CN103289917B - A bdellovibrio bacteriovorus preparation, and a fermentation method and applications thereof - Google Patents

A bdellovibrio bacteriovorus preparation, and a fermentation method and applications thereof Download PDF

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CN103289917B
CN103289917B CN201310103758.3A CN201310103758A CN103289917B CN 103289917 B CN103289917 B CN 103289917B CN 201310103758 A CN201310103758 A CN 201310103758A CN 103289917 B CN103289917 B CN 103289917B
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bdellovibrio
preparation
bdellovibrio bacteriovorus
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bacteriovorus preparation
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CN103289917A (en
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蔡俊鹏
陈小红
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South China University of Technology SCUT
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Abstract

The invention discloses a bdellovibrio bacteriovorus preparation, and a fermentation method and applications thereof. The fermentation method comprises the following steps of: (1) preparation of host bacteria suspension; (2) preparation of bdellovibrio bacteriovorus swimming body concentrated solution; and (3) preparation of the bdellovibrio bacteriovorus preparation. Host bacteria adopted in the fermentation method are Gram positive bacteria which are beneficial or harmless to the environment, the method can keep and even improve the capability of bdellovibrio bacteriovorus for cracking certain Gram positive bacteria, and the bdellovibrio bacteriovorus preparation prepared by using the method has certain improvement on the cracking capability of pathogenic bacteria; and the prepared bdellovibrio bacteriovorus preparation can be directly used without more fermentation post treatment, so the use range of the preparation is greatly increased. The method relatively shortens the fermentation period at the same time of obtaining high-concentration bdellovibrio bacteriovorus bacterial solution so as to effectively solve the problems of over-high energy consumption and over-high productioncost caused by overlong fermentation time; and the obtained bdellovibrio bacteriovorus preparation has low cost and high activity.

Description

A kind of bdellovibrio bacteriovorus preparation and fermentation process thereof and application
The patent application of the present invention divisional application that to be application number be " 201010270772.9 ", the applying date of original application is " on August 31st, 2010 ", application number is " 201010270772.9 ", and denomination of invention is " a kind of bdellovibrio bacteriovorus preparation and fermentation process and application " thereof.
Technical field
The present invention relates to fermentation technical field, be specifically related to a kind of bdellovibrio bacteriovorus preparation and fermentation process thereof and application.
Background technology
The Main Means of current control, elimination pathogenic bacterium is still the method for various physics and/or chemistry, comprises various antibiotic use.The method of physics and/or chemistry all also exists obvious drawback, and first, microbiotic is abused in a large number, can cause the generation of some side effects, as the resistance, suppression profitable strain etc. of pathogenic bacteria.Secondly, though the method for acid-alkali treatment can reach certain sterilizing/sterilization effect, after harmful bacteria forms microbial film, the effect of these methods is just extremely undesirable.Finally, the method for physics can only be applied to a certain link of association area to the eliminating effect of various pathogenic bacterium, and is difficult to all links expanding to whole field.The method of biological control then can have powerful superiority, very likely makes the process etc. of large-scale water in its disease control of serving daily life and biological warfare and the attack of terrorism, food source contact scar.
The research and development of microbial ecological preparation are in recent years subject to each side and pay close attention to, and particularly the research of Bdellovibrio is subject to the favor of people.Since Bdellovibrio was found from 1963, because it is a kind of bacterial parasite, can the gram negative pathogenic bacteria such as cracking intestinal bacteria, Salmonellas, Aeromonas hydrophila, vibrios and harmless to humans and animals, and receive much concern.
Key Bdellovibrio being applied to control pathogenic bacterium obtains the bdellovibrio bacteriovorus preparation that concentration is high, cracking ability is strong.For this reason, people have carried out unremitting research to the preparation method of bdellovibrio bacteriovorus preparation, for example number of patent application is 93111749.6, name is called that intestinal bacteria kill by the national inventing patent application proposition high temperature (70 ~ 150 DEG C) of " bactericide prepared from biology and production method thereof " or chemicals (chloroform etc.), manufacture the Host Strains of deactivation, then cultivate Bdellovibrio with it, obtain bdellovibrio bacteriovorus preparation; Number of patent application 200810145709.5, name are called, and " production method of bdellovibrio bacteriovorus ecological preparation " provides the production method of bdellovibrio bacteriovorus ecological preparation, and main and traditional method uses unlike host e. coli being lyophilized into bacterium powder.Above-mentioned two parts of patent applications all adopt intestinal bacteria alive as Host Strains, finally understand influence ecological environment.And the present invention's Host Strains used is the bacterium of probiotics or environmental sound, can not threaten environment structure.The national inventing patent application that number of patent application 200810202809.7, name are called " fermentation method for producing of dual-purpose bdellovibrio " provides a kind of fermentation method for producing of dual-purpose bdellovibrio, namely first fermentation is for the suspension of Host Strains, then adds host strain turbid liquor and Bdellovibrio liquid ferments in the nutrient solution prepared.Although the Bdellovibrio content that this production method is produced is higher by (5 × 10 8pfu/mL), but its Host Strains has selected pathogenic Aeromonas hydrophila, and fermentation time longer (72 ~ 120h), can cause the increase of its production cost.In addition, this technology also may also exist the not high problem of the Bdellovibrio lytic activity of gained.And the present invention adopts be gram-positive microorganism is host, experiment proves, can maintain, even improve the cracking ability to other Gram-negative bacterias and positive bacteria with the Bdellovibrio that this host's fermentation obtains.
Summary of the invention
In order to overcome the problem that bdellovibrio bacteriovorus preparation content is low and lytic activity is low prepared by existing method, primary and foremost purpose of the present invention is the fermentation process providing a kind of bdellovibrio bacteriovorus preparation.
Another object of the present invention is to provide a kind of bdellovibrio bacteriovorus preparation prepared by above-mentioned fermentation process.
Another object of the present invention is the application providing above-mentioned bdellovibrio bacteriovorus preparation.
Object of the present invention is achieved through the following technical solutions: a kind of fermentation process of bdellovibrio bacteriovorus preparation, comprises the following steps:
(1) preparation of host bacteria suspension
Host Strains is cultured to logarithmic phase, and collect thalline, then adjust concentration with phosphate buffered saline buffer, obtaining concentration is 10 17~ 10 22the Host Strains suspension of cfu/mL, is then placed in 2 ~ 15 DEG C and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In phosphate buffered saline buffer, add Host Strains suspension prepared by step (1), the concentration of adjustment Host Strains is 10 10~ 10 15cfu/mL, access Bdellovibrio spot again, then this nutrient solution is cultivated 20 ~ 60h at 20 ~ 40 DEG C, then at 2 ~ 15 DEG C, the centrifugal 15 ~ 35min of 5000 ~ 8000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 2 ~ 15 DEG C, the centrifugal 15 ~ 40min of 12000 ~ 20000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, the concentration adding phosphate buffered saline buffer adjustment Bdellovibrio nectophore in precipitation is 10 6~ 10 9pfu/mL, obtains Bdellovibrio nectophore concentrated solution, is placed in 2 ~ 15 DEG C and saves backup;
(3) preparation of bdellovibrio bacteriovorus preparation:
Be dissolved in by sodium-chlor in solvent and form the sodium chloride solution that mass volume ratio concentration is 0 ~ 30g/L, sterilizing, obtains fermention medium; Add Host Strains suspension prepared by step (1) and Bdellovibrio nectophore concentrated solution prepared by step (2) in the fermentation medium, make Host Strains initial concentration be 10 10~ 10 15cfu/mL, Bdellovibrio nectophore initial concentration is 10 1~ 10 3pfu/mL, ferments; Leavening temperature controls at 28 ~ 30 DEG C, and pH value controls 7.2 ~ 7.6; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 20% ~ 30%; Add 1 ~ 3 Host Strains suspension in fermenting process, add 12 ~ 18h interval time of Host Strains suspension at every turn, the amount at every turn adding Host Strains suspension with the Host Strains newly added concentration in the fermentation medium for 10 10~ 10 15cfu/mL is as the criterion; Namely fermentation culture 36 ~ 48h makes bdellovibrio bacteriovorus preparation; Described solvent is DNB liquid nutrient medium, distilled water or phosphate buffered saline buffer.
Step (1) described Host Strains is gram-positive microorganism that is useful or environmental sound, be preferably subtilis (Bacillus subtilis), bacillus natto (Bacillus natto), bifidumbacterium bifidum (Bifidobacterium bifidum), enterococcus faecalis (Enterococcus faecalis), lactobacillus bulgaricus (Lactobacillus bulgaricus), pediococcus acidilactici (Pediococcus acidilactici), streptococcus acidi lactici (Streptococcus lactis), faecium (Enterococcus faecium), Bacillus licheniformis (Bacillus licheniformis), Lactobacterium acidophilum (Lactobacillus acidophilus), lactobacterium casei (Lactobacillus casei), lactobacillus lactis (Lactobacillus lactis), plant lactobacillus (Lactobacillus plantarum) or Pediococcus pentosaceus (Pediococcus pentasaceus),
Step (2) described Bdellovibrio is BDF01, BDF02, BDF03, BDJ01, BDJ02, BDM01, BDS01, BDS02, BDSM08 or BDFM05.
