precipitation, pours in bottle,suction, and sucking filtration stays filter cake, prepares chitosan, dry, standby heavy；
B, the preparation method of epsilon-polylysine:
The streptomyces albus (Streptomyces albulus) that bacterium numbering is 4.121 is inoculated in containing 5% glycerol, 1% sulfur
Acid ammonium, 0.5% yeast extract, 0.05% Magnesium sulfate heptahydrate, 0.003% ferrous sulfate heptahydrate, 0.004% zinc sulphate heptahydrate, pH is 6.8
Fluid medium in, at a temperature of 30 DEG C, cultivate 48 hours continuously, obtain the epsilon-polylysine that content is 0.38 grams per liter, standby
With；
C, the preparation method of lysozyme:
The preparation of Ovum Gallus domesticus album: the egg two ends that 4～5 fresh are respectively struck a duck eye, makes Ovum Gallus domesticus album flow out, tune Ovum Gallus domesticus album pH value is
8, it is gently mixed 5 minutes so that it is homogenous consistency, uses two-layer filtered through gauze, standby；
Ovum Gallus domesticus album crude product separates: the Ovum Gallus domesticus album of above-mentioned filtration measures 100 milliliters and is slowly stirred while add isopyknic deionization
Water, uniformly after adjust pH value to 7 at the hydrochloric acid solution being stirred continuously lower 1 mol/L, filter with absorbent cotton, collect filtrate, prepared
Egg white solution, standby；
D152 Macroporous weak acid cation exchange resin chromatographs:
1) D152 resin treatment: first with distilled water, D152 resin is washed away foreign material, leaches, with 1 mol/L sodium hydroxide solution
Soak, and stir 4～8 hours, sucking filtration, be washed till pH7.5, sucking filtration with distilled water, then press upper with the hydrochloric acid solution of 1 mol/L
Stating the method that 1 mol/L sodium hydroxide solution processes resin, until being all transformed into Hydrogen, sucking filtration, being washed till with distilled water
PH5.5, keeps overnight, sucking filtration, with 2 mol/L sodium hydroxide solutions by the method for above-mentioned process resin, is allowed to be changed into sodium
Type, pH value to 6.5, blots solution, adds the phosphate buffer balance resin of 0.02 mol/L of pH6.5, prepare resin and suspend
Liquid；
2) dress post: 1.6 centimetres of cut-off footpath, the chromatographic column of a length of 30 centimetres, injects treated resin suspension, closes from top
Close chromatographic column outlet, after resin settles, release the solution of excess, add resin suspension, to pitch deposition to 15～20
Cm height, continuously adds the 0.02 mol/L phosphate buffer balance resin of pH6.5, makes effluent in chromatographic column top
Till pH is 6.5, close chromatography column outlet, keep liquid level to exceed resin surface 1 centimetre；
3) upper prop absorption: above-mentioned egg white solution is added to resin top, opens outlet and make it flow slowly in post, flow velocity is 1 milli
Liter/min；
4) eluting: with the phosphate buffer eluting foreign protein of 0.02 mol/L of pH6.5, during collecting eluent,
By the elution profile of effective ultraviolet spectrophotometer inspection foreign protein, after baseline starts to walk and puts down, use instead containing 1.0 mol/L
The concentration of the pH value 6.5 of sodium chloride is 0.02 mol/L sodium phosphate buffer eluting, collects eluent；
5) Polyethylene Glycol concentrates: is merged by above-mentioned eluent and loads in bag filter, puts in container, and outside, coated with Polyethylene Glycol, holds
Device is added a cover, and the Polyethylene Glycol that the water in enzyme liquid is dialyzed outside film is absorbed, and when being concentrated to 5 milliliters, washes away dialysis with distilled water
Polyethylene Glycol outside film, takes out concentrated solution；
6) dialysis desalination: distilled water dialysis desalination 24 hours, prepares Ovum Gallus domesticus album crude product；
Sephadex G50 molecular sieve column chromatography:
1) dress post: first the Sephadex G50 chromatographic column sucking filtration preserved with 20% ethanol is removed ethanol, with 6 grams per liter sodium chloride
