CN105853465A - Method for inhibiting reproduction of vibrio parahaemolyticus in mussel bodies through biological antagonism of lactic acid bacteria - Google Patents
Method for inhibiting reproduction of vibrio parahaemolyticus in mussel bodies through biological antagonism of lactic acid bacteria Download PDFInfo
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- CN105853465A CN105853465A CN201610182359.4A CN201610182359A CN105853465A CN 105853465 A CN105853465 A CN 105853465A CN 201610182359 A CN201610182359 A CN 201610182359A CN 105853465 A CN105853465 A CN 105853465A
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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Abstract
The invention discloses a method for inhibiting reproduction of vibrio parahaemolyticus in mussel bodies through biological antagonism of lactic acid bacteria. The method comprises the following steps: cultivating lactococcus lactis subsp. Lactis CICC 6242, so that a high-density bacterial suspension which ranges from 109cfu/mL to 1010cfu/mL in density; and adding the high-density bacterial suspension to a water body in which mussels are bred, so that the reproduction of the vibrio parahaemolyticus in the mussel bodies is inhibited. According to the method disclosed by the invention, a bacteriocin, namely Nisin, which is harmless to human bodies and is strong in effect on killing bacteria, is generated by virtue of the lactococcus lactis subsp. Lactis CICC 6242, so that the reproduction of the vibrio parahaemolyticus in the mussel bodies is inhibited; therefore, the reproduction of the vibrio parahaemolyticus in the mussel bodies is inhibited through the biological antagonism and the eating safety of the mussels is guaranteed.
Description
Technical field
The present invention relates to field of food safety, be specifically related to a kind of by lactic acid bacteria biological antagonism suppression mussel
The method of internal vibrio parahaemolytious breeding.
Background technology
Nearly 1,000,000 tons of mussel year cultivation amount of China, Zhejiang Province is mussel major production areas, accounts for whole nation total output
More than 15%.Over nearly 30 years, CHINESE OFFSHORE pollutes aggravation, adds the factors such as global warming,
In sea water, vibrio parahaemolytious (Vibrio parahaemolyticus, Vp) content is on the rise.VP's
Influence factor of causing a disease is more, such as aggressivity, hemotoxin, the most again with Thermostable direc t hemolysin
(Thermalstable Directheamolysin, TDH) is mostly important.The food that VP pollutes can cause
Acute gastroenteritis, patients symptomatic mainly shows as suffering from diarrhoea, vomits, has a headache, feels sick, abdominal colic and low
Burning, the course of disease is generally 2～4d, and the persistent period is shorter, and can spontaneous recovery.Serious symptom also can cause wound infection
And septicemia, other tissue of health also can produce pathological changes, such as skin bulla infringement, reactive arthritis
With rheumatoid arthritis etc..
And mussel is filter food, Vp can be enriched with from environment in a large number, make mussel become Vp diarrhoea
Important food source.Monitoring Data according to country of China food origin disease monitoring net shows, Vp cause
Alimentary toxicosis in bacterial food poisoning, already take up critical positions.
Mussel is after fishing for disembarkation, for keeping its freshness, often continues to support at normal temperatures at sales section
Grow rather than carry out cold preservation or freezing.Vp may proceed to breeding at normal temperatures.Therefore, for ensureing mussel food
By safety, in needing exploitation suppression mussel body at normal temperatures, Vp breeds and don't affects the skill of mussel meat
Art.
Summary of the invention
The invention provides a kind of by vibrio parahaemolytious (Vp) in lactic acid bacteria biological antagonism suppression mussel body
The method bred and ensure mussel edible safety, by the lactococcus lactis Lactococcus by producing Nisin
Lactis subsp.lactis (CICC 6242) adds in the water body of cultivation mussel, plays in mussel body
The inhibitory action of Vp, thus ensure mussel edible safety.
A kind of by lactic acid bacteria biological antagonism suppression mussel body in vibrio parahaemolytious breeding method, including with
Lower step:
1) lactococcus lactis Lactococcus lactis subsp.Lactis CICC 6242 is cultivated,
Making density is 109～1010The high density bacteria suspension of cfu/mL；
2) by step 1) in 109～1010The high density bacteria suspension of cfu/mL joins the water body of cultivation mussel
In, vibrio parahaemolytious breeding in suppression shellfish body.