Described BDF01 is preserved in the China typical culture collection center in Wuhan University of Wuhan, China city on January 13rd, 2008, deposit number is CCTCC NO:M208008; Described BDF02 is preserved in the China typical culture collection center in Wuhan University of Wuhan, China city on January 13rd, 2008, deposit number is CCTCC NO:M208009; Described BDF03 is preserved in the China typical culture collection center in Wuhan University of Wuhan, China city on January 13rd, 2008, deposit number is CCTCC NO:M208010; Described BDJ01 is preserved in the China typical culture collection center in Wuhan University of Wuhan, China city on January 14th, 2008, deposit number is CCTCC NO:M208011; Described BDJ02 is preserved in the China typical culture collection center in Wuhan University of Wuhan, China city on January 14th, 2008, deposit number is CCTCC NO:M208012; Described BDM01 is preserved in the China typical culture collection center in Wuhan University of Wuhan, China city on April 28th, 2008, deposit number is CCTCC NO:M208066; Described BDS01 is preserved in the China typical culture collection center in Wuhan University of Wuhan, China city on August 7th, 2009, deposit number is CCTCC NO:M209169; Described BDS02 is preserved in the China typical culture collection center in Wuhan University of Wuhan, China city on August 7th, 2009, deposit number is CCTCC NO:M209170; Described BDSM08 is preserved in the China typical culture collection center in Wuhan University of Wuhan, China city on August 7th, 2009, deposit number is CCTCC NO:M209171; Described BDFM05 is preserved in the China typical culture collection center in Wuhan University of Wuhan, China city on August 7th, 2009, deposit number is CCTCC NO:M209172.
The mode of the described collection thalline of step (1) obtains preferably by centrifugation, and centrifugal condition is 2 ~ 15 DEG C, the centrifugal 15 ~ 35min of 5000 ~ 8000rpm;
The concentration of the described Host Strains suspension of step (1) is preferably 10 17~ 10 22cfu/mL;
The described Bdellovibrio spot of step (2) adopts ordinary method (be 200910042274.6 according to application number, the patent application that name is called " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ") to carry out separation and obtains.
Described in step (1) ~ any one of (3), phosphate buffered saline buffer is preferably concentration to be 0.1 ~ 0.3mol/L, pH be 7.2 ~ 7.6 phosphate buffered saline buffer;
The concentration of the described Bdellovibrio nectophore concentrated solution of step (2) is preferably 10 6~ 10 9pfu/mL;
When step (3) described Bdellovibrio derives from salt water environment, described solvent is DNB liquid nutrient medium or distilled water, and the mass volume ratio concentration of described sodium chloride solution is 25 ~ 30g/L;
When step (3) described Bdellovibrio derives from salt-fresh water environment, described solvent is DNB liquid nutrient medium or distilled water, and the mass volume ratio concentration of described sodium chloride solution is 5 ~ 10g/L;
When step (3) described Bdellovibrio derives from fresh water environment, do not add sodium-chlor;
The condition optimization of step (3) described sterilizing is 121 DEG C of sterilizing 20min;
The concentration of step (3) described bdellovibrio bacteriovorus preparation is 10 8~ 10 12pfu/mL;
The described DNB liquid nutrient medium of step (3) is dissolved in 1000mL distilled water by nutrient broth 0.8g, caseinic acid hydrolyzate 0.5g and yeastex extract 0.1g, adjust ph to 7.2 ~ 7.6.
A kind of bdellovibrio bacteriovorus preparation, is prepared by the fermentation process of described bdellovibrio bacteriovorus preparation;
Described bdellovibrio bacteriovorus preparation can reach the object of control germ evil by other pathogenic bacterium of cracking or potentially pathogenic organism.Described bdellovibrio bacteriovorus preparation not only directly can make preparation, also by further centrifugal, makes simple telotroch preparation, leech liposome preparation and their preparation be mixed in proportion.
The present invention has following advantage and effect relative to prior art:
(1) Host Strains adopted in fermentation process of the present invention is gram-positive microorganism that is useful or environmental sound.Like this, on the one hand, can keep, even improve the ability of some Gram-negative bacterias of Bdellovibrio cracking, and experiment proves, the cracking ability of the bdellovibrio bacteriovorus preparation obtained by the method to pathogenic bacterium improves; On the other hand, obtained bdellovibrio bacteriovorus preparation can directly use, without the need to through aftertreatment of more fermenting; Moreover because host is the bacterial strain of probiotics or environmental sound, the use range of said preparation also increases greatly.
(2) the present invention is while obtaining high density Bdellovibrio bacterium liquid, relatively also shorten fermentation period, effectively like this solve that fermentation time is long and energy consumption that is that cause is excessive, the problem that production cost is too high, the bdellovibrio bacteriovorus preparation obtained not only cost is low, and active high.
Accompanying drawing explanation
Fig. 1 is the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (subtilis and intestinal bacteria) fermentation to the cracking ability-time diagram of Salmonella typhimurium and streptococcus aureus.
Fig. 2 is the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (bacillus natto and intestinal bacteria) fermentation to the cracking ability-time diagram of listeria monocytogenes and Salmonella choleraesuls.
Fig. 3 is the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (bifidumbacterium bifidum and Salmonella typhimurium) fermentation to the cracking ability-time diagram of streptococcus suis II and streptococcus uberis.
Fig. 4 is that the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (enterococcus faecalis and Salmonella choleraesuls) fermentation is to staphylococcus epidermidis and colibacillary cracking ability-time diagram.
Fig. 5 is the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (lactobacillus bulgaricus and intestinal bacteria) fermentation to the cracking ability-time diagram of streptococcus equisimilis and Salmonella typhimurium.
Fig. 6 is that the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (pediococcus acidilactici and Aeromonas hydrophila) fermentation is to streptococcus uberis and colibacillary cracking ability-time diagram.
Fig. 7 is the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (streptococcus acidi lactici and pig hammer II type) fermentation to the cracking ability-time diagram of Salmonella choleraesuls and staphylococcus epidermidis.
Fig. 8 is the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (faecium and intestinal bacteria) fermentation to the cracking ability-time diagram of Salmonella typhimurium and listeria monocytogenes.
Fig. 9 is the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (Bacillus licheniformis and intestinal bacteria) fermentation to the cracking ability-time diagram of Aeromonas hydrophila and streptococcus equisimilis.
Figure 10 is the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (Lactobacterium acidophilum and intestinal bacteria) fermentation to the cracking ability-time diagram of streptococcus aureus and Salmonella choleraesuls.
Figure 11 is that the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (lactobacterium casei and Aeromonas hydrophila) fermentation is to streptococcus equisimilis and colibacillary cracking ability-time diagram.
Figure 12 is the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (lactobacillus lactis and pig hammer II type) fermentation to the cracking ability-time diagram of staphylococcus epidermidis and Aeromonas hydrophila.
Figure 13 is the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (plant lactobacillus and Salmonella choleraesuls) fermentation to the cracking ability-time diagram of streptococcus agalactiae and Salmonella typhimurium.
Figure 14 is the bdellovibrio bacteriovorus preparation that obtains with two kinds of different hosts (Pediococcus pentosaceus and intestinal bacteria) fermentation to the cracking ability-time diagram of streptococcus uberis and suis II type.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
Respectively with subtilis (bacterium numbering: GIM1.136, derive from Guangdong Province's Culture Collection) and intestinal bacteria (Escherichia coli, bacterium numbering: GIM1.137, derive from Guangdong Province's Culture Collection) prepare bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B for Host Strains, detailed process is as follows:
(1) preparation of bacillus subtilis bacteria suspension
Subtilis is inoculated in (peptone 10g in nutrient broth medium, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2), be placed in 33 DEG C of shaking tables and cultivate 18h, make it be in logarithmic phase, nutrient solution under 4 DEG C of conditions, the centrifugal 25min of 5000rpm, retain precipitation abandoning supernatant, in precipitation, add potassium-sodium phosphates salt buffer (concentration is 0.2mol/L, pH7.6), obtaining concentration is 10 19the bacillus subtilis suspended liquid of cfu/mL, is then placed in 4 DEG C of refrigerators and saves backup;
The same method, preparation concentration is 10 19the intestinal bacteria suspension of cfu/mL, is placed in 4 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In potassium-sodium phosphates salt buffer (concentration is 0.2mol/L, pH7.6), add E. coli suspension prepared by step (1), adjusting colibacillary concentration is 10 12cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treat plaque appears in the double-deck agar plate containing Host Strains) cultivate the Bdellovibrio BDJ02(of separation out from seawater and be preserved in China typical culture collection center in Wuhan University of Wuhan, China city on January 14th, 2008, deposit number is CCTCC NO:M208012) spot, then this nutrient solution is cultivated 48h at 30 DEG C, again at 4 DEG C, the centrifugal 35min of 5000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 4 DEG C, the centrifugal 30min of 13000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.2mol/L is added in precipitation, the potassium-sodium phosphates salt buffer of pH7.6, the concentration of adjustment Bdellovibrio nectophore is 10 8pfu/mL, obtains Bdellovibrio BDJ02 telotroch concentrated solution, is placed in 2 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
In DNB liquid nutrient medium, add sodium-chlor, add that quality is DNB liquid nutrient medium volume 2.5% of sodium-chlor, load fermentor tank, 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add bacillus subtilis suspended liquid prepared by step (1) and Bdellovibrio BDJ02 telotroch concentrated solution prepared by step (2), the initial concentration of the subtilis in fermention medium is 10 12cfu/mL, Bdellovibrio initial concentration is 10 2pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 28 DEG C; The fermention medium pH value during the fermentation adding bacillus subtilis suspended liquid and Bdellovibrio nectophore concentrated solution controls 7.6; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 28%; Add twice bacillus subtilis suspended liquid in culturing process, add 15h interval time of bacillus subtilis suspended liquid at every turn, the amount at every turn adding bacillus subtilis suspended liquid with the subtilis newly added concentration in the fermentation medium for 10 12cfu/mL is as the criterion, and namely fermentation culture 48h makes concentration is 1 × 10 9the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add E. coli suspension prepared by step (1) and Bdellovibrio BDJ02 telotroch concentrated solution prepared by step (2), obtained concentration is 1 × 10 9the bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all has good lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, streptococcus aureuses.Respectively with Salmonella typhimurium (the Salmonella typhimurium that is feminine gender to gram, strain number: GIM1.237, derive from Guangdong Province's Culture Collection) and be positive streptococcus aureus (Staphylococcus aureus to gram, strain number: GIM1.142, derives from Guangdong Province's Culture Collection) be cracked into example.Be equipped with the potassium-sodium phosphates salt buffer that 4 bottles are respectively equipped with 50mL, add the bacterial sediment that conventional culture methods obtains Salmonella typhimurium and streptococcus aureus respectively, often kind of a bacterium has two bottles.Regulate the cell concentration consistent (10 of 4 bottles of damping fluids 11cfu/mL).In two bottles of buffer systems containing Salmonella typhimurium, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, in two bottles of buffer systems containing streptococcus aureus, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these nine moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h -1~ 10 -11extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in Figure 1.As can be seen from Figure 1, compared with the bdellovibrio bacteriovorus preparation B obtained with Escherichia coli fermentation, be not only that negative Salmonella typhimurium has stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of fermentation of bacillus subtilis, and be that the lytic effect of positive streptococcus aureus is also good than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by subtilis not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 2