Solution stirring Sephadex G50 several minutes, then sucking filtration, repeated multiple times till without alcohol taste, add colloid amass 6 grams of 1/4/
Rise sodium chloride solution, be sufficiently stirred for, ultrasonic removing bubble, load the glass chromatography column of 1.6 × 50 centimetres, post bed 45 centimetres；
2) loading: the Ovum Gallus domesticus album crude product of above-mentioned preparation is added chromatographic column；
3) eluting: after sample stream is complete, adds the sodium chloride solution 50 milliliters of 6 grams per liters, the sample on eluting post jamb the most by several times, connects
Connecing constant flow pump, making flow velocity is 0.5 ml/min, collects with fraction collector, every 10 minutes one pipes；
4) Polyethylene Glycol concentrates: merges Peak Activity solution, when being concentrated into 5 milliliters with Polyethylene Glycol, washes away dialyzer with distilled water
Outer Polyethylene Glycol, takes out concentrated solution；
5) dialysis desalination: distilled water dialysis desalination 24 hours, collects dialysis solution, measures volume, prepare lysozyme, standby；
B, the preparation of composite bacteria agent capable:
Prepare the bacterium solution of described streptomyces hygroscopicus, the bacterium solution of streptomyces microflavus, the fermentation process of bacterium solution of gold streptomyces chromogenes:
A, slant strains activation culture: take Gause I synthetic fluid culture medium, access the slant culture of a kind of mentioned microorganism
Thing, under the conditions of 27～28 DEG C on shaking table shaken cultivation 96～120 hours, speed of agitator is 60～120 revs/min, then transfers
Cultivating in identical culture medium, method is transferred 3～5 times continuously like this, is applied to flat board solid after being diluted by the culture fluid of acquisition
In body culture medium, cultivating 4～5 days under the conditions of 27～28 DEG C, after growing bacterium colony, picking macrocolony is at same solid plate
Ruling in culture medium, picking individual colonies is transferred to solid slant culture base, cultivates and obtains naturalized strain, saves backup；B, two grades
Shaking flask spawn culture: take Gause I synthetic fluid culture medium, accesses primary inclined plane strain, cultivate at 27～28 DEG C 96～
120 hours, speed of agitator was 60～120 revs/min, obtained second-level shake flask seed liquor；According to production scale, use seed tank further
Carry out the amplification culture of seed；
C, the fermenting and producing of microbial inoculum: take Gause I synthetic fluid culture medium, access the fermentation seed liquid of above-mentioned preparation,
In pH7.2～7.4, temperature 27～28 DEG C, ferment 4～5 days, to obtain final product, can be standby as producing bacterium solution；
Prepare the fermentation process of the bacterium solution of the bacterium solution of described cereuisiae fermentum, the bacterium solution of Paecilomyces lilacinus, trichoderma harzianum:
A, slant strains activation culture: take PDA fluid medium, access the slant culture of a kind of mentioned microorganism, 25～28
Under the conditions of DEG C on shaking table shaken cultivation 48～72 hours, speed of agitator is 60～120 revs/min, is then transferred to identical training
Supporting in base and cultivate, method is transferred 3～5 times continuously like this, is applied on Solid media for plates after being diluted by the culture fluid of acquisition,
Cultivating 3～5 days under the conditions of 25～28 DEG C, rotating speed is 60～120 revs/min, and after growing sturdy mycelia, the sturdy mycelia of picking is same
Rule in the solid plate culture medium of sample, select sturdy mycelia and be transferred to solid slant culture base, cultivate and obtain naturalized strain, protect
Deposit standby；
B, second-level shake flask spawn culture: take PDA fluid medium, access primary inclined plane strain, cultivate at 25～28 DEG C 48～
72 hours, speed of agitator was 60～120 revs/min, obtained second-level shake flask seed liquor；According to production scale, use seed tank further
Carry out the amplification culture of seed；
C, the fermenting and producing of microbial inoculum: take PDA fluid medium, access the fermentation seed liquid of above-mentioned preparation, in pH7.0～
