CN108208317A - A kind of viable bacteria antistaling agent and its application in cultured fishes are fresh-keeping - Google Patents
A kind of viable bacteria antistaling agent and its application in cultured fishes are fresh-keeping Download PDFInfo
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- CN108208317A CN108208317A CN201810171244.4A CN201810171244A CN108208317A CN 108208317 A CN108208317 A CN 108208317A CN 201810171244 A CN201810171244 A CN 201810171244A CN 108208317 A CN108208317 A CN 108208317A
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- fish
- antistaling agent
- bacteria
- bacillus subtilis
- viable bacteria
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The present invention provides a kind of viable bacteria antistaling agent and its application in cultured fishes are fresh-keeping, using live bacillus subtilis preparation to the growth inhibition of fish body spoilage organisms, so as to extend the purpose of the refrigerated shelf phase of fish, the bacillus subtilis is bacillus subtilis BSh0851, it is advantageous that bacillus subtilis antistaling agent of the present invention inhibits the growth of fish body spoilage organisms, adherency and growth of the spoilage organisms on fish intestines and the fish gill are alleviated from source, so as to achieve the purpose that prolong storage period.
Description
Technical field
The invention belongs to microbial preparation fields, and in particular to a kind of viable bacteria antistaling agent and its in cultured fishes are fresh-keeping
Using.
Background technology
Preservation technology is an important ring in aquatic products industry, be to maintain high price, Shelf-life, expand sale radius with
And extend the key of process time, there is important economic implications to the development of aquatic products.At present, applied to the fresh-keeping of aquatic products
Technology mainly has preservation by low temperature, fresh chemically, gas preservation, radiation preservation and biological preservation etc..Biological preservation is with its safety
The advantage high, low energy consumption, which becomes, develops new hot spot.Plant source preservative mainly plays antioxidation；Animal based bacteriostat such as milt
Albumen；Common microbial source bacteriostatic agent mainly has the lactic acid bacteria lactein generated, its mycin etc. of receiving in aquatic products.It is dynamic/to plant
The antistaling agent in object source is limited by raw material, higher price；And the microbial source antistaling agent of industrialization at present is microbial metabolism
Object, high processing costs are used only in high level product.If the thinking of probiotic products can be used for reference, exploitation probiotics are used to protect
It is fresh, production cost will be effectively reduced, expands the application range of bio-preservative.Some bacillus subtilis strains have suppression
Pathogen processed and the activity of microalgae, are allowed to use in fruits and vegetables, fish meal.
There is the bacillus subtilis for significantly inhibiting fish body spoilage organisms activity in the present invention using one plant（Preserving number：
BSh0851）The fresh-keeping preparation of viable bacteria of the bacterial strain is developed in innovation, is launched with Feed forms into aquaculture pond, by field planting in the fish gill and
Fish intestines position forms mycoderm and antibacterial substance, changes Bacterial community, reduces the spoilage organisms of fish enteron aisle, the fish gill and fish-skin attachment, prolongs
Long freshness date.The use of said preparation reduces fresh-keeping cost for the quality and foodsafety of raising fish.
Invention content
The purpose of the present invention is to provide a kind of viable bacteria antistaling agent and its applications in cultured fishes are fresh-keeping.
To achieve the above object, the present invention adopts the following technical scheme that：
Heretofore described bacillus subtilis is bacillus subtilis（Bacillus subtulis）BSh0851, preservation date
For on December 13rd, 2009, depositary institution was China Microbiological bacterial strain preservation administration committee common micro-organisms center, preservation
Location is BeiChen West Road, Chaoyang District, BeiJing City 1, and Institute of Microorganism, Academia Sinica, preserving number is CGMCC No.3502.The bacterium
Strain can significantly inhibit source of fish spoilage organisms, including Xi Washi and pseudomonad, as shown in table 1.
The bacillus subtilis（Bacillus subtilis）For gram-positive bacteria, gemma ellipse to column is located at
Thalline is central or slightly inclined, and thalline does not expand after sporulation, and bacterium colony rough surface is opaque, and dirty white or yellowish is commonly formed
Wrinkle mould；Aerobic bacteria, available protein, a variety of sugar and starch, V-P are positive.
