CN103320341A - Bdellovibrio bacteriovorus preparation, and fermentation method and applications thereof - Google Patents

Bdellovibrio bacteriovorus preparation, and fermentation method and applications thereof Download PDF

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CN103320341A
CN103320341A CN2013101021640A CN201310102164A CN103320341A CN 103320341 A CN103320341 A CN 103320341A CN 2013101021640 A CN2013101021640 A CN 2013101021640A CN 201310102164 A CN201310102164 A CN 201310102164A CN 103320341 A CN103320341 A CN 103320341A
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bdellovibrio
bdellovibrio bacteriovorus
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CN103320341B (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 invention discloses a bdellovibrio bacteriovorus preparation and a fermentation method and application thereof. The fermentation method comprises the following steps of: (1) preparation of a host bacteria suspension; (2) preparation of a bdellovibrio bacteriovorus telotroch 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 for pathogenic bacteria; and the prepared bdellovibrio bacteriovorus preparation can be directly used without more fermentation post treatment, and 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
Patent application of the present invention is that application number is dividing an application of " 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 thereof and application ".
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 at present control, elimination pathogenic bacterium is still the method for various physics and/or chemistry, comprises various antibiotic uses.The method of physics and/or chemistry all exists obvious drawback, and at first, microbiotic is abused in a large number, can cause the generation of some side effects, such as the resistance of pathogenic bacteria, suppress profitable strain etc.Secondly, though the method for acid-alkali treatment can reach certain sterilization/sterilization effect, after harmful bacteria formed microbial film, the effect of these methods was just extremely undesirable.At last, the method for physics can only be applied to a certain link of association area to the elimination effect of various pathogenic bacterium, and is difficult to expand to all links in whole field.The method of biological control then can have powerful superiority, very likely makes the processing of large-scale water in its disease control of serving daily life and biological warfare and the attack of terrorism, food source contact scar etc.
The in recent years research and development of microbial ecological preparation are subject to each side and pay close attention to, and particularly the research of Bdellovibrio is subject to people's favor.Bdellovibrio since 1963 are found, because it is a kind of bacterial parasite, can cracking the gram negative pathogenic bacterias and harmless to humans and animals such as intestinal bacteria, Salmonellas, Aeromonas hydrophila, vibrios, and receive much concern.
The key that Bdellovibrio is applied to prevent and treat pathogenic bacterium is to obtain 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, the national inventing patent application that for example number of patent application is 93111749.6, name is called " bactericide prepared from biology and production method thereof " proposes with high temperature (70～150 ℃) or chemicals (chloroform etc.) intestinal bacteria to be killed, make the Host Strains of deactivation, then cultivate Bdellovibrio with it, obtain bdellovibrio bacteriovorus preparation; Number of patent application 200810145709.5, name are called the production method that " production method of bdellovibrio bacteriovorus ecological preparation " provides bdellovibrio bacteriovorus ecological preparation, and mainly different with traditional method is host e. coli to be lyophilized into the bacterium powder use.Above-mentioned two parts of patent applications all adopt intestinal bacteria alive as Host Strains, finally understand influence ecological environment.And the used Host Strains of the present invention is probiotics or the bacterium of 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, the i.e. first suspension of fermentation preparation Host Strains adds host strain turbid liquor again and Bdellovibrio liquid ferments in the nutrient solution for preparing.Although the Bdellovibrio content that this production method is produced is higher by (5 * 10 8Pfu/mL), but its Host Strains has been selected pathogenic Aeromonas hydrophila, and fermentation time long (72～120h), can cause the increase of its production cost.In addition, this technology also may exist the not high problem of Bdellovibrio lytic activity of gained.Be that gram-positive microorganism is the host and the present invention adopts, experiment showed, that the Bdellovibrio that makes with this host's fermentation can keep, even improve the cracking ability to other Gram-negative bacterias and positive bacteria.