Be that Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B with bacillus natto (bacterium numbering: CGMCC1.1086 derives from China Committee for Culture Collection of Microorganisms's common micro-organisms center) and intestinal bacteria, detailed process is as follows:
(1) preparation of bacillus natto suspension
Bacillus natto is inoculated in nutrient broth medium (peptone 10g, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2) in, be placed in 35 DEG C of shaking tables and cultivate 15h, make it be in logarithmic phase, nutrient solution under 4 DEG C of conditions, the centrifugal 25min of 6000rpm, retain precipitation abandoning supernatant, in precipitation, add sodium phosphate buffer (concentration is 0.1mol/L, pH7.4), obtaining concentration is 10 18the bacillus natto suspension of cfu/mL, is then placed in 2 DEG C of refrigerators and saves backup;
The same method, preparation concentration is 10 18the intestinal bacteria suspension of cfu/mL, is placed in 2 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In sodium phosphate buffer (concentration is 0.1mol/L, pH7.4), add E. coli suspension prepared by step (1), adjusting colibacillary concentration is 10 13cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treat plaque appears in the double-deck agar plate containing Host Strains) cultivate the Bdellovibrio BDF02(of separation out from fresh water and be preserved in China typical culture collection center in Wuhan University of Wuhan, China city on January 13rd, 2008, deposit number is CCTCC NO:M208009) spot, then this nutrient solution is cultivated 45h at 30 DEG C, again at 4 DEG C, the centrifugal 25min of 6000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 4 DEG C, the centrifugal 28min of 14000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.1mol/L is added in precipitation, the sodium phosphate buffer of pH7.4, the concentration of adjustment Bdellovibrio nectophore is 10 9pfu/mL, obtains Bdellovibrio BDF02 telotroch concentrated solution, is placed in 4 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
DNB liquid nutrient medium is loaded fermentor tank, and 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add bacillus natto suspension prepared by step (1) and Bdellovibrio BDF02 telotroch concentrated solution prepared by step (2), the initial concentration of the bacillus natto in fermention medium is 10 13cfu/mL, Bdellovibrio initial concentration is 10 2pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 29 DEG C; The fermention medium pH value during the fermentation adding bacillus natto suspension and Bdellovibrio nectophore concentrated solution controls 7.4; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 28%; Add twice bacillus natto suspension in culturing process, add 16h interval time of bacillus natto suspension at every turn, the amount at every turn adding bacillus natto suspension is the bacillus natto concentration in the fermentation medium newly added is 10 13cfu/mL is as the criterion, and namely fermentation culture 45h makes concentration is 1 × 10 10the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add E. coli suspension prepared by step (1) and Bdellovibrio BDF02 telotroch concentrated solution prepared by step (2), obtained concentration is 1 × 10 10the bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B has good lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, listeria monocytogenes.Respectively with listeria monocytogenes (the Listeria monocytohenes that is the positive to gram, strain number: GIM1.228, derive from Guangdong Province's Culture Collection) cracking and be negative Salmonella choleraesuls (Salmonella enterica to gram, strain number: GIM1.244, derives from Guangdong Province's Culture Collection) be cracked into example.Being equipped with 4 bottles is respectively equipped with in the potassium-sodium phosphates salt buffer of 50mL, adds the bacterial sediment that conventional culture methods obtains listeria monocytogenes and Salmonella choleraesuls respectively, each two bottles of often kind of bacterium, regulates the cell concentration consistent (10 of 4 bottles of damping fluids 10cfu/mL).In two bottles of buffer systems containing listeria monocytogenes, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, in two bottles of buffer systems containing Salmonella choleraesuls, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1m respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these eight moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h -1~ 10 -10extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in Figure 2.As can be seen from Figure 2, compared with the bdellovibrio bacteriovorus preparation B with Escherichia coli fermentation, be not only that negative Salmonella choleraesuls have stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of bacillus natto to ferment, and be that the lytic effect of positive listeria monocytogenes is also better than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by bacillus natto not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 3
Be that Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B with bifidumbacterium bifidum (bacterium numbering: GIM1.169 derives from Guangdong Province's Culture Collection) and Salmonella typhimurium respectively, detailed process is as follows:
(1) preparation of bifidumbacterium bifidum suspension
Bifidumbacterium bifidum is inoculated in PTYG substratum, be placed in 37 DEG C of shaking tables and cultivate 24h, it is made to be in logarithmic phase, nutrient solution is under 8 DEG C of conditions, the centrifugal 20min of 7000rpm, retains precipitation abandoning supernatant, and in precipitation, adding potassium phosphate salt damping fluid, (concentration is 0.3mol/L, pH7.5), obtaining concentration is 10 22the bifidumbacterium bifidum suspension of cfu/mL, is then placed in 8 DEG C of refrigerators and saves backup;
The same method, preparing concentration with nutrient broth medium is 10 22the Salmonella typhimurium suspension of cfu/mL, is placed in 8 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In potassium phosphate salt damping fluid (concentration is 0.3mol/L, pH7.5), add Salmonella typhimurium suspension prepared by step (1), the concentration of adjustment Salmonella typhimurium is 10 10cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treat plaque appears in the double-deck agar plate containing Host Strains) cultivate the Bdellovibrio BDS02(of separation out from soil and be preserved in China typical culture collection center in Wuhan University of Wuhan, China city on August 7th, 2009, deposit number is CCTCC NO:M209170) spot, then this nutrient solution is cultivated 50h at 30 DEG C, again at 8 DEG C, the centrifugal 20min of 7000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 8 DEG C, the centrifugal 20min of 16000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.3mol/L is added in precipitation, the potassium phosphate salt damping fluid of pH7.5, the concentration of adjustment Bdellovibrio nectophore is 10 9pfu/mL, obtains Bdellovibrio BDS02 telotroch concentrated solution, is placed in 15 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
Add sodium-chlor at DNB liquid nutrient medium, add that quality is DNB liquid nutrient medium volume 0.7% of sodium-chlor, load fermentor tank, 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add bifidumbacterium bifidum suspension prepared by step (1) and Bdellovibrio BDS02 telotroch concentrated solution prepared by step (2), the initial concentration of the bifidumbacterium bifidum in fermention medium is 10 15cfu/mL, Bdellovibrio initial concentration is 10 3pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 30 DEG C; The fermention medium pH value during the fermentation adding bifidumbacterium bifidum suspension and Bdellovibrio nectophore concentrated solution controls 7.2; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 22%; Add a bifidumbacterium bifidum suspension in culturing process, add 12h interval time of bifidumbacterium bifidum suspension at every turn, the amount at every turn adding bifidumbacterium bifidum suspension with the bifidumbacterium bifidum newly added concentration in the fermentation medium for 10 15cfu/mL is as the criterion, and namely fermentation culture 36h makes concentration is 5 × 10 11the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add Salmonella typhimurium suspension prepared by step (1) and Bdellovibrio BDS02 telotroch concentrated solution prepared by step (2), obtained concentration is 5 × 10 11the bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all has lytic effect to pathogenic bacterium such as streptococcus uberis, Salmonellas, swine streptococcus.Respectively with streptococcus suis II (the Streptococcus suis that is feminine gender to gram, strain number: CVCC3306, derive from National Veterinary Culture Collection) and be positive streptococcus uberis (Streptococcus uberis to gram, strain number: 700407, derives from Shanghai three and steps on Science and Technology Ltd.) be cracked into example.Being equipped with 4 bottles is respectively equipped with in the potassium-sodium phosphates salt buffer of 50mL, adds the bacterial sediment that conventional culture methods obtains streptococcus suis II and streptococcus uberis respectively, each two bottles of often kind of bacterium, regulates the cell concentration consistent (10 of 4 bottles of damping fluids 11cfu/mL).In two bottles of buffer systems containing streptococcus suis II, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, in two bottles of buffer systems containing streptococcus uberis, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these nine moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h -1~ 10 -11extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in Figure 3.As can be seen from Figure 3, compared with the bdellovibrio bacteriovorus preparation B fermented with Salmonella typhimurium, be not only that negative streptococcus suis II has stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of bifidumbacterium bifidum fermentation, and be that the lytic effect of positive streptococcus uberis is also better than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by bifidumbacterium bifidum not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 4
Respectively with enterococcus faecalis (Enterococcus faecalis, bacterium numbering: CGMCC1.131, derive from China Committee for Culture Collection of Microorganisms's common micro-organisms center) and Salmonella choleraesuls be that Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B, detailed process is as follows:
(1) preparation of enterococcus faecalis suspension
Enterococcus faecalis is inoculated in nutrient broth medium (peptone 10g, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2) in, be placed in 30 DEG C of shaking tables and cultivate 24h, make it be in logarithmic phase, nutrient solution under 4 DEG C of conditions, the centrifugal 20min of 6000rpm, retain precipitation abandoning supernatant, in precipitation, add sodium phosphate buffer (concentration is 0.2mol/L, pH7.4), obtaining concentration is 10 20the enterococcus faecalis suspension of cfu/mL, is then placed in 2 ~ 4 DEG C of refrigerators and saves backup;