7.2, temperature 25～28 DEG C ferment 3～5 days, to obtain final product, and as producing, bacterium solution is standby；
Prepare the fermentation process of the bacterium solution of the bacterium solution of described streptococcus acidi lactici, the bacterium solution of bacillus subtilis, Bacillus licheniformis:
A, slant strains activation culture: take beef extract-peptone fluid medium, access the slant culture of a kind of mentioned microorganism
Thing, under the conditions of 30～40 DEG C on shaking table shaken cultivation 20～48 hours, rotating speed is 60～120 revs/min, is then transferred to phase
Cultivating in same culture medium, method is transferred 3～5 times continuously like this, is applied to flat-plate solid training after being diluted by the culture fluid of acquisition
Supporting on base, cultivate 1～2 day under the conditions of 30～40 DEG C, rotating speed is 60～120 revs/min, and after growing bacterium colony, picking macrocolony exists
Ruling in same solid plate culture medium, picking individual colonies is transferred to solid slant culture base, cultivates and obtains naturalized strain, protects
Deposit standby；
B, second-level shake flask spawn culture: take beef extract-peptone fluid medium, access primary inclined plane strain, at 30～40 DEG C
Cultivating 20～48 hours, rotating speed is 60～120 revs/min, obtains second-level shake flask seed liquor；According to production scale, use kind further
Sub-tank carries out the amplification culture of seed；
C, the fermenting and producing of microbial inoculum: take beef extract-peptone fluid medium, access the fermentation seed liquid of above-mentioned preparation,
In pH7.2～8.5, temperature 30～40 DEG C, fermenting 1～2 day, to obtain final product, as producing, bacterium solution is standby；
C, the preparation of liquid complex biological preservative:
According to the parts by weight of raw materials proportioning of composite bacteria agent capable described in claim 1～8, the various bacterium solution above-mentioned step B prepared are mixed
Closing, make composite bacteria agent capable, through inspection, make to reach 200,000,000/milliliter containing total living bacteria count, the quantity of each effective bacterium must not be lacked
More than 20,000,000/milliliter, meet standard GB/T-20287-2006, according still further to the raw material group described in claim 1～8
The weight divided, chitosan above-mentioned steps prepared, epsilon-polylysine, lysozyme are mixed homogeneously with composite bacteria agent capable, i.e.
?.
10. the purposes of the liquid complex biological preservative described in a claim 1～8, it is characterised in that it is protected as fruit
Fresh dose is applied to Nanguo Pear, wild actinidia arguta or Fructus Fragariae Ananssae.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610222937.2A CN105875820A (en) | 2016-04-12 | 2016-04-12 | Liquid compound biological preservative and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610222937.2A CN105875820A (en) | 2016-04-12 | 2016-04-12 | Liquid compound biological preservative and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105875820A true CN105875820A (en) | 2016-08-24 |
Family
ID=57012578
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610222937.2A Pending CN105875820A (en) | 2016-04-12 | 2016-04-12 | Liquid compound biological preservative and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105875820A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108157493A (en) * | 2017-12-12 | 2018-06-15 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of compound bio antisepsis antistaling agent based on the zymotic fluid containing epsilon-polylysine |
| CN109090430A (en) * | 2018-09-20 | 2018-12-28 | 贵州省贵福菌业发展有限公司 | A kind of fresh fried flour mushroom is fresh-keeping to use lactic acid bacteria fresh-keeping liquid |
| CN109090224A (en) * | 2018-07-09 | 2018-12-28 | 湖州吴兴道场城乡建设发展有限公司 | A kind of microorganism fruit antistaling agent |
| CN110055203A (en) * | 2019-04-23 | 2019-07-26 | 沈阳农业大学 | A kind of Antiphytoviral epsilon-polylysine superior strain and preparation process |