Summary of the invention
In order to overcome the existing prepared problem that bdellovibrio bacteriovorus preparation content is low and lytic activity is low of method, primary and foremost purpose of the present invention is to provide a kind of fermentation process of bdellovibrio bacteriovorus preparation.
Another object of the present invention is to provide a kind of bdellovibrio bacteriovorus preparation that is prepared by above-mentioned fermentation process.
A further object of the present invention is to provide the application of above-mentioned bdellovibrio bacteriovorus preparation.
Purpose of the present invention is achieved through the following technical solutions: a kind of fermentation process of bdellovibrio bacteriovorus preparation may further comprise the steps:
(1) preparation of host bacteria suspension
Host Strains is cultured to logarithmic phase, collects thalline, then adjust concentration with phosphate buffered saline buffer, obtaining concentration is 10 17～10 22Then the Host Strains suspension of cfu/mL place 2～15 ℃ to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
The Host Strains suspension that adds step (1) preparation in phosphate buffered saline buffer, the concentration of adjusting Host Strains is 10 10～10 15Cfu/mL, access again the Bdellovibrio spot, then this nutrient solution is cultivated 20～60h at 20～40 ℃, again at 2～15 ℃, the centrifugal 15～35min of 5000～8000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 2～15 ℃, the centrifugal 15～40min of 12000～20000rpm keeps the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, the concentration that adds phosphate buffered saline buffer adjustment Bdellovibrio nectophore in precipitation is 10 6～10 9Pfu/mL obtains the Bdellovibrio nectophore concentrated solution, places 2～15 ℃ to save backup;
(3) preparation of bdellovibrio bacteriovorus preparation:
Sodium-chlor is dissolved in the solvent to form mass volume ratio concentration be the sodium chloride solution of 0～30g/L, sterilization obtains fermention medium; Add the Host Strains suspension of step (1) preparation and the Bdellovibrio nectophore concentrated solution of step (2) preparation in fermention medium, making the Host Strains initial concentration is 10 10～10 15Cfu/mL, Bdellovibrio nectophore initial concentration are 10 1～10 3Pfu/mL ferments; Leavening temperature is controlled at 28～30 ℃, and the pH value is controlled at 7.2～7.6; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 20%～30%; Add Host Strains suspension in the fermenting process 1～3 time, add 12～18h pitch time of Host Strains suspension at every turn, the amount that at every turn adds Host Strains suspension is take the concentration of Host Strains in fermention medium of new adding as 10 10～10 15Cfu/mL is as the criterion; Fermentation culture 36～48h namely makes bdellovibrio bacteriovorus preparation; Described solvent is DNB liquid nutrient medium, distilled water or phosphate buffered saline buffer.
The described Host Strains of step (1) is gram-positive microorganism useful or environmental sound, is 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);
The described Bdellovibrio of step (2) is BDF01, BDF02, BDF03, BDJ01, BDJ02, BDM01, BDS01, BDS02, BDSM08 or BDFM05.
Described BDF01 is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on January 13rd, 2008, and deposit number is CCTCC NO:M208008; Described BDF02 is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on January 13rd, 2008, and deposit number is CCTCC NO:M208009; Described BDF03 is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on January 13rd, 2008, and deposit number is CCTCC NO:M208010; Described BDJ01 is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on January 14th, 2008, and deposit number is CCTCC NO:M208011; Described BDJ02 is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on January 14th, 2008, and deposit number is CCTCC NO:M208012; Described BDM01 is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on April 28th, 2008, and deposit number is CCTCC NO:M208066; Described BDS01 is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on August 7th, 2009, and deposit number is CCTCC NO:M209169; Described BDS02 is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on August 7th, 2009, and deposit number is CCTCC NO:M209170; Described BDSM08 is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on August 7th, 2009, and deposit number is CCTCC NO:M209171; Described BDFM05 is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on August 7th, 2009, and deposit number is CCTCC NO:M209172.