The same method, preparation concentration is 10 20the Salmonella choleraesuls suspension of cfu/mL, is placed in 2 ~ 4 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In sodium phosphate buffer (concentration is 0.2mol/L, pH7.4), add Salmonella choleraesuls suspension prepared by step (1), the concentration of adjustment Salmonella choleraesuls is 10 12cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treat plaque appears in the double-deck agar plate containing Host Strains) cultivate the Bdellovibrio BDFM05(of separation out from soil and be preserved in China typical culture collection center in Wuhan University of Wuhan, China city on August 7th, 2009, deposit number is CCTCC NO:M209172) spot, then this nutrient solution is cultivated 38h at 30 DEG C, again at 4 DEG C, the centrifugal 20min of 6000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 4 DEG C, the centrifugal 22min of 15000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.2mol/L is added in precipitation, the sodium phosphate buffer of pH7.4, the concentration of adjustment Bdellovibrio nectophore is 10 9pfu/mL, obtains Bdellovibrio BDFM05 telotroch concentrated solution, is placed in 4 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
Sodium phosphate buffer (concentration is 0.2mol/L, pH7.6), load fermentor tank, 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add enterococcus faecalis suspension prepared by step (1) and Bdellovibrio BDFM05 telotroch concentrated solution prepared by step (2), the initial concentration of the enterococcus faecalis in fermention medium is 10 14cfu/mL, Bdellovibrio initial concentration is 10 2pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 30 DEG C; The fermention medium pH value during the fermentation adding enterococcus faecalis suspension and Bdellovibrio nectophore concentrated solution controls 7.2; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 30%; Add twice enterococcus faecalis suspension in culturing process, add 18h interval time of enterococcus faecalis suspension at every turn, the amount at every turn adding enterococcus faecalis suspension with the enterococcus faecalis newly added concentration in the fermentation medium for 10 14cfu/mL is as the criterion, and namely fermentation culture 48h makes concentration is 5 × 10 10the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add Salmonella choleraesuls suspension prepared by step (1) and Bdellovibrio BDFM05 telotroch concentrated solution prepared by step (2), obtained concentration is 5 × 10 10the bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all has lytic effect to pathogenic bacterium such as intestinal bacteria, Salmonellas, staphylococcus epidermidiss.Be negative be colibacillaryly cracked into example with the staphylococcus epidermidis (Staphylococcus epidermidis, strain number: GIM1.143 derive from Guangdong Province's Culture Collection) that is the positive to gram with to gram respectively.Being equipped with 4 bottles is respectively equipped with in the potassium-sodium phosphates salt buffer of 50mL, respectively adds conventional culture methods respectively and obtains staphylococcus epidermidis and colibacillary bacterial sediment, often kind of bacterium two bottles, regulates the cell concentration consistent (10 of 4 bottles of damping fluids 12cfu/mL).In two bottles of buffer systems containing staphylococcus epidermidis, add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, at two bottles containing in colibacillary buffer system, add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these nine moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h -1~ 10 -12extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in Figure 4.As can be seen from Figure 4, compared with the bdellovibrio bacteriovorus preparation B fermented with Salmonella choleraesuls, be not only that negative intestinal bacteria have stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of Enterococcus faecalis fermentation, and be that the lytic effect of positive staphylococcus epidermidis is also better than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by enterococcus faecalis not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 5
Be that Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B with lactobacillus bulgaricus (bacterium numbering: GIM1.80 derives from Guangdong Province's Culture Collection) and intestinal bacteria respectively, detailed process is as follows:
(1) preparation of lactobacillus bulgaricus suspension
Lactobacillus bulgaricus is inoculated in nutrient broth medium (peptone 10g, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2) in, be placed in 37 DEG C of shaking tables and cultivate 20h, make it be in logarithmic phase, nutrient solution under 10 DEG C of conditions, the centrifugal 25min of 6000rpm, retain precipitation abandoning supernatant, in precipitation, add potassium phosphate salt damping fluid (concentration is 0.3mol/L, pH7.3), obtaining concentration is 10 17the lactobacillus bulgaricus suspension of cfu/mL, is then placed in 2 ~ 4 DEG C of refrigerators and saves backup;
The same method, preparation concentration is 10 17the intestinal bacteria suspension of cfu/mL, is placed in 2 ~ 4 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In potassium phosphate salt damping fluid (concentration is 0.3mol/L, pH7.3), add E. coli suspension prepared by step (1), adjusting colibacillary concentration is 10 13cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treat plaque appears in the double-deck agar plate containing Host Strains) cultivate the Bdellovibrio BDJ02(of separation out from seawater and be preserved in China typical culture collection center in Wuhan University of Wuhan, China city on January 14th, 2008, deposit number is CCTCC NO:M208012) spot, then this nutrient solution is cultivated 40h at 30 DEG C, again at 10 DEG C, the centrifugal 25min of 6000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 15 DEG C, the centrifugal 20min of 15000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.3mol/L is added in precipitation, the potassium phosphate salt damping fluid of pH7.3, the concentration of adjustment Bdellovibrio nectophore is 10 8pfu/mL, obtains Bdellovibrio BDJ02 telotroch concentrated solution, is placed in 2 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
In distilled water, add sodium-chlor, add that quality is distilled water volume 2.8% of sodium-chlor, load fermentor tank, 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add lactobacillus bulgaricus suspension prepared by step (1) and Bdellovibrio BDJ02 telotroch concentrated solution prepared by step (2), the initial concentration of the lactobacillus bulgaricus in fermention medium is 10 13cfu/mL, Bdellovibrio initial concentration is 10pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 28 DEG C; The fermention medium pH value during the fermentation adding lactobacillus bulgaricus suspension and Bdellovibrio nectophore concentrated solution controls 7.6; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 21%; Add 2 lactobacillus bulgaricus suspension in culturing process, add 18h interval time of lactobacillus bulgaricus suspension at every turn, the amount at every turn adding lactobacillus bulgaricus suspension with the lactobacillus bulgaricus newly added concentration in the fermentation medium for 10 13cfu/mL is as the criterion, and namely fermentation culture 40h makes concentration is 1 × 10 11the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add E. coli suspension prepared by step (1) and Bdellovibrio BDJ02 telotroch concentrated solution prepared by step (2), obtained concentration is 1 × 10 11the bdellovibrio bacteriovorus preparation B of pfu/mL.
This preparation has good lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, streptococcus equisimilises.Respectively with the streptococcus equisimilis (Streptococcus equinus, strain number: cvcc1925 derive from National Veterinary Culture Collection) that is the positive to gram and to gram be negative Salmonella typhimurium be cracked into example.Being equipped with 4 bottles is respectively equipped with in the potassium-sodium phosphates salt buffer of 50mL, respectively adds the bacterial sediment that conventional culture methods obtains streptococcus equisimilis and Salmonella typhimurium respectively, often kind of bacterium two bottles, regulates the cell concentration consistent (10 of two bottles of damping fluids 11cfu/mL).In two bottles of buffer systems containing streptococcus equisimilis, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, in two bottles of buffer systems containing Salmonella typhimurium, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL bacterium respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these eight moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h -1~ 10 -10extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in Figure 5.As can be seen from Figure 5, compared with the bdellovibrio bacteriovorus preparation B with Escherichia coli fermentation, be not only that negative Salmonella typhimurium has stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of fermentation using lactobacillus bulgaricus, and be that positive streptococcus equisimilis streptococcus equisimilis lytic effect is also better than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by lactobacillus bulgaricus not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 6
Respectively with pediococcus acidilactici (bacterium numbering: GIM1.263, derive from Guangdong Province's Culture Collection) and Aeromonas hydrophila (Aeromonas hydrophila, bacterium numbering: GIM1.172, derive from Guangdong Province's Culture Collection) prepare bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B for Host Strains, detailed process is as follows:
(1) preparation of pediococcus acidilactici suspension
Pediococcus acidilactici is inoculated in nutrient broth medium (peptone 10g, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2) in, be placed in 30 DEG C of shaking tables and cultivate 18h, make it be in logarithmic phase, nutrient solution under 5 DEG C of conditions, the centrifugal 15min of 7000rpm, retain precipitation abandoning supernatant, in precipitation, add sodium phosphate buffer (concentration is 0.1mol/L, pH7.4), obtaining concentration is 10 22the pediococcus acidilactici suspension of cfu/mL, is then placed in 15 DEG C of refrigerators and saves backup;
The same method, preparation concentration is 10 22the Aeromonas hydrophila suspension of cfu/mL, is placed in 15 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In sodium phosphate buffer (concentration is 0.1mol/L, pH7.4), add Aeromonas hydrophila suspension prepared by step (1), the concentration of adjustment Aeromonas hydrophila is 10 15cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treat plaque appears in the double-deck agar plate containing Host Strains) cultivate the Bdellovibrio BDF03(of separation out from fresh water and be preserved in China typical culture collection center in Wuhan University of Wuhan, China city on January 13rd, 2008, deposit number is CCTCC NO:M208010) spot, then this nutrient solution is cultivated 54h at 30 DEG C, again at 5 DEG C, the centrifugal 20min of 7000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 5 DEG C, the centrifugal 25min of 15000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.1mol/L is added in precipitation, the sodium phosphate buffer of pH7.4, the concentration of adjustment Bdellovibrio nectophore is 10 9pfu/mL, obtains Bdellovibrio BDF03 telotroch concentrated solution, is placed in 10 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
Distilled water is loaded fermentor tank, and 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add pediococcus acidilactici suspension prepared by step (1) and Bdellovibrio BDF03 telotroch concentrated solution prepared by step (2), the initial concentration of the pediococcus acidilactici in fermention medium is 10 14cfu/mL, Bdellovibrio initial concentration is 10pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 30 DEG C; The fermention medium pH value during the fermentation adding pediococcus acidilactici suspension and Bdellovibrio nectophore concentrated solution controls 7.4; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 24%; Add twice pediococcus acidilactici suspension in culturing process, add 15h interval time of pediococcus acidilactici suspension at every turn, the amount at every turn adding pediococcus acidilactici suspension with the pediococcus acidilactici newly added concentration in the fermentation medium for 10 14namely cfu/mL, fermentation culture 45h make concentration is 5 × 10 8bdellovibrio bacteriovorus preparation A.