1 bacillus subtilis BSh0851 of table is to the growth inhibition effect of part bacterium
Note："+", and to represent fungistatic effect more apparent
Viable bacteria antistaling agent making of the present invention containing bacillus subtilis specifically includes following steps：
（1）It is prepared by seed liquor：The bacterial strain for being preserved in 4 DEG C of refrigerators is taken out, one ring bacterium of picking accesses 100 mL beef extract-peptones
In solid medium, 37 DEG C, 200r/min, 1d is cultivated；Beef-protein medium：Beef extract 2.5g, sodium chloride 2.5g, egg
White peptone 5g, distilled water 500mL, pH7.0~7.2；
（2）It is prepared by the liquid culture medium that ferments：Yeast extract 14g, DEXTROSE ANHYDROUS 14g, MgSO41g, distilled water 1000mL, pH7.0；
（3）Preparation of the bacillus subtilis without thalline zymotic fluid：Seed liquor is seeded to step with 5% ratio（2）Fermentation training
Base is supported, triangular flask liquid amount is 100 mL, and 37 DEG C, 150 r/min, 48 h of culture obtain zymocyte liquid；
（3）Centrifugation obtains thalline：Using 10000 r/min, 15 DEG C of 15 min of centrifugation；The bacterium mud equivalent being collected into is sterile
Brine centrifuges again, is repeated 2 times；With the sterile physiological saline of equivalent again suspension thalline, bacteria suspension is made；This
When, bacteria suspension concentration 109A/mL；
（4）Spray drying：Arabic gum is selected as protective agent, by gummi arabicum pulveratum dissolving in deionized water, mass concentration
15%, it is configured to wall material solution, homogeneous 15 min, 90 DEG C of 5 min of heating water bath；Wall material solution is sufficiently uniformly dissolved, is put into and kills
Bacterium pot sterilization；At room temperature, bacteria suspension and wall material are mixed in equal volume, continued stirring until with 500 r/min it is uniformly mixed, through compacted
It is dynamic to pump uniform sample introduction to spray dryer, charging rate：550 mL/h, 150 DEG C of inlet temperature, 84 DEG C of outlet temperature, atomization pressure
0.1 mpa of power collects product；Final viable bacteria antistaling agent bacterial content is 5 × 108A/g.
Application of the viable bacteria antistaling agent in fish are fresh-keeping, is sprayed on feed surface by active bacteria formulation, is caught by fish
Food enters fish enteron aisle, inhibits the growth of spoilage organisms in enteron aisle, reduces fish refrigeration period spoilage organisms initial concentration, fresh-keeping so as to extend
Phase.Bacillus subtilis BSh0851 bacterium powders are added to plain particles shape feed by the bacterium amount of 10g/kg.Bacterium powder is dissolved in sterile
It in water, is uniformly sprayed onto on normal diet, dry 24 h in 45 DEG C of baking ovens, 4 DEG C of preservations after encapsulation.
It is caught the last week in fish, feeds the primary feed daily.The viable bacteria antistaling agent can effectively extend fish in refrigeration and ice
Freshness date when fresh.
Beneficial effects of the present invention are as follows：
There is the bacillus subtilis for significantly inhibiting fish body spoilage organisms activity in the present invention using one plant（Preserving number：BSh0851）
The fresh-keeping preparation of viable bacteria of the bacterial strain is developed in innovation, is launched with Feed forms into aquaculture pond, by being colonized in the fish gill and fish intestines position,
Mycoderm and antibacterial substance are formed, changes Bacterial community, the spoilage organisms of fish enteron aisle, the fish gill and fish-skin attachment is reduced, prolongs storage period.
The use of said preparation reduces fresh-keeping cost for the quality and foodsafety of raising fish.