The mode of the described collection thalline of step (1) is preferably by centrifugation and obtains, and centrifugal condition is 2～15 ℃, 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, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ") to separate and obtains.
It is 0.1～0.3mol/L that each described phosphate buffered saline buffer of step (1)～(3) is preferably concentration, and pH is 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 the described Bdellovibrio of step (3) derived from salt water environment, described solvent was DNB liquid nutrient medium or distilled water, and the mass volume ratio concentration of described sodium chloride solution is 25～30g/L;
When the described Bdellovibrio of step (3) derived from degree of saltiness water surrounding, described solvent was DNB liquid nutrient medium or distilled water, and the mass volume ratio concentration of described sodium chloride solution is 5～10g/L;
When the described Bdellovibrio of step (3) derives from fresh water environment, do not add sodium-chlor;
The condition optimization of the described sterilization of step (3) is 121 ℃ of sterilization 20min;
The concentration of the described bdellovibrio bacteriovorus preparation of step (3) is 10 8～10 12Pfu/mL;
The described DNB liquid nutrient medium of step (3) is that nutrient broth 0.8g, caseinic acid hydrolyzate 0.5g and yeast extract 0.1g are dissolved in the 1000mL distilled water, regulates pH value 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 by other pathogenic bacterium of cracking or potentially pathogenic organism the purpose of control germ evil.Described bdellovibrio bacteriovorus preparation not only can directly be made preparation, also can by further centrifugal, make simple telotroch preparation, leech liposome preparation and their preparation that is mixed in proportion.
The present invention has following advantage and effect with respect to prior art:
(1) Host Strains that adopts in the fermentation process of the present invention is gram-positive microorganism 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 showed, that the bdellovibrio bacteriovorus preparation that makes with the method improves to the cracking ability of pathogenic bacterium; On the other hand, prepared bdellovibrio bacteriovorus preparation can directly use, and need not through more fermentation aftertreatment; Moreover because the host is probiotics or the bacterial strain of environmental sound, the use range of said preparation also increases greatly.
(2) the present invention is when obtaining high density Bdellovibrio bacterium liquid, relatively also shortened fermentation period, effectively like this solved that fermentation time is long and energy consumption that cause is excessive, the problem that production cost is too high, the bdellovibrio bacteriovorus preparation that obtains not only cost is low, and active high.
Description of drawings
Fig. 1 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (subtilis and intestinal bacteria) fermentations is to cracking ability-time diagram of Salmonella typhimurium and streptococcus aureus.
Fig. 2 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (bacillus natto and intestinal bacteria) fermentations is to cracking ability-time diagram of listeria monocytogenes and Salmonella choleraesuls.
Fig. 3 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (bifidumbacterium bifidum and Salmonella typhimurium) fermentations is to cracking ability-time diagram of streptococcus suis II and streptococcus uberis.
Fig. 4 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (enterococcus faecalis and Salmonella choleraesuls) fermentations is to staphylococcus epidermidis and colibacillary cracking ability-time diagram.
Fig. 5 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (lactobacillus bulgaricus and intestinal bacteria) fermentations is to cracking ability-time diagram of streptococcus equisimilis and Salmonella typhimurium.
Fig. 6 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (pediococcus acidilactici and Aeromonas hydrophila) fermentations is to streptococcus uberis and colibacillary cracking ability-time diagram.
Fig. 7 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (streptococcus acidi lactici and pig hammer II type) fermentations is to the cracking ability-time diagram of Salmonella choleraesuls and staphylococcus epidermidis.
Fig. 8 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (faecium and intestinal bacteria) fermentations is to the cracking ability-time diagram of Salmonella typhimurium and listeria monocytogenes.
Fig. 9 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (Bacillus licheniformis and intestinal bacteria) fermentations is to the cracking ability-time diagram of Aeromonas hydrophila and streptococcus equisimilis.
Figure 10 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (Lactobacterium acidophilum and intestinal bacteria) fermentations is to cracking ability-time diagram of streptococcus aureus and Salmonella choleraesuls.