Same method, in the fermention medium of 30 DEG C, add Aeromonas hydrophila suspension prepared by step (1) and Bdellovibrio BDF03 telotroch concentrated solution prepared by step (2), obtained concentration is 5 × 10 8bdellovibrio bacteriovorus preparation B.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B has good lytic effect to pathogenic bacterium such as intestinal bacteria, Salmonellas, streptococcus uberises.Respectively with to streptococcus uberis be colibacillaryly cracked into example.Being equipped with 4 bottles is respectively equipped with in the potassium-sodium phosphates salt buffer of 50mL, respectively adds conventional culture methods respectively and obtains streptococcus uberis and colibacillary bacterial sediment, often kind of bacterium 2 bottles, regulates the cell concentration consistent (10 of 4 bottles of damping fluids 10cfu/mL).In two bottles of buffer systems containing streptococcus uberis, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, at two bottles containing in colibacillary buffer system, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these nine moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h -1~ 10 -10extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in Figure 6.As can be seen from Figure 6, compared with the bdellovibrio bacteriovorus preparation B fermented with Aeromonas hydrophila, be not only that negative intestinal bacteria have stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of pediococcus acidilactici fermentation, and be that the lytic effect of positive streptococcus uberis is also better than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by pediococcus acidilactici not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 7
Be that Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B with streptococcus acidi lactici (bacterium numbering: GIM1.156 derives from Guangdong Province's Culture Collection) and streptococcus suis II respectively, detailed process is as follows:
(1) preparation of nisin bacteria suspension
Streptococcus acidi lactici is inoculated in nutrient broth medium (peptone 10g, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2) in, be placed in 37 DEG C of shaking tables and cultivate 18h, make it be in logarithmic phase, nutrient solution under 4 DEG C of conditions, the centrifugal 27min of 6000rpm, retain precipitation abandoning supernatant, in precipitation, add potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), obtaining concentration is 10 20the streptococcus acidi lactici suspension of cfu/mL, is then placed in 4 DEG C of refrigerators and saves backup;
The same method, preparation concentration is 10 20the streptococcus suis II suspension of cfu/mL, is placed in 4 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), add streptococcus suis II suspension prepared by step (1), the concentration of adjustment streptococcus suis II is 10 15cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treating plaque appears in the double-deck agar plate containing Host Strains) the Bdellovibrio BDSM08(of separation soil that cultivates out is preserved in China typical culture collection center in Wuhan University of Wuhan, China city on August 7th, 2009, deposit number is CCTCC NO:M209171) spot, then this nutrient solution is cultivated 60h at 30 DEG C, again at 4 DEG C, the centrifugal 20min of 6000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 4 DEG C, the centrifugal 25min of 16000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.2mol/L is added in precipitation, the potassium phosphate salt damping fluid of pH7.5, the concentration of adjustment Bdellovibrio nectophore is 10 7pfu/mL, obtains Bdellovibrio BDSM08 telotroch concentrated solution, is placed in 4 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
Be add sodium-chlor in the DNB liquid nutrient medium of 7.2 in pH value, add that quality is DNB liquid nutrient medium volume 1.5% of sodium-chlor, load fermentor tank, 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add streptococcus acidi lactici suspension prepared by step (1) and Bdellovibrio BDSM08 telotroch concentrated solution prepared by step (2), the initial concentration of the streptococcus acidi lactici in fermention medium is 10 13cfu/mL, Bdellovibrio initial concentration is 10 2pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 30 DEG C; The fermention medium pH value during the fermentation adding streptococcus acidi lactici suspension and Bdellovibrio nectophore concentrated solution controls 7.5; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 27%; Add 2 streptococcus acidi lactici suspension in culturing process, add 14h interval time of streptococcus acidi lactici suspension at every turn, the amount at every turn adding streptococcus acidi lactici suspension with the streptococcus acidi lactici newly added concentration in the fermentation medium for 10 13cfu/mL is as the criterion, and namely fermentation culture 42h makes concentration is 5 × 10 9the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add streptococcus suis II suspension prepared by step (1) and Bdellovibrio BDSM08 telotroch concentrated solution prepared by step (2), obtained concentration is 5 × 10 9the bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all has lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, staphylococcus epidermidiss.Respectively to be cracked into example to Salmonella choleraesuls and staphylococcus epidermidis.Being equipped with 4 bottles is respectively equipped with in the potassium-sodium phosphates salt buffer of 50mL, respectively adds the bacterial sediment that conventional culture methods obtains Salmonella choleraesuls and staphylococcus epidermidis respectively, often kind of bacterium 2 bottles, regulates the cell concentration consistent (10 of 4 bottles of damping fluids 10cfu/mL).In two bottles of buffer systems containing Salmonella choleraesuls, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, in two bottles of buffer systems containing staphylococcus epidermidis, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these nine moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h -1~ 10 -10extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in Figure 7.As can be seen from Figure 7, compared with the bdellovibrio bacteriovorus preparation B fermented by streptococcus suis II, be not only that negative Salmonella choleraesuls have stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of streptococcus acidi lactici fermentation, and be that the lytic effect of positive staphylococcus epidermidis is also better than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by streptococcus acidi lactici not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 8
Be that Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B with faecium (bacterium numbering: CGMCC1.2136 derives from China Committee for Culture Collection of Microorganisms's common micro-organisms center) and intestinal bacteria respectively, detailed process is as follows:
(1) preparation of faecium suspension
Faecium is inoculated in nutrient broth medium (peptone 10g, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2) in, be placed in 32 DEG C of shaking tables and cultivate 24h, make it be in logarithmic phase, nutrient solution under 4 DEG C of conditions, the centrifugal 20min of 5000rpm, retain precipitation abandoning supernatant, in precipitation, add potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), obtaining concentration is 10 18the faecium suspension of cfu/mL, is then placed in 4 DEG C of refrigerators and saves backup;
The same method, preparation concentration is 10 18the intestinal bacteria suspension of cfu/mL, is placed in 4 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), add E. coli suspension prepared by step (1), adjusting colibacillary concentration is 10 15cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treat plaque appears in the double-deck agar plate containing Host Strains) cultivate the Bdellovibrio BDJ02(of separation out from seawater and be preserved in China typical culture collection center in Wuhan University of Wuhan, China city on January 14th, 2008, deposit number is CCTCC NO:M208012) spot, then this nutrient solution is cultivated 60h at 30 DEG C, again at 4 DEG C, the centrifugal 20min of 6000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 4 DEG C, the centrifugal 25min of 16000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.2mol/L is added in precipitation, the potassium phosphate salt damping fluid of pH7.5, the concentration of adjustment Bdellovibrio nectophore is 10 6pfu/mL, obtains Bdellovibrio BDJ02 telotroch concentrated solution, is placed in 4 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
Be add sodium-chlor in the DNB liquid nutrient medium of 7.2 in pH value, add that quality is DNB liquid nutrient medium volume 2.7% of sodium-chlor, load fermentor tank, 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add faecium suspension prepared by step (1) and Bdellovibrio BDJ02 telotroch concentrated solution prepared by step (2), the initial concentration of the faecium in fermention medium is 10 13cfu/mL, Bdellovibrio initial concentration is 10 3pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 30 DEG C; The fermention medium pH value during the fermentation adding faecium suspension and Bdellovibrio nectophore concentrated solution controls 7.3; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 26%; Add twice faecium suspension in culturing process, add 14h interval time of faecium suspension at every turn, the amount at every turn adding faecium suspension with the faecium newly added concentration in the fermentation medium for 10 13cfu/mL is as the criterion, and namely fermentation culture 42h makes concentration is 1 × 10 9the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add E. coli suspension prepared by step (1) and Bdellovibrio BDJ02 telotroch concentrated solution prepared by step (2), obtained concentration is 1 × 10 9the bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all has lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, listeria monocytogenes.Respectively to be cracked into example to listeria monocytogenes and Salmonella typhimurium.Being equipped with 4 bottles is respectively equipped with in the potassium-sodium phosphates salt buffer of 50mL, respectively adds the bacterial sediment that conventional culture methods obtains listeria monocytogenes and Salmonella typhimurium respectively, often kind of bacterium 2 bottles, regulates the cell concentration consistent (10 of 4 bottles of damping fluids 11cfu/mL).In two bottles of buffer systems containing listeria monocytogenes, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, in two bottles of buffer systems containing Salmonella typhimurium, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these eight moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h -1~ 10 -11extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in Figure 8.As can be seen from Figure 8, compared with the bdellovibrio bacteriovorus preparation B with Escherichia coli fermentation, be not only that negative Salmonella typhimurium has stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of faecium fermentation, and be that the lytic effect of positive listeria monocytogenes is also better than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by faecium not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 9