Specific embodiment
Embodiment 1
Viable bacteria antistaling agent making of the present invention containing bacillus subtilis specifically includes following steps：
（1）It is prepared by seed liquor：The bacterial strain for being preserved in 4 DEG C of refrigerators is taken out, one ring bacterium of picking accesses 100 mL beef extract-peptones
In solid medium, 37 DEG C, 200r/min, 1d is cultivated；Beef-protein medium：Beef extract 2.5g, sodium chloride 2.5g, egg
White peptone 5g, distilled water 500mL, pH7.0~7.2；
（2）It is prepared by the liquid culture medium that ferments：Yeast extract 14g, DEXTROSE ANHYDROUS 14g, MgSO41g, distilled water 1000mL, pH7.0；
（3）Preparation of the bacillus subtilis without thalline zymotic fluid：Seed liquor is seeded to step with 5% ratio（2）Fermentation training
Base is supported, triangular flask liquid amount is 100 mL, and 37 DEG C, 150 r/min, 48 h of culture obtain zymocyte liquid；
（3）Centrifugation obtains thalline：Using 10000 r/min, 15 DEG C of 15 min of centrifugation；The bacterium mud equivalent being collected into is sterile
Brine centrifuges again, is repeated 2 times；With the sterile physiological saline of equivalent again suspension thalline, bacteria suspension is made；This
When, bacteria suspension concentration 109A/mL；
（4）Spray drying：Arabic gum is selected as protective agent, by gummi arabicum pulveratum dissolving in deionized water, mass concentration
15%, it is configured to wall material solution, homogeneous 15 min, 90 DEG C of 5 min of heating water bath；Wall material solution is sufficiently uniformly dissolved, is put into and kills
Bacterium pot sterilization；At room temperature, bacteria suspension and wall material are mixed in equal volume, continued stirring until with 500 r/min it is uniformly mixed, through compacted
It is dynamic to pump uniform sample introduction to spray dryer, charging rate：550 mL/h, 150 DEG C of inlet temperature, 84 DEG C of outlet temperature, atomization pressure
0.1 mpa of power collects product；Final viable bacteria antistaling agent bacterial content is 5 × 108A/g.
Embodiment 2
（1）The nursing of Tilapia mossambica.130 20-30g Rofes juvenile fish cultivate according to following 4 groups of feed modes.Every group about 30~35
Juvenile tilapia, mean allocation raise the chest in two about 75L.Per the control of case water temperature at 28 ± 1 DEG C, half is changed daily
Water.By daily 1% body weight ratio feed for nursing, after one week laundering period, start with chow diet according to following processing group
7d is fed in grouping.
Processing group 1：In normal water body, normal diet nursing group.By daily 1% body weight ratio feed for nursing.
Processing group 2：In normal water body, probiotic feed nursing group.Bacillus subtilis is fed by daily 1% body weight ratio
Bacterium feed.
Processing group 3：In corrupt water body, normal diet nursing group is added.In corrupt water body, 1d additions 10 are fed8A/mL
Spoilage organisms Shewanella and each 5ml of pseudomonad, by daily 1% body weight ratio feed for nursing.
Processing group 4：In corrupt water body, probiotic feed nursing group is added.In corrupt water body, 1d additions 10 are fed8A/
ML spoilage organisms Shewanella and each 5ml of pseudomonad feed bacillus subtilis feed by daily 1% body weight ratio.
Probiotics is the viable bacteria antistaling agent containing bacillus subtilis prepared in embodiment 1, by the fresh-keeping of 10g/kg
Bacterium powder is added to plain particles shape feed by agent.
（2）Sample freshness determination.After 7d is fed, each group fish is caught, it is cold to be placed on 4 DEG C of refrigerator for trash ice urgency jelly immediately
It hides.The sample after 24 ~ 48h is taken to carry out freshness detection.
Volatile Base Nitrogen（TVB-N）Measure.This experiment is using the automatic kjeldahl determinations of GB5009.228-2016
Instrument method measures Volatile Base Nitrogen（TVB-N）, according to GB2733-2015《National food safety standard --- fresh, jelly animality
Aquatic products》Defined Volatile Base Nitrogen index judges freshness, and Volatile Base Nitrogen is less than 30mg/100g.