In the present invention, by lactococcus lactis Lactococcus lactis subsp.Lactis (CICC 6242)
Produce a kind of harmless and antibacterial is had the bacteriocin Nisin of strong killing action, it is possible to suppression is made a gift of
Vibrio parahaemolytious (Vp) breeding, lactococcus lactis Lactococcus lactis subsp.lactis in shellfish body
(CICC 6242) is bred by Vp in biological antagonist suppression mussel body and ensures mussel edible safety.
Step 1) in, lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242)
Use prior art, use commercially available prod, from Chinese industrial Microbiological Culture Collection administrative center
(CICC), address: No. 6 building of Road, Jiuxianqiao, Chaoyang District, Beijing City 24 institute；Preservation date is 2010
Year, strain keeps numbered 6242.
As preferably, lactococcus lactis Lactococcus lactis subsp.Lactis CICC 6242 is carried out
Cultivate, including: setting out of lactococcus lactis Lactococcus lactis subsp.Lactis CICC 6242 is close
Degree is 106～107Individual/mL, being seeded to inoculum concentration in fluid medium is 1%～5%, 33 DEG C～39 DEG C vibrations
Cultivate 20～28h, be 10 to bacterium solution density9～1010The high density bacteria suspension of cfu/mL.
Further preferably, lactococcus lactis Lactococcus lactis subsp.Lactis CICC 6242 is entered
Row is cultivated, including: lactococcus lactis Lactococcus lactis subsp.Lactis CICC 6242 sets out
Density is 106～107Individual/mL, being seeded to inoculum concentration in fluid medium is 2%～3%, and 36 DEG C～37 DEG C are shaken
Swing cultivation 22～26h, be 10 to bacterium solution density9～1010The high density bacteria suspension of cfu/mL.
Described fluid medium, in terms of 1L, including following components:
The pH value of this fluid medium regulates to 6.0～6.6.
Further preferably, described fluid medium, in terms of 1L, including following components:
The pH value of this fluid medium regulates to 6.3, and fluid medium is at 121 DEG C of sterilizing 20min.
Step 2) in, as preferably, high density bacteria suspension adds in the water body of room temperature cultivation mussel, presses
Take a picture than the ratio addition that water body is 1～3%.The i.e. volume of the water body of high density bacteria suspension and cultivation mussel
Ratio is 1～3:100, and further preferably, high density bacteria suspension adds in the water body of room temperature cultivation mussel, presses
Take a picture and add than the ratio that water body is 2%.The i.e. volume ratio of the water body of high density bacteria suspension and cultivation mussel
For 2:100.
After high density bacteria suspension being added in the water body of room temperature cultivation mussel, lactococcus lactis
Lactococcus lactis subsp.lactis (CICC 6242) continues natural propagation to play mussel body
The inhibitory action of interior vibrio parahaemolytious.
Compared with prior art, present invention have the advantage that
In the present invention, by lactococcus lactis Lactococcus lactis subsp.Lactis (CICC 6242)
Produce a kind of harmless and antibacterial is had the bacteriocin Nisin of strong killing action, it is possible to suppression is made a gift of
Vibrio parahaemolytious (Vp) breeding, lactococcus lactis Lactococcus lactis subsp.lactis in shellfish body
(CICC 6242) is bred by Vp in biological antagonist suppression mussel body and ensures mussel edible safety,
Be conducive to marketization utilization and extention, possess wide application prospect.
Detailed description of the invention
Embodiment 1
1) lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242) of Nisin is produced
High Density Cultivation, the density of setting out of CICC 6242 is 1.10 × 106Individual/mL, is seeded to fluid medium
(in terms of 1L, peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast extract 5.0g, diammonium hydrogen citrate 2.0g,
Glucose 20.0g, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.58g, manganese sulfate 0.25g,
Tween 80 1.0mL, water surplus；PH=6.3,121 DEG C of sterilizing 20min) in, inoculum concentration is 1%, 37 DEG C
Shaken cultivation 24h, bacterium solution density is 2.25 × 109Cfu/mL, obtains high density bacteria suspension.
2) by lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242) density it is
2.25×109The high density bacteria suspension of cfu/mL joins room temperature 25 DEG C according to comparing the ratio that water body is 1%
In the water body of cultivation mussel, in 25 DEG C of water bodys of room temperature, lactococcus lactis Lactococcus lactis subsp.
Lactis (CICC 6242) is bred by vibrio parahaemolytious in biological antagonist suppression mussel body and ensures mussel
Edible safety.
3) through step 1) and step 2) process after (after 24hr), in mussel, Vp density is subject to
To substantially suppression, specifically refer to table 1.
Lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242) is from Chinese industrial
Microbiological Culture Collection administrative center (CICC), strain keeps numbered 6242.
In mussel, Vp measures: according to standard SN 0173-2010, " export food vibrio parahaemolyticus is checked
Method " most probable number (most probable numer, MPN) 9 tube method.
Table 1
Embodiment 2
1) lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242) of Nisin is produced
High Density Cultivation, the density of setting out of CICC 6242 is 1.23 × 106Individual/mL, is seeded to fluid medium
(in terms of 1L, peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast extract 5.0g, diammonium hydrogen citrate 2.0g,
Glucose 20.0g, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.58g, manganese sulfate 0.25g,
Tween 80 1.0mL, water surplus；PH=6.3,121 DEG C of sterilizing 20min) in, inoculum concentration is 5%, 37 DEG C
Shaken cultivation 24h, bacterium solution density is 2.63 × 109Cfu/mL, obtains high density bacteria suspension.
2) by lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242) density it is
2.63×109The high density bacteria suspension of cfu/mL joins room temperature 25 DEG C according to comparing the ratio that water body is 3%
In the water body of cultivation mussel, in 25 DEG C of water bodys of room temperature, lactococcus lactis Lactococcus lactis subsp.
Lactis (CICC 6242) is bred by vibrio parahaemolytious in biological antagonist suppression mussel body and ensures mussel
Edible safety.
3) through step 1) and step 2) process after (after 24hr), in mussel, Vp density is subject to
To substantially suppression, specifically refer to table 2.
Lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242) is from Chinese industrial
Microbiological Culture Collection administrative center (CICC), strain keeps numbered 6242.
In mussel, Vp measures: according to standard SN 0173-2010, " export food vibrio parahaemolyticus is checked
Method " most probable number (most probable numer, MPN) 9 tube method.
Table 2
Embodiment 3
1) lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242) of Nisin is produced
High Density Cultivation, the density of setting out of CICC 6242 is 1.34 × 106Individual/mL, is seeded to fluid medium
(peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast extract 5.0g, diammonium hydrogen citrate 2.0g, glucose
20.0g, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.58g, manganese sulfate 0.25g, tween
80 1.0mL, water surplus；PH=6.3,121 DEG C of sterilizing 20min) in, inoculum concentration is 2%, and 37 DEG C are shaken
Swinging cultivation 24h, bacterium solution density is 2.52 × 109Cfu/mL, obtains high density bacteria suspension.
2) by lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242) density it is
2.52×109The high density bacteria suspension of cfu/mL joins room temperature 25 DEG C according to comparing the ratio that water body is 2%
In the water body of cultivation mussel, in 25 DEG C of water bodys of room temperature, lactococcus lactis Lactococcus lactis subsp.
Lactis (CICC 6242) is bred by vibrio parahaemolytious in biological antagonist suppression mussel body and ensures mussel
Edible safety.
3) through step 1) and step 2) process after (after 24hr), in mussel, Vp density is subject to
To substantially suppression, specifically refer to table 3.
Lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242) is from Chinese industrial
Microbiological Culture Collection administrative center (CICC), strain keeps numbered 6242.
In mussel, Vp measures: according to standard SN 0173-2010, " export food vibrio parahaemolyticus is checked
Method " most probable number (most probable numer, MPN) 9 tube method.
Table 3
Embodiment 4
1) lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242) of Nisin is produced
High Density Cultivation, the density of setting out of CICC 6242 is 1.07 × 106Individual/mL, is seeded to fluid medium
(peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast extract 5.0g, diammonium hydrogen citrate 2.0g, glucose
20.0g, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.58g, manganese sulfate 0.25g, tween
80 1.0mL, water surplus；PH=6.3,121 DEG C of sterilizing 20min) in, inoculum concentration is 3%, and 37 DEG C are shaken
Swinging cultivation 24h, bacterium solution density is 2.55 × 109Cfu/mL, obtains high density bacteria suspension.
2) by lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242) density it is
2.55×109The high density bacteria suspension of cfu/mL joins room temperature 25 DEG C according to comparing the ratio that water body is 2%
In the water body of cultivation mussel, in 25 DEG C of water bodys of room temperature, lactococcus lactis Lactococcus lactis subsp.
Lactis (CICC 6242) is bred by vibrio parahaemolytious in biological antagonist suppression mussel body and ensures mussel
Edible safety.
3) through step 1) and step 2) process after (after 24hr), in mussel, Vp density is subject to
To substantially suppression, specifically refer to table 4.