Be that Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B with Bacillus licheniformis (bacterium numbering: GIM1.182 derives from Guangdong Province's Culture Collection) and intestinal bacteria respectively, detailed process is as follows:
(1) preparation of Bacillus licheniformis suspension
Bacillus licheniformis is inoculated in (peptone 10g in nutrient broth medium, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2), be placed in 33 DEG C of shaking tables and cultivate 18h, make it be in logarithmic phase, nutrient solution under 4 DEG C of conditions, the centrifugal 25min of 5000rpm, retain precipitation abandoning supernatant, in precipitation, add potassium-sodium phosphates salt buffer (concentration is 0.2mol/L, pH7.6), obtaining concentration is 10 19the Bacillus licheniformis suspension of cfu/mL, is then placed in 4 DEG C of refrigerators and saves backup;
The same method, preparation concentration is 10 19the intestinal bacteria suspension of cfu/mL, is placed in 4 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In potassium-sodium phosphates salt buffer (concentration is 0.2mol/L, pH7.6), add E. coli suspension prepared by step (1), adjusting colibacillary concentration is 10 12cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treat plaque appears in the double-deck agar plate containing Host Strains) cultivate the Bdellovibrio BDM01(of separation out from seawater and be preserved in China typical culture collection center in Wuhan University of Wuhan, China city on April 28th, 2008, deposit number is CCTCC NO:M208066) spot, then this nutrient solution is cultivated 48h at 30 DEG C, again at 4 DEG C, the centrifugal 30min of 5000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 4 DEG C, the centrifugal 30min of 13000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.2mol/L is added in precipitation, the potassium-sodium phosphates salt buffer of pH7.6, the concentration of adjustment Bdellovibrio nectophore is 10 8pfu/mL, obtains Bdellovibrio BDM01 telotroch concentrated solution, is placed in 4 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
In DNB liquid nutrient medium, add sodium-chlor, add that quality is DNB liquid nutrient medium volume 3.0% of sodium-chlor, load fermentor tank, 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add Bacillus licheniformis suspension prepared by step (1) and Bdellovibrio BDM01 telotroch concentrated solution prepared by step (2), the initial concentration of the Bacillus licheniformis in fermention medium is 10 12cfu/mL, Bdellovibrio initial concentration is 10 2pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 30 DEG C; The fermention medium pH value during the fermentation adding Bacillus licheniformis suspension and Bdellovibrio nectophore concentrated solution controls 7.2; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 28%; Add twice Bacillus licheniformis suspension in culturing process, add 15h interval time of Bacillus licheniformis suspension at every turn, the amount at every turn adding Bacillus licheniformis suspension with the Bacillus licheniformis newly added concentration in the fermentation medium for 10 12cfu/mL is as the criterion, and namely fermentation culture 48h makes concentration is 1 × 10 12the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add E. coli suspension prepared by step (1) and Bdellovibrio BDM01 telotroch concentrated solution prepared by step (2), obtained concentration is 1 × 10 12the bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all has good lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, streptococcus aureuses.Example is cracked into respectively with the Aeromonas hydrophila that is feminine gender to gram and the streptococcus equisimilis (Streptococcus equinus, strain number: cvcc1925 derive from National Veterinary Culture Collection) that is the positive to gram.Be equipped with the potassium-sodium phosphates salt buffer that 4 bottles are respectively equipped with 50mL, add the bacterial sediment that conventional culture methods obtains Aeromonas hydrophila and streptococcus equisimilis respectively, often kind of a bacterium has two bottles.Regulate the cell concentration consistent (10 of 4 bottles of damping fluids 10cfu/mL).In two bottles of buffer systems containing Aeromonas hydrophila, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, in two bottles of buffer systems containing streptococcus equisimilis, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these nine moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h -1~ 10 -10extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in Figure 9.As can be seen from Figure 9, compared with the bdellovibrio bacteriovorus preparation B obtained with Escherichia coli fermentation, be not only that negative Aeromonas hydrophila has stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of the lichen bacillus ferments, and be that the lytic effect of positive streptococcus equisimilis is also good than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by Bacillus licheniformis not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 10
Be that Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B with Lactobacterium acidophilum (bacterium numbering: GIM1.208 derives from Guangdong Province's Culture Collection) and intestinal bacteria respectively, detailed process is as follows:
(1) preparation of Lactobacterium acidophilum
Lactobacterium acidophilum is inoculated in nutrient broth medium (peptone 10g, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2) in, be placed in 32 DEG C of shaking tables and cultivate 24h, make it be in logarithmic phase, nutrient solution under 4 DEG C of conditions, the centrifugal 20min of 5000rpm, retain precipitation abandoning supernatant, in precipitation, add potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), obtaining concentration is 10 17the Lactobacterium acidophilum suspension of cfu/mL, is then placed in 8 DEG C of refrigerators and saves backup;
The same method, preparation concentration is 10 17the intestinal bacteria suspension of cfu/mL, is placed in 8 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), add E. coli suspension prepared by step (1), adjusting colibacillary concentration is 10 15cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treat plaque appears in the double-deck agar plate containing Host Strains) cultivate the Bdellovibrio BDS02(of separation out from soil and be preserved in China typical culture collection center in Wuhan University of Wuhan, China city on August 7th, 2009, deposit number is CCTCC NO:M209170) spot, then this nutrient solution is cultivated 60h at 30 DEG C, again at 4 DEG C, the centrifugal 20min of 6000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 15 DEG C, the centrifugal 20min of 13000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.2mol/L is added in precipitation, the potassium phosphate salt damping fluid of pH7.5, the concentration of adjustment Bdellovibrio nectophore is 10 7pfu/mL, obtains Bdellovibrio BDS02 telotroch concentrated solution, is placed in 6 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
Be load fermentor tank in the DNB liquid nutrient medium of 7.2 by pH value, 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add Lactobacterium acidophilum suspension prepared by step (1) and Bdellovibrio BDS02 telotroch concentrated solution prepared by step (2), the initial concentration of the Lactobacterium acidophilum in fermention medium is 10 10cfu/mL, Bdellovibrio initial concentration is 10pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 30 DEG C; The fermention medium pH value during the fermentation adding Lactobacterium acidophilum suspension and Bdellovibrio nectophore concentrated solution controls 7.5; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 20%; Add twice Lactobacterium acidophilum suspension in culturing process, add 14h interval time of Lactobacterium acidophilum suspension at every turn, the amount at every turn adding Lactobacterium acidophilum suspension with the Lactobacterium acidophilum newly added concentration in the fermentation medium for 10 13cfu/mL is as the criterion, and namely fermentation culture 42h makes concentration is 1 × 10 8the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add E. coli suspension prepared by step (1) and Bdellovibrio BDS02 telotroch concentrated solution prepared by step (2), obtained concentration is 1 × 10 8the bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all has lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, streptococcus aureuses.Respectively to be cracked into example to streptococcus aureus and Salmonella choleraesuls.Being equipped with 4 bottles is respectively equipped with in the potassium-sodium phosphates salt buffer of 50mL, respectively adds the bacterial sediment that conventional culture methods obtains streptococcus aureus and Salmonella choleraesuls respectively, often kind of bacterium 2 bottles, regulates the cell concentration consistent (10 of 4 bottles of damping fluids 11cfu/mL).In two bottles of buffer systems containing streptococcus aureus, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, in two bottles of buffer systems containing Salmonella choleraesuls, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these eight moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h -1~ 10 -11extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in Figure 10.As can be seen from Figure 10, compared with the bdellovibrio bacteriovorus preparation B with Escherichia coli fermentation, be not only that negative Salmonella choleraesuls have stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of Lactobacterium acidophilum fermentation, and be that the lytic effect of positive streptococcus aureus is also better than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by Lactobacterium acidophilum not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 11
Be that Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B with lactobacterium casei (bacterium numbering: GIM1.159 derives from Guangdong Province's Culture Collection) and Aeromonas hydrophila respectively, detailed process is as follows:
(1) preparation of lactobacterium casei
Lactobacterium casei is inoculated in nutrient broth medium (peptone 10g, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2) in, be placed in 30 DEG C of shaking tables and cultivate 18h, make it be in logarithmic phase, nutrient solution under 5 DEG C of conditions, the centrifugal 15min of 8000rpm, retain precipitation abandoning supernatant, in precipitation, add sodium phosphate buffer (concentration is 0.1mol/L, pH7.4), obtaining concentration is 10 22the lactobacterium casei suspension of cfu/mL, is then placed in 15 DEG C of refrigerators and saves backup;
The same method, preparation concentration is 10 22the Aeromonas hydrophila suspension of cfu/mL, is placed in 15 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In sodium phosphate buffer (concentration is 0.1mol/L, pH7.4), add Aeromonas hydrophila suspension prepared by step (1), the concentration of adjustment Aeromonas hydrophila is 10 15cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treat plaque appears in the double-deck agar plate containing Host Strains) cultivate the Bdellovibrio BDF02(of separation out from fresh water and be preserved in China typical culture collection center in Wuhan University of Wuhan, China city on January 13rd, 2008, deposit number is CCTCC NO:M208009) spot, then this nutrient solution is cultivated 54h at 30 DEG C, again at 5 DEG C, the centrifugal 20min of 7000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 5 DEG C, the centrifugal 15min of 20000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.1mol/L is added in precipitation, the sodium phosphate buffer of pH7.4, the concentration of adjustment Bdellovibrio nectophore is 10 7pfu/mL, obtains Bdellovibrio BDF02 telotroch concentrated solution, is placed in 2 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