| CN111838298A (en) * | 2020-07-29 | 2020-10-30 | 四川大学 | Ripening accelerating method for shortening after-ripening time of kiwi fruits and product thereof |
| CN112120077A (en) * | 2019-06-09 | 2020-12-25 | 王虎 | Green and environment-friendly waxberry preservative formula and preparation method thereof |
| CN113796414A (en) * | 2021-09-23 | 2021-12-17 | 临沂大学 | Preservation method of fresh blueberry fruits |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101619308A (en) * | 2009-08-14 | 2010-01-06 | 湖北神地农业科贸有限公司 | Preparation method for extracting lysozyme from egg white |
| CN101870959A (en) * | 2010-06-11 | 2010-10-27 | 河北省科学院生物研究所 | Application of bsacillus subtilis, microbial inoculum thereof and preparations thereof to field of fruit freshness keeping |
| CN102188034A (en) * | 2010-03-03 | 2011-09-21 | 天津科技大学 | Compound biological antiseptic and preservation agent for food |
| CN102617755A (en) * | 2012-04-20 | 2012-08-01 | 江南大学 | Simple and easy process for preparing chitosan by utilizing shrimp shells or processed residues of shrimp shells |
| CN103125590A (en) * | 2011-11-21 | 2013-06-05 | 南通中科睿智科技服务有限公司 | Grape biological fresh-keeping agent and preparation method thereof |
| CN103651774A (en) * | 2013-12-03 | 2014-03-26 | 河北省科学院生物研究所 | Biological storage agent for edible fungi, preparation method of biological storage agent and edible fungi preservative paper |
| CN104082830A (en) * | 2014-06-11 | 2014-10-08 | 南通昊友食品添加剂有限公司 | Composite biological antiseptic antistaling agent |
| CN104496707A (en) * | 2015-01-11 | 2015-04-08 | 鞍山禾瑞生物科技有限公司 | Liquid composite biological multi-control fertilizer, and preparation method and application thereof |
| CN104542727A (en) * | 2015-01-27 | 2015-04-29 | 辽宁玉泉生态农业科技有限公司 | Liquid-state composite biological multi-control insecticide and preparation method thereof |
| CN104611144A (en) * | 2014-12-17 | 2015-05-13 | 山东省药学科学院 | Natural fruit and vegetable cleaning fresh-keeping agent and sue method thereof |
-
2016
- 2016-04-12 CN CN201610222937.2A patent/CN105875820A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101619308A (en) * | 2009-08-14 | 2010-01-06 | 湖北神地农业科贸有限公司 | Preparation method for extracting lysozyme from egg white |
| CN102188034A (en) * | 2010-03-03 | 2011-09-21 | 天津科技大学 | Compound biological antiseptic and preservation agent for food |
| CN101870959A (en) * | 2010-06-11 | 2010-10-27 | 河北省科学院生物研究所 | Application of bsacillus subtilis, microbial inoculum thereof and preparations thereof to field of fruit freshness keeping |
| CN103125590A (en) * | 2011-11-21 | 2013-06-05 | 南通中科睿智科技服务有限公司 | Grape biological fresh-keeping agent and preparation method thereof |
| CN102617755A (en) * | 2012-04-20 | 2012-08-01 | 江南大学 | Simple and easy process for preparing chitosan by utilizing shrimp shells or processed residues of shrimp shells |
| CN103651774A (en) * | 2013-12-03 | 2014-03-26 | 河北省科学院生物研究所 | Biological storage agent for edible fungi, preparation method of biological storage agent and edible fungi preservative paper |
| CN104082830A (en) * | 2014-06-11 | 2014-10-08 | 南通昊友食品添加剂有限公司 | Composite biological antiseptic antistaling agent |
| CN104611144A (en) * | 2014-12-17 | 2015-05-13 | 山东省药学科学院 | Natural fruit and vegetable cleaning fresh-keeping agent and sue method thereof |
| CN104496707A (en) * | 2015-01-11 | 2015-04-08 | 鞍山禾瑞生物科技有限公司 | Liquid composite biological multi-control fertilizer, and preparation method and application thereof |
| CN104542727A (en) * | 2015-01-27 | 2015-04-29 | 辽宁玉泉生态农业科技有限公司 | Liquid-state composite biological multi-control insecticide and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 个人分享: "《教学研究：壳聚糖制备工艺》", 23 February 2016, 豆丁网 * |
| 贾士儒: "《生物防腐剂》", 30 September 2009, 北京：中国轻工业出版社 * |
| 陈庆森 等: "《实用生物化学实验技术指导》", 30 September 2013, 杭州：浙江大学出版社 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108157493A (en) * | 2017-12-12 | 2018-06-15 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of compound bio antisepsis antistaling agent based on the zymotic fluid containing epsilon-polylysine |
| CN109090224A (en) * | 2018-07-09 | 2018-12-28 | 湖州吴兴道场城乡建设发展有限公司 | A kind of microorganism fruit antistaling agent |