Freshness K values measure.Uniform about 1 g of flesh of fish sample of grinding is weighed, is put into 50 mL centrifuge tubes, adds 10% high chlorine
10 mL of acid solution, vortex vibrate 1 min, and 8000 r/min centrifuge 10 min at 4 DEG C, take out supernatant.Again with 5% perchloric acid
Determinand in 5 mL extraction sediments, 8000 r/min centrifuge 10 min at 4 DEG C.It is primary to repeat aforesaid operations, in merging
Clear liquid.With 10 mol/L sodium hydroxide solution tune supernatants pH 4.0 or so, then with 1 mol/L sodium hydroxide solution tune pH extremely
6.0-6.4.This solution is transferred in 25 mL volumetric flasks of precooling, 4 DEG C of deionized water constant volumes, at 4 DEG C 8000 r/min from
10 min of the heart, 0.22 um membrane filtrations, filtrate are stored in be measured in 4 DEG C of refrigerators.High performance liquid chromatography detects, mobile phase A
For 20 mmol/L potassium dihydrogen phosphates and 20 mmol/L dipotassium hydrogen phosphates（1:1）Solution is pure methanol with phosphoric acid tune pH to 5.8, B
Solution；Column temperature:30℃；Sample size:10μL；Detection wavelength:254 nm；Flow velocity:1.0 mL/min；
K values are the percentage that the decomposition product HxR of ATP and Hx total amounts account for ATP related compounds total amounts, and K values are lower, illustrate HxR
The ratio that whole decomposition products are accounted for Hx is lower, illustrates that the freshness of fish is better.It is formulated as：
In formula:MATP, MADP, MAMP, MIMP, MHxR, MHx are the content of ATP, AMP, ADP, IMP, Hx, HxR in sample,
Unit is every gram of micromole (μm ol/g).
（3）Different disposal group fresh target change.
Tilapia mossambica Volatile Base Nitrogen TVB-N values variation in cold storage procedure is as shown in table 2.Four groups of flesh of fish sample TVB-
There is different degrees of growth with cold preservation time in N values.After 4 DEG C of refrigerations for 24 hours, the ability of TVB-N is produced between different disposal group
With significant difference（p<0.05）.The degree that corruption occurs is followed successively by processing group 3 from high to low>Processing group 1>Processing group 4>Place
Reason group 2.The water body for adding in spoilage organisms is easiest to make fish corrupt（Processing group 3）, by the nursing of probiotic feed, the anti-corruption of fish
Ability significantly improves, either the fish in corrupt water body（Processing group 4）Or the fish of normal water body（Processing group 2）, corruption
When degree is all than being not added with probiotic feed（Processing group 1 and 3）Significantly slowed down.After 4 DEG C of refrigeration 48h, processing group 3 is close
The terminal of corruption（30mg/100g）, degree of spoilage is still processing group 3 from high to low>Processing group 1>Processing group 4>Processing group 2.
The fish corruption fed by probiotic feed is significantly slow, and normal water body group fish when degree of spoilage is with refrigerating for 24 hours is not aobvious
Write difference.
TVB-N values variation (mg/100g) in 2 four groups of processing flesh of fish cold storage procedures of table
3 four groups of Tilapia mossambica flesh of fish sample K values of table measure
The 48h of the K values of four groups of Tilapia mossambica flesh of fish after release from storage has reached maximum value, illustrates that freshness is significantly deteriorated（Table 3）.24h
When, the seafood degree of 4 kinds of processing modes is not significantly different；After 48h, the seafood degree in normal water body and corrupt water body significantly becomes
Difference；The fish of probiotic feed nursing is added after 48h is stored, although freshness, which has than for 24 hours, notable loss, than without prebiotic
The fish of bacterium raising, freshness loss have significant alleviation（p<0.05）.
The present invention can delay the corruption of fish body at low ambient temperatures, apply using Shewanella and pseudomonad as advantage corruption
It loses in refrigeration or the chilled preservation process of the cultivation fresh water or fish of bacterium.The equivalent change that claim is done is with repairing
Decorations should all belong to the covering scope of the present invention.