Distilled water is loaded fermentor tank, and 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add lactobacterium casei suspension prepared by step (1) and Bdellovibrio BDF02 telotroch concentrated solution prepared by step (2), the initial concentration of the lactobacterium casei in fermention medium is 10 14cfu/mL, Bdellovibrio initial concentration is 10 3pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 29 DEG C; The fermention medium pH value during the fermentation adding lactobacterium casei suspension and Bdellovibrio nectophore concentrated solution controls 7.2; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 30%; Add twice lactobacterium casei suspension in culturing process, add 15h interval time of lactobacterium casei suspension at every turn, the amount at every turn adding lactobacterium casei suspension with the lactobacterium casei newly added concentration in the fermentation medium for 10 14cfu/mL is as the criterion, and namely fermentation culture 45h makes concentration is 1 × 10 9the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add Aeromonas hydrophila suspension prepared by step (1) and Bdellovibrio BDF02 telotroch concentrated solution prepared by step (2), obtained concentration is 1 × 10 9the bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B has good lytic effect to pathogenic bacterium such as intestinal bacteria, Salmonellas, streptococcus equisimilises.Respectively with to streptococcus equisimilis be colibacillaryly cracked into example.Being equipped with 4 bottles is respectively equipped with in the potassium-sodium phosphates salt buffer of 50mL, respectively adds conventional culture methods respectively and obtains streptococcus equisimilis and colibacillary bacterial sediment, often kind of bacterium 2 bottles, regulates the cell concentration consistent (10 of 4 bottles of damping fluids 12cfu/mL).In two bottles of buffer systems containing streptococcus equisimilis, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, at two bottles containing in colibacillary buffer system, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these eight moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h -1~ 10 -12extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in figure 11.As can be seen from Figure 11, compared with the bdellovibrio bacteriovorus preparation B fermented with Aeromonas hydrophila, be not only that negative intestinal bacteria have stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of lactobacterium casei fermentation, and be that the lytic effect of positive streptococcus equisimilis is also better than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A that Cheesecake Bacterium lacticum obtains not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 12
Be that Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B with lactobacillus lactis (bacterium numbering: ATCC12315 derives from American Type Culture Collection) and streptococcus suis II respectively, detailed process is as follows:
(1) preparation of lactobacillus lactis suspension
Lactobacillus lactis is inoculated in nutrient broth medium (peptone 10g, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2) in, be placed in 37 DEG C of shaking tables and cultivate 18h, make it be in logarithmic phase, nutrient solution under 4 DEG C of conditions, the centrifugal 20min of 6000rpm, retain precipitation abandoning supernatant, in precipitation, add potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), obtaining concentration is 10 20the lactobacillus lactis suspension of cfu/mL, is then placed in 4 DEG C of refrigerators and saves backup;
The same method, preparation concentration is 10 20the streptococcus suis II suspension of cfu/mL, is placed in 4 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), add streptococcus suis II suspension prepared by step (1), the concentration of adjustment streptococcus suis II is 10 15cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treats plaque appears in the double-deck agar plate containing Host Strains.) the Bdellovibrio BDS01(that cultivates separation soil is out preserved in China typical culture collection center in Wuhan University of Wuhan, China city on August 7th, 2009, deposit number is CCTCC NO:M209169) spot, then this nutrient solution is cultivated 20h at 40 DEG C, again at 4 DEG C, the centrifugal 20min of 6000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 4 DEG C, the centrifugal 40min of 12000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.2mol/L is added in precipitation, the potassium phosphate salt damping fluid of pH7.5, the concentration of adjustment Bdellovibrio nectophore is 10 8pfu/mL, obtains Bdellovibrio BDS01 telotroch concentrated solution, is placed in 4 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
Be add sodium-chlor in the DNB liquid nutrient medium of 7.2 in pH value, add that quality is DNB liquid nutrient medium volume 0.5% of sodium-chlor, load fermentor tank, 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add lactobacillus lactis suspension prepared by step (1) and Bdellovibrio BDS01 telotroch concentrated solution prepared by step (2), the initial concentration of the lactobacillus lactis in fermention medium is 10 13cfu/mL, Bdellovibrio initial concentration is 10 2pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 30 DEG C; The fermention medium pH value during the fermentation adding lactobacillus lactis suspension and Bdellovibrio nectophore concentrated solution controls 7.2 ~ 7.6; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 23%; Add 1 lactobacillus lactis suspension in culturing process, add 18h interval time of lactobacillus lactis suspension at every turn, the amount at every turn adding lactobacillus lactis suspension with the lactobacillus lactis newly added concentration in the fermentation medium for 10 13cfu/mL is as the criterion, and namely fermentation culture 42h makes concentration is 5 × 10 9the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add streptococcus suis II suspension prepared by step (1) and Bdellovibrio BDS01 telotroch concentrated solution prepared by step (2), obtained concentration is 5 × 10 9the bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all has lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, staphylococcus epidermidiss.Respectively to be cracked into example to Aeromonas hydrophila and staphylococcus epidermidis.Being equipped with 4 bottles is respectively equipped with in the potassium-sodium phosphates salt buffer of 50mL, respectively adds the bacterial sediment that conventional culture methods obtains Aeromonas hydrophila and staphylococcus epidermidis respectively, often kind of bacterium 2 bottles, regulates the cell concentration consistent (10 of 4 bottles of damping fluids 11cfu/mL).In two bottles of buffer systems containing Aeromonas hydrophila, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, in two bottles of buffer systems containing staphylococcus epidermidis, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these nine moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h -1~ 10 -11extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in figure 12.As can be seen from Figure 12, compared with the bdellovibrio bacteriovorus preparation B fermented by streptococcus suis II, be not only that negative Aeromonas hydrophila has stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of lactobacillus lactis fermentation, and be that the lytic effect of positive staphylococcus epidermidis is also better than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by lactobacillus lactis not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 13
Be that Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B with plant lactobacillus (bacterium numbering: GIM1.140 derives from Guangdong Province's Culture Collection) and Salmonella choleraesuls respectively, detailed process is as follows:
(1) preparation of plant lactobacillus
Plant lactobacillus is inoculated in nutrient broth medium (peptone 10g, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2) in, be placed in 30 DEG C of shaking tables and cultivate 24h, make it be in logarithmic phase, nutrient solution under 8 DEG C of conditions, the centrifugal 20min of 6000rpm, retain precipitation abandoning supernatant, in precipitation, add sodium phosphate buffer (concentration is 0.2mol/L, pH7.4), obtaining concentration is 10 20the plant lactobacillus suspension of cfu/mL, is then placed in 4 DEG C of refrigerators and saves backup;
The same method, the concentration of preparation is 10 20cfu/mL Salmonella choleraesuls suspension, is placed in 4 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In sodium phosphate buffer (concentration is 0.2mol/L, pH7.4), add Salmonella choleraesuls suspension prepared by step (1), the concentration of adjustment Salmonella choleraesuls is 10 12cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treat plaque appears in the double-deck agar plate containing Host Strains) cultivate the Bdellovibrio BDF01(of separation out from fresh water and be preserved in China typical culture collection center in Wuhan University of Wuhan, China city on January 13rd, 2008, deposit number is CCTCC NO:M208008) spot, then this nutrient solution is cultivated 20h at 30 DEG C, again at 2 DEG C, the centrifugal 15min of 8000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 4 DEG C, the centrifugal 22min of 15000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.2mol/L is added in precipitation, the sodium phosphate buffer of pH7.4, the concentration of adjustment Bdellovibrio nectophore is 10 6pfu/mL, obtains Bdellovibrio BDF01 telotroch concentrated solution, is placed in 15 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
Sodium phosphate buffer (concentration is 0.2mol/L, pH7.6), load fermentor tank, 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add plant lactobacillus suspension prepared by step (1) and Bdellovibrio BDF01 telotroch concentrated solution prepared by step (2), the initial concentration of the plant lactobacillus in fermention medium is 10 10cfu/mL, Bdellovibrio initial concentration is 10 2pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 30 DEG C; The fermention medium pH value during the fermentation adding plant lactobacillus suspension and Bdellovibrio nectophore concentrated solution controls 7.4; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 30%; Add twice plant lactobacillus suspension in culturing process, add 18h interval time of plant lactobacillus suspension at every turn, the amount at every turn adding plant lactobacillus suspension with the plant lactobacillus newly added concentration in the fermentation medium for 10 10cfu/mL is as the criterion, and namely fermentation culture 48h makes concentration is 5 × 10 10the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add Salmonella choleraesuls suspension prepared by step (1) and Bdellovibrio BDF01 telotroch concentrated solution prepared by step (2), obtained concentration is 5 × 10 10the bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all has lytic effect to pathogenic bacterium such as intestinal bacteria, Salmonellas, streptococcus agalactiaes.Respectively to be cracked into example to streptococcus agalactiae and Salmonella typhimurium.Being equipped with 4 bottles is respectively equipped with in the potassium-sodium phosphates salt buffer of 50mL, respectively adds the bacterial sediment that conventional culture methods obtains streptococcus agalactiae and Salmonella typhimurium respectively, often kind of bacterium two bottles, regulates the cell concentration consistent (10 of 4 bottles of damping fluids 10cfu/mL).In two bottles of buffer systems containing streptococcus agalactiae, add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, in two bottles of buffer systems containing Salmonella typhimurium, add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these 98 moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h -1~ 10 -10extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in figure 13.As can be seen from Figure 13, compared with the bdellovibrio bacteriovorus preparation B fermented with Salmonella choleraesuls, be not only that negative Salmonella typhimurium has stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of plant lactobacillus fermentation, and be that the lytic effect of positive streptococcus agalactiae is also better than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by plant lactobacillus not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Embodiment 14