Lactococcus lactis Lactococcus lactis subsp.lactis (CICC 6242) is from Chinese industrial
Microbiological Culture Collection administrative center (CICC), strain keeps numbered 6242.
In mussel, Vp measures: according to standard SN 0173-2010, " export food vibrio parahaemolyticus is checked
Method " most probable number (most probable numer, MPN) 9 tube method.
Table 4
Claims (7)
1. by a method for vibrio parahaemolytious breeding in lactic acid bacteria biological antagonism suppression mussel body, its
It is characterised by, comprises the following steps:
1) lactococcus lactis Lactococcus lactis subsp.Lactis CICC 6242 is cultivated,
Making density is 109～1010The high density bacteria suspension of cfu/mL；
2) by step 1) in 109～1010The high density bacteria suspension of cfu/mL joins the water body of cultivation mussel
In, vibrio parahaemolytious breeding in suppression shellfish body.
The most according to claim 1 by secondary haemolysis arc in lactic acid bacteria biological antagonism suppression mussel body
The method of bacterium breeding, it is characterised in that step 1) in, by lactococcus lactis Lactococcus lactis subsp.
Lactis CICC 6242 cultivates, including: lactococcus lactis Lactococcus lactis subsp.Lactis
The density of setting out of CICC 6242 is 106～107Individual/mL, being seeded to inoculum concentration in fluid medium is
1%～5%, 33 DEG C～39 DEG C of shaken cultivation 20～28h, be 10 to bacterium solution density9～1010Cfu/mL's is highly dense
Degree bacteria suspension.
The most according to claim 2 by secondary haemolysis arc in lactic acid bacteria biological antagonism suppression mussel body
The method of bacterium breeding, it is characterised in that by lactococcus lactis Lactococcus lactis subsp.Lactis
CICC 6242 cultivates, including: lactococcus lactis Lactococcus lactis subsp.Lactis CICC
The density of setting out of 6242 is 106～107Individual/mL, being seeded to inoculum concentration in fluid medium is 2%～3%,
36 DEG C～37 DEG C of shaken cultivation 22～26h, be 10 to bacterium solution density9～1010The high density bacteria suspension of cfu/mL.
4. according to described in Claims 2 or 3 by secondary molten in lactic acid bacteria biological antagonism suppression mussel body
The method of blood vibrio breeding, it is characterised in that described fluid medium, in terms of 1L, including following
Component:
The pH value of this fluid medium regulates to 6.0～6.6.
The most according to claim 4 by secondary haemolysis arc in lactic acid bacteria biological antagonism suppression mussel body
The method of bacterium breeding, described fluid medium, in terms of 1L, including following components:
The pH value of this fluid medium regulates to 6.3, and this fluid medium is at 121 DEG C of sterilizing 20min.
The most according to claim 1 by secondary haemolysis arc in lactic acid bacteria biological antagonism suppression mussel body
The method of bacterium breeding, step 2) in, the volume of the water body of described high density bacteria suspension and cultivation mussel
Ratio is 1～3:100.
The most according to claim 6 by secondary haemolysis arc in lactic acid bacteria biological antagonism suppression mussel body
The method of bacterium breeding, step 2) in, the volume of the water body of described high density bacteria suspension and cultivation mussel
Ratio is 2:100.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114369612A (en) * | 2021-12-27 | 2022-04-19 | 甘肃奥林贝尔生物科技集团有限公司 | Construction method of genetic engineering bacteria capable of efficiently secreting Nisin, engineering bacteria and application thereof |
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| CN102150925A (en) * | 2011-01-10 | 2011-08-17 | 扬州大学 | Bacteriostat prepared by fermentation of lactobacillus and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102150925A (en) * | 2011-01-10 | 2011-08-17 | 扬州大学 | Bacteriostat prepared by fermentation of lactobacillus and preparation method thereof |
Non-Patent Citations (3)
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| S.D. SHEHANE等: "Isolation and preliminary characterization of bacteriocins", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| 刘君等: "水产源乳酸菌的多样性及抑菌活性研究", 《水产科学》 * |
| 陶静等: "固定化细胞发酵生产乳链菌肽工艺研究", 《中国食品添加剂》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114369612A (en) * | 2021-12-27 | 2022-04-19 | 甘肃奥林贝尔生物科技集团有限公司 | Construction method of genetic engineering bacteria capable of efficiently secreting Nisin, engineering bacteria and application thereof |
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