Figure 11 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (lactobacterium casei and Aeromonas hydrophila) fermentations is to streptococcus equisimilis and colibacillary cracking ability-time diagram.
Figure 12 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (lactobacillus lactis and pig hammer II type) fermentations is to cracking ability-time diagram of staphylococcus epidermidis and Aeromonas hydrophila.
Figure 13 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (plant lactobacillus and Salmonella choleraesuls) fermentations is to cracking ability-time diagram of streptococcus agalactiae and Salmonella typhimurium.
Figure 14 is that the bdellovibrio bacteriovorus preparation that makes with two kinds of different hosts (Pediococcus pentosaceus and intestinal bacteria) fermentations is to the cracking ability-time diagram of streptococcus uberis and suis II type.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
Embodiment 1
Respectively with subtilis (bacterium numbering: GIM1.136, derive from microbial strains preservation center, Guangdong Province) and intestinal bacteria (Escherichia coli, bacterium numbering: GIM1.137, derive from microbial strains preservation center, Guangdong Province) for Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B, detailed process is as follows:
(1) preparation of bacillus subtilis bacteria suspension
Subtilis is inoculated in (peptone 10g in the nutrient broth medium, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2), place 33 ℃ of shaking tables to cultivate 18h, make it be in logarithmic phase, nutrient solution under 4 ℃ of conditions, the centrifugal 25min of 5000rpm, keep the precipitation abandoning supernatant, add potassium-sodium phosphates salt buffer (concentration is 0.2mol/L, pH7.6) in precipitation, obtaining concentration is 10 19Then the bacillus subtilis suspended liquid of cfu/mL place 4 ℃ of refrigerators to save backup;
The same method, preparation concentration is 10 19The intestinal bacteria suspension of cfu/mL places 4 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the intestinal bacteria suspension of step (1) preparation in potassium-sodium phosphates salt buffer (concentration is 0.2mol/L, pH7.6), adjusting colibacillary concentration is 10 12Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the separation of cultivating out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University from the Bdellovibrio BDJ02(of seawater on January 14th, 2008, deposit number is CCTCC NO:M208012) spot, then this nutrient solution is cultivated 48h at 30 ℃, again at 4 ℃, the centrifugal 35min of 5000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 4 ℃, the centrifugal 30min of 13000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.2mol/L, the potassium-sodium phosphates salt buffer of pH7.6, the concentration of adjusting Bdellovibrio nectophore is 10 8Pfu/mL obtains Bdellovibrio BDJ02 telotroch concentrated solution, places 2 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
Add sodium-chlor in the DNB liquid nutrient medium, the adding quality of sodium-chlor is 2.5% of DNB liquid nutrient medium volume, the fermentor tank of packing into, and 121 ℃ of sterilization 20min obtain fermention medium; Add the bacillus subtilis suspended liquid of step (1) preparation and the Bdellovibrio BDJ02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the subtilis in the fermention medium is 10 12Cfu/mL, Bdellovibrio initial concentration are 10 2Pfu/mL ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 28 ℃; The fermention medium pH value during the fermentation that adds bacillus subtilis suspended liquid and Bdellovibrio nectophore concentrated solution is controlled at 7.6; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 28%; Add bacillus subtilis suspended liquid in the culturing process twice, add 15h pitch time of bacillus subtilis suspended liquid at every turn, the amount that at every turn adds bacillus subtilis suspended liquid is take the concentration of subtilis in fermention medium of new adding as 10 12Cfu/mL is as the criterion, and it is 1 * 10 that fermentation culture 48h namely makes concentration 9The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the intestinal bacteria suspension of step (1) preparation and the Bdellovibrio BDJ02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 1 * 10 9The bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all have good lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, streptococcus aureuses.Respectively with to the negative Salmonella typhimurium of gram (Salmonella typhimurium, strain number: GIM1.237, derive from microbial strains preservation center, Guangdong Province) and to the positive streptococcus aureus of gram (Staphylococcus aureus, strain number: GIM1.142 derives from microbial strains preservation center, Guangdong Province) be cracked into example.Be equipped with 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, add