Be that Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B with Pediococcus pentosaceus (bacterium numbering: CGMCC1.2695 derives from China Committee for Culture Collection of Microorganisms's common micro-organisms center) and intestinal bacteria respectively, detailed process is as follows:
(1) preparation of Pediococcus pentosaceus
Pediococcus pentosaceus is inoculated in nutrient broth medium (peptone 10g, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2) in, be placed in 32 DEG C of shaking tables and cultivate 24h, make it be in logarithmic phase, nutrient solution under 4 DEG C of conditions, the centrifugal 20min of 5000rpm, retain precipitation abandoning supernatant, in precipitation, add potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), obtaining concentration is 10 18the Pediococcus pentosaceus suspension of cfu/mL, is then placed in 8 DEG C of refrigerators and saves backup;
The same method, preparation concentration is 10 18the intestinal bacteria suspension of cfu/mL, is placed in 8 DEG C of refrigerators and saves backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), add E. coli suspension prepared by step (1), adjusting colibacillary concentration is 10 15cfu/mL, access ordinary method again (is 200910042274.6 according to application number, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof preventing and treating mastadenitis of cow ", be specially and adopt the double-layer agar technique of Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and the mixing of 0.5mL E. coli suspension, this mixed solution again with DNB upper strata substratum (the distilled water 500mL of 3-3.5mL50 ° of C, pH7.2, agar powder 3.5g) mix, be poured into DNB lower floor flat board (nutrient broth 0.8g, tyrosine acid hydrolysis thing 0.5g, yeastex extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Double-layer plate is placed in 28 ° of C constant incubators to cultivate, treat plaque appears in the double-deck agar plate containing Host Strains) cultivate the Bdellovibrio BDJ01(of separation out from seawater and be preserved in China typical culture collection center in Wuhan University of Wuhan, China city on January 14th, 2008, deposit number is CCTCC NO:M208011) spot, then this nutrient solution is cultivated 60h at 30 DEG C, again at 8 DEG C, the centrifugal 30min of 5000rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 4 DEG C, the centrifugal 25min of 16000rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, 0.2mol/L is added in precipitation, the potassium phosphate salt damping fluid of pH7.5, the concentration of adjustment Bdellovibrio nectophore is 10 9pfu/mL, obtains Bdellovibrio BDJ01 telotroch concentrated solution, is placed in 4 DEG C of Refrigerator stores for subsequent use,
(3) preparation of bdellovibrio bacteriovorus preparation
Be add sodium-chlor in the DNB liquid nutrient medium of 7.2 in pH value, add that quality is DNB liquid nutrient medium volume 2.5% of sodium-chlor, load fermentor tank, 121 DEG C of sterilizing 20min, obtain fermention medium; In the fermention medium of 30 DEG C, add Pediococcus pentosaceus suspension prepared by step (1) and Bdellovibrio BDJ01 telotroch concentrated solution prepared by step (2), the initial concentration of the Pediococcus pentosaceus in fermention medium is 10 13cfu/mL, Bdellovibrio initial concentration is 10 3pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature controls at 30 DEG C; The fermention medium pH value during the fermentation adding Pediococcus pentosaceus suspension and Bdellovibrio nectophore concentrated solution controls 7.6; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 25%; Add 3 Pediococcus pentosaceus suspension in culturing process, add 12h interval time of Pediococcus pentosaceus suspension at every turn, the amount at every turn adding Pediococcus pentosaceus suspension with the Pediococcus pentosaceus newly added concentration in the fermentation medium for 10 13cfu/mL is as the criterion, and namely fermentation culture 48h makes concentration is 1 × 10 9the bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method, in the fermention medium of 30 DEG C, add E. coli suspension prepared by step (1) and Bdellovibrio BDJ01 telotroch concentrated solution prepared by step (2), obtained concentration is 1 × 10 9the bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all has lytic effect to pathogenic bacterium such as streptococcus uberis, Salmonellas, streptococcus suis II.Respectively to be cracked into example to streptococcus uberis and streptococcus suis II.Being equipped with 4 bottles is respectively equipped with in the potassium-sodium phosphates salt buffer of 50mL, respectively adds the bacterial sediment that conventional culture methods obtains streptococcus uberis and streptococcus suis II respectively, often kind of bacterium 2 bottles, regulates the cell concentration consistent (10 of 4 bottles of damping fluids 10cfu/mL).In two bottles of buffer systems containing streptococcus uberis, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively; Equally, in two bottles of buffer systems containing streptococcus suis II, respectively add bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL respectively, in 30 DEG C, the shaking table of 200rpm is cultivated.Sample respectively, by 10 in these eight moment of 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h -1~ 10 -10extension rate dilute, then the diluent respectively getting 100 μ L carry out respectively flat board coating, after cultivating 24h under 30 DEG C of conditions, calculate colony number.Experimental result as shown in figure 14.As can be seen from Figure 14, compared with the bdellovibrio bacteriovorus preparation B with Escherichia coli fermentation, be not only that negative streptococcus suis II has stronger lytic effect to gram with the bdellovibrio bacteriovorus preparation A of Pediococcus pentosaceus fermentation, and be that the lytic effect of positive streptococcus uberis is also better than bdellovibrio bacteriovorus preparation B to gram.In addition, the bdellovibrio bacteriovorus preparation A obtained by Pediococcus pentosaceus not only directly can make preparation, also by further centrifugal, makes simple telotroch, leech liposome preparation.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (5)

1. a fermentation process for bdellovibrio bacteriovorus preparation, is characterized in that comprising the following steps:
(1) preparation of host bacteria suspension:
Host Strains is cultured to logarithmic phase, and collect thalline, adjust concentration with phosphate buffered saline buffer, obtaining concentration is 10 17~ 10 22the Host Strains suspension of cfu/mL, is then placed in 2 ~ 15 DEG C and saves backup; Described Host Strains be bacillus natto ( bacillus natto) CGMCC 1.1086 or lactobacterium casei ( lactobacillus casei) GIM1.159;
(2) preparation of Bdellovibrio nectophore concentrated solution:
In phosphate buffered saline buffer, add Host Strains suspension prepared by step (1), the concentration of adjustment Host Strains is 10 10~ 10 15cfu/mL, access Bdellovibrio spot again, then this nutrient solution is cultivated 20 ~ 60h at 20 ~ 40 DEG C, then at 2 ~ 15 DEG C, the centrifugal 15 ~ 35min of 5000 ~ 8000 rpm, retain supernatant liquor and discard precipitation, then by supernatant liquor at 2 ~ 15 DEG C, centrifugal 15 ~ 40 min of 12000 ~ 20000 rpm, retain precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, the concentration adding phosphate buffered saline buffer adjustment Bdellovibrio nectophore in precipitation is 10 6~ 10 9pfu/mL, obtains Bdellovibrio nectophore concentrated solution, is placed in 2 ~ 15 DEG C and saves backup; Described Bdellovibrio is BDF02, BDF02 are preserved in Wuhan University of Wuhan, China city China typical culture collection center on January 13rd, 2008, and deposit number is CCTCC NO:M 208009;
(3) preparation of bdellovibrio bacteriovorus preparation:
By solvent sterilizing, obtain fermention medium; Add Host Strains suspension prepared by step (1) and Bdellovibrio nectophore concentrated solution prepared by step (2) in the fermentation medium, make Host Strains initial concentration be 10 10~ 10 15cfu/mL, Bdellovibrio nectophore initial concentration is 10 1~ 10 3pfu/mL, ferments; Leavening temperature controls at 28 ~ 30 DEG C, and pH value controls 7.2 ~ 7.6; Mixing speed is set and dissolved oxygen links, makes dissolved oxygen level control 20% ~ 30%; Add 1 ~ 3 Host Strains suspension in fermenting process, add 12 ~ 18h interval time of Host Strains suspension at every turn, the amount at every turn adding Host Strains suspension with the Host Strains newly added concentration in the fermentation medium for 10 10~ 10 15cfu/mL is as the criterion; Namely fermentation culture 36 ~ 48h makes bdellovibrio bacteriovorus preparation; Described solvent is DNB liquid nutrient medium, distilled water or phosphate buffered saline buffer.
2. the fermentation process of a kind of bdellovibrio bacteriovorus preparation according to claim 1, is characterized in that: the mode of the described collection thalline of step (1) is for obtain by centrifugation, and centrifugal condition is 2 ~ 15 DEG C, the centrifugal 15 ~ 35min of 5000 ~ 8000rpm; Described in step (1) ~ any one of (3), the concentration of phosphate buffered saline buffer is 0.1 ~ 0.3 mol/L, pH is 7.2 ~ 7.6.
3. the fermentation process of a kind of bdellovibrio bacteriovorus preparation according to claim 1, is characterized in that: the condition of step (3) described sterilizing is 121 DEG C of sterilizing 20min; The concentration of described bdellovibrio bacteriovorus preparation is 10 8~ 10 12pfu/mL; Described DNB liquid nutrient medium is dissolved in 1000mL distilled water by nutrient broth 0.8g, caseinic acid hydrolyzate 0.5g and yeastex extract 0.1g, adjust ph to 7.2 ~ 7.6.
4. a bdellovibrio bacteriovorus preparation, is prepared by method described in any one of claims 1 to 3.
5. bdellovibrio bacteriovorus preparation according to claim 4 is at preparation control listeria monocytogenes and Salmonella choleraesuls medicine, or prevents and treats the application in streptococcus equisimilis and intestinal bacteria medicine.
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