Claims (4)
1. a kind of viable bacteria antistaling agent, which is bacillus subtilis BSh0851, and preservation date is on December 13rd, 2009, preservation
Unit is China Microbiological bacterial strain preservation administration committee common micro-organisms center, and preserving number is CGMCC No.3502.
2. a kind of preparation method of viable bacteria antistaling agent as described in claim 1, it is characterised in that：Include the following steps：
（1）It is prepared by seed liquor：The bacterial strain for being preserved in 4 DEG C of refrigerators is taken out, one ring bacterium of picking accesses 100 mL beef extract-peptones
In solid medium, 37 DEG C, 200r/min, 1d is cultivated；Beef-protein medium：Beef extract 2.5g, sodium chloride 2.5g, egg
White peptone 5g, distilled water 500mL, pH7.0~7.2；
（2）It is prepared by the liquid culture medium that ferments：Yeast extract 14g, DEXTROSE ANHYDROUS 14g, MgSO41g, distilled water 1000mL, pH7.0；
（3）Preparation of the bacillus subtilis without thalline zymotic fluid：Seed liquor is seeded to step with 5% ratio（2）Fermentation training
Base is supported, triangular flask liquid amount is 100 mL, and 37 DEG C, 150 r/min, 48 h of culture obtain zymocyte liquid；
（3）Centrifugation obtains thalline：Using 10000 r/min, 15 DEG C of 15 min of centrifugation；The bacterium mud equivalent being collected into is sterile
Brine centrifuges again, is repeated 2 times；With the sterile physiological saline of equivalent again suspension thalline, bacteria suspension is made；This
When, bacteria suspension concentration 109A/mL；
（4）Spray drying：Arabic gum is selected as protective agent, by gummi arabicum pulveratum dissolving in deionized water, mass concentration
15%, it is configured to wall material solution, homogeneous 15 min, 90 DEG C of 5 min of heating water bath；Wall material solution is sufficiently uniformly dissolved, is put into and kills
Bacterium pot sterilization；At room temperature, bacteria suspension and wall material are mixed in equal volume, continued stirring until with 500 r/min it is uniformly mixed, through compacted
It is dynamic to pump uniform sample introduction to spray dryer, charging rate：550 mL/h, 150 DEG C of inlet temperature, 84 DEG C of outlet temperature, atomization pressure
0.1 mpa of power collects product；Final viable bacteria antistaling agent bacterial content is 5 × 108A/g.
3. application of the viable bacteria antistaling agent as described in claim 1 in fish are fresh-keeping.
4. application according to claim 3, it is characterised in that：Active bacteria formulation is sprayed on feed surface, feeding fish, and feed is pressed
The active bacteria formulation dosage of 10g/kg is added to plain particles shape feed, and bacterium powder is dissolved in sterile water, is uniformly sprayed onto common feeding
On material, dry 24 h, are packaged in 4 DEG C of preservations in 45 DEG C of baking ovens.
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| CN201810171244.4A CN108208317B (en) | 2018-03-01 | 2018-03-01 | Live bacterium preservative and application thereof in preservation of cultured fishes |
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Cited By (3)
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| CN108783462A (en) * | 2018-07-02 | 2018-11-13 | 哈尔滨美华生物技术股份有限公司 | A kind of industrial process of beneficial bacteria of intestinal tract preparation |
| CN112725248A (en) * | 2021-03-05 | 2021-04-30 | 福州大学 | Bacillus subtilis spray-drying composite protective agent and preparation method thereof |
| RU2797684C1 (en) * | 2022-08-23 | 2023-06-07 | Ирина Васильевна Ткачева | Method for keeping live fish during transportation and storage |
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| RU2797684C1 (en) * | 2022-08-23 | 2023-06-07 | Ирина Васильевна Ткачева | Method for keeping live fish during transportation and storage |
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| CN108208317B (en) | 2021-07-13 |
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