respectively the bacterial sediment that the cellar culture method obtains Salmonella typhimurium and streptococcus aureus, every kind of bacterium has two bottles.Regulate the cell concentration consistent (10 of 4 bottles of damping fluids 11Cfu/mL).In two bottles of buffer systems that contain Salmonella typhimurium, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, in two bottles of buffer systems that contain streptococcus aureus, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, these nine moment of 48h, by 10 -1～10 -11Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in Figure 1.As can be seen from Figure 1, compare with the bdellovibrio bacteriovorus preparation B that obtains with Escherichia coli fermentation, bdellovibrio bacteriovorus preparation A with fermentation of bacillus subtilis not only has stronger lytic effect to the negative Salmonella typhimurium of gram, and also good than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive streptococcus aureus of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by subtilis not only can directly make preparation, also can by further centrifugal, make 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 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, place 35 ℃ of shaking tables to cultivate 15h, make it be in logarithmic phase, nutrient solution under 4 ℃ of conditions, the centrifugal 25min of 6000rpm, keep the precipitation abandoning supernatant, add sodium phosphate salt damping fluid (concentration is 0.1mol/L, pH7.4) in precipitation, obtaining concentration is 10 18Then the bacillus natto suspension of cfu/mL place 2 ℃ of refrigerators to save backup;
The same method, preparation concentration is 10 18The intestinal bacteria suspension of cfu/mL places 2 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the intestinal bacteria suspension of step (1) preparation in sodium phosphate salt damping fluid (concentration is 0.1mol/L, pH7.4), adjusting colibacillary concentration is 10 13Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the separation of cultivating out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University from the Bdellovibrio BDF02(of fresh water on January 13rd, 2008, deposit number is CCTCC NO:M208009) spot, then this nutrient solution is cultivated 45h at 30 ℃, again at 4 ℃, the centrifugal 25min of 6000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 4 ℃, the centrifugal 28min of 14000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.1mol/L, the sodium phosphate salt damping fluid of pH7.4, the concentration of adjusting Bdellovibrio nectophore is 10 9Pfu/mL obtains Bdellovibrio BDF02 telotroch concentrated solution, places 4 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
With the DNB liquid nutrient medium fermentor tank of packing into, 121 ℃ of sterilization 20min obtain fermention medium; Add the bacillus natto suspension of step (1) preparation and the Bdellovibrio BDF02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the bacillus natto in the fermention medium is 10 13Cfu/mL, Bdellovibrio initial concentration are 10 2Pfu/mL ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 29 ℃; The fermention medium pH value during the fermentation that adds bacillus natto suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.4; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 28%; Add bacillus natto suspension in the culturing process twice, add 16h pitch time of bacillus natto suspension at every turn, the amount that at every turn adds bacillus natto suspension is for take the concentration of bacillus natto in fermention medium of new adding as 10 13Cfu/mL is as the criterion, and it is 1 * 10 that fermentation culture 45h namely makes concentration 10The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the intestinal bacteria suspension of step (1) preparation and the Bdellovibrio BDF02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 1 * 10 10The bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B have good lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, listeria monocytogenes.Respectively with to the positive listeria monocytogenes of gram (Listeria monocytohenes, strain number: GIM1.228, derive from microbial strains preservation center, Guangdong Province) cracking and to the negative Salmonella choleraesuls of gram (Salmonella enterica, strain number: GIM1.244 derives from microbial strains preservation center, Guangdong Province) be cracked into example.Be equipped with in 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, add respectively the bacterial sediment that the cellar culture method obtains listeria monocytogenes and Salmonella choleraesuls, each two bottles of every kind of bacterium are regulated the cell concentration consistent (10 of 4 bottles of damping fluids 10Cfu/mL).In two bottles of buffer systems that contain listeria monocytogenes, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, in two bottles of buffer