| CN109090430A (en) * | 2018-09-20 | 2018-12-28 | 贵州省贵福菌业发展有限公司 | A kind of fresh fried flour mushroom is fresh-keeping to use lactic acid bacteria fresh-keeping liquid |
| CN110055203A (en) * | 2019-04-23 | 2019-07-26 | 沈阳农业大学 | A kind of Antiphytoviral epsilon-polylysine superior strain and preparation process |
| CN110055203B (en) * | 2019-04-23 | 2021-04-20 | 沈阳农业大学 | High-yield strain resisting plant virus epsilon-polylysine and preparation process thereof |
| CN112120077A (en) * | 2019-06-09 | 2020-12-25 | 王虎 | Green and environment-friendly waxberry preservative formula and preparation method thereof |
| CN111838298A (en) * | 2020-07-29 | 2020-10-30 | 四川大学 | Ripening accelerating method for shortening after-ripening time of kiwi fruits and product thereof |
| CN111838298B (en) * | 2020-07-29 | 2022-12-06 | 四川大学 | Ripening accelerating method for shortening after-ripening time of kiwi fruits and product thereof |
| CN113796414A (en) * | 2021-09-23 | 2021-12-17 | 临沂大学 | Preservation method of fresh blueberry fruits |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105875820A (en) | Liquid compound biological preservative and preparation method and application thereof | |
| CN103478439B (en) | Shrimp feed addictive composition and manufacture method thereof | |
| CN107446852B (en) | One lactobacillus plantarum and its application in terms of fermented feed | |
| CN106399159B (en) | A kind of bacillus firmus microbial inoculum and the preparation method and application thereof | |
| CN105919110B (en) | Black tea composite enzyme and preparation method thereof | |
| CN107593955A (en) | Ferment edible mushroom tea preparation process and fermentation edible mushroom tea | |
| CN108611287A (en) | The Pichia membranaefaciens of one plant of control peach fruit postharvest disease | |
| CN105613707A (en) | Bacillus subtilis biological preservative and application thereof to preservation of large yellow croakers | |
| CN106234895A (en) | A kind of Fructus Nitraria schoberi lactacidase fermenting beverage and preparation method thereof | |
| KR20160136718A (en) | Astragalus membranaceus Fermentation product and its manufacturing method | |
| CN105981581A (en) | Artificial culture method of cordyceps sobolifera | |
| KR101701827B1 (en) | Method for producing fermented grain without soaking | |
| KR20200136964A (en) | Method for producing a composition for treating hair loss | |
| CN108157065A (en) | A kind of mycelial cultural method of Antrodia camphorata and its application | |
| CN110547384B (en) | Small yellow croaker ossein antibacterial peptide and application thereof | |
| CN107027516A (en) | A kind of selenium-rich Periostracum cicadae, its cultural method and application | |
| CN105794963A (en) | Saccharomyces cerevisiae degrading zearalenone toxins and application thereof | |
| CN110527639A (en) | A kind of U.S. pole plum surprise yeast and its application | |
| CN109628334A (en) | Method, preparation and the used medium that yeast inhibits fruit disease effect are improved using L-PROLINE Fiber differentiation | |
| CN104988183A (en) | Preparation method of pleurotus ostreatus fermentation broth and application of fermentation broth in degradation of aflatoxin B1 | |
| CN102630480B (en) | Iron-rich black fungus and production method thereof | |
| CN106072518B (en) | Spondias axillaris ferment and preparation method thereof | |
| KR102233304B1 (en) | Medium composition comprising a large amount of human immune improving and physiological active substance for stable cultivating vegetable worms and the method of cultivating vegetable worms using the medium and the vegetable worms cultivated by using the method | |
| CN1536069B (en) | Method for large-scale production of catepillar fungus and lucid ganoderma | |
| CN103931665A (en) | Mixed formulation preparation for prevention and control of nilaparvata lugens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 114041 Liaoning, Anshan, Qianshan District, Tun Town, Tun Tun Village Applicant after: Liaoning Hei Bio Technology Co., Ltd. Address before: 114041 Liaoning, Anshan, Qianshan District, Tun Town, Tun Tun Village Applicant before: ANSHAN HERUI BIOTECHNOLOGY CO., LTD. |
|
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160824 |