systems that contain Salmonella choleraesuls, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1m, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, these eight moment of 36h, 42h, by 10 -1～10 -10Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in Figure 2.As can be seen from Figure 2, compare with the bdellovibrio bacteriovorus preparation B with Escherichia coli fermentation, bdellovibrio bacteriovorus preparation A with bacillus natto to ferment not only has stronger lytic effect to the negative Salmonella choleraesuls of gram, and also better than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive listeria monocytogenes of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by bacillus natto not only can directly make preparation, also can by further centrifugal, make 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 microbial strains preservation center, Guangdong Province) and Salmonella typhimurium respectively, detailed process is as follows:
(1) preparation of bifidumbacterium bifidum suspension
Bifidumbacterium bifidum is inoculated in the PTYG substratum, place 37 ℃ of shaking tables to cultivate 24h, make it be in logarithmic phase, nutrient solution is under 8 ℃ of conditions, the centrifugal 20min of 7000rpm keeps the precipitation abandoning supernatant, and (concentration is 0.3mol/L to adding potassium phosphate salt damping fluid in the precipitation, pH7.5), obtaining concentration is 10 22Then the bifidumbacterium bifidum suspension of cfu/mL place 8 ℃ of refrigerators to save backup;
The same method, preparing concentration with nutrient broth medium is 10 22The Salmonella typhimurium suspension of cfu/mL places 8 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the Salmonella typhimurium suspension of step (1) preparation in potassium phosphate salt damping fluid (concentration is 0.3mol/L, pH7.5), the concentration of adjusting Salmonella typhimurium is 10 10Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the separation of cultivating out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University from the Bdellovibrio BDS02(of soil on August 7th, 2009, deposit number is CCTCC NO:M209170) spot, then this nutrient solution is cultivated 50h at 30 ℃, again at 8 ℃, the centrifugal 20min of 7000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 8 ℃, the centrifugal 20min of 16000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.3mol/L, the potassium phosphate salt damping fluid of pH7.5, the concentration of adjusting Bdellovibrio nectophore is 10 9Pfu/mL obtains Bdellovibrio BDS02 telotroch concentrated solution, places 15 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
Add sodium-chlor at the DNB liquid nutrient medium, the adding quality of sodium-chlor is 0.7% of DNB liquid nutrient medium volume, the fermentor tank of packing into, and 121 ℃ of sterilization 20min obtain fermention medium; Add the bifidumbacterium bifidum suspension of step (1) preparation and the Bdellovibrio BDS02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the bifidumbacterium bifidum in the fermention medium is 10 15Cfu/mL, Bdellovibrio initial concentration are 10 3Pfu/mL ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 30 ℃; The fermention medium pH value during the fermentation that adds bifidumbacterium bifidum suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.2; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 22%; Add bifidumbacterium bifidum suspension in the culturing process one time, add 12h pitch time of bifidumbacterium bifidum suspension at every turn, the amount that at every turn adds bifidumbacterium bifidum suspension is take the concentration of bifidumbacterium bifidum in fermention medium of new adding as 10 15Cfu/mL is as the criterion, and it is 5 * 10 that fermentation culture 36h namely makes concentration 11The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the Salmonella typhimurium suspension of step (1) preparation and the Bdellovibrio BDS02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 5 * 10 11The bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all have lytic effect to pathogenic bacterium such as streptococcus uberis, Salmonellas, swine streptococcus.Respectively with to the negative streptococcus suis II of gram (Streptococcus suis, strain number: CVCC3306, derive from national veterinary microorganism DSMZ) and to the positive streptococcus uberis of gram (Streptococcus uberis, strain number: 700407, derive from Shanghai three and step on Science and Technology Ltd.) be cracked into example.Be equipped with in 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, add respectively the bacterial sediment that the cellar culture method obtains streptococcus suis II and streptococcus uberis, each two bottles of every kind of bacterium are regulated the cell concentration consistent (10 of 4 bottles of damping fluids 11Cfu/mL).In two bottles of buffer systems that contain streptococcus suis II, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, in two bottles of buffer systems that contain streptococcus uberis, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, these nine moment of 48h, by 10 -1～10 -11Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in Figure 3.As can be seen from Figure 3, compare with the bdellovibrio bacteriovorus preparation B with the Salmonella typhimurium fermentation, bdellovibrio bacteriovorus preparation A with the bifidumbacterium bifidum fermentation not only has stronger lytic effect to the negative streptococcus suis II of gram, and also better than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive streptococcus uberis of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by bifidumbacterium bifidum not only can directly make preparation, also can by further centrifugal, make 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 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, place 30 ℃ of shaking tables to cultivate 24h, make it be in logarithmic phase, nutrient solution under 4 ℃ of conditions, the centrifugal 20min of 6000rpm, keep the precipitation abandoning supernatant, add sodium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.4) in precipitation, obtaining concentration is 10 20Then the enterococcus faecalis suspension of cfu/mL place 2～4 ℃ of refrigerators to save backup;
The same method, preparation concentration is 10 20The Salmonella choleraesuls suspension of cfu/mL places 2～4 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the Salmonella choleraesuls suspension of step (1) preparation in sodium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.4), the concentration of adjusting Salmonella choleraesuls is 10 12Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the separation of cultivating out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University from the Bdellovibrio BDFM05(of soil on August 7th, 2009, deposit number is CCTCC NO:M209172) spot, then this nutrient solution is cultivated 38h at 30 ℃, again at 4 ℃, the centrifugal 20min of 6000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 4 ℃, the centrifugal 22min of 15000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.2mol/L, the sodium phosphate salt damping fluid of pH7.4, the concentration of adjusting Bdellovibrio nectophore is 10 9Pfu/mL obtains Bdellovibrio BDFM05 telotroch concentrated solution, places 4 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
Sodium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.6), the fermentor tank of packing into, 121 ℃ of sterilization 20min obtain fermention medium; Add the enterococcus faecalis suspension of step (1) preparation and the Bdellovibrio BDFM05 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the enterococcus faecalis in the fermention medium is 10 14Cfu/mL, Bdellovibrio initial concentration are 10 2Pfu/mL ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 30 ℃; The fermention medium pH value during the fermentation that adds enterococcus faecalis suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.2; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 30%; Add enterococcus faecalis suspension in the culturing process twice, add 18h pitch time of enterococcus faecalis suspension at every turn, the amount that at every turn adds enterococcus faecalis suspension is take the concentration of enterococcus faecalis in fermention medium of new adding as 10 14Cfu/mL is as the criterion, and it is 5 * 10 that fermentation culture 48h namely makes concentration 10The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the Salmonella choleraesuls suspension of step (1) preparation and the Bdellovibrio BDFM05 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 5 * 10 10The bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all have lytic effect to pathogenic bacterium such as intestinal bacteria, Salmonellas, staphylococcus epidermidiss.Respectively with to the positive staphylococcus epidermidis of gram (Staphylococcus epidermidis, strain number: GIM1.143 derive from microbial strains preservation center, Guangdong Province) with to the negative colibacillary example that is cracked into of gram.Be equipped with in 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, respectively add respectively the cellar culture method and obtain staphylococcus epidermidis and colibacillary bacterial sediment, two bottles of every kind of bacterium are regulated the cell concentration consistent (10 of 4 bottles of damping fluids 12Cfu/mL).In two bottles of buffer systems that contain staphylococcus epidermidis, add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, contain in the colibacillary buffer system at two bottles, add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, these nine moment of 48h, by 10 -1～10 -12Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in Figure 4.As can be seen from Figure 4, compare with the bdellovibrio bacteriovorus preparation B with the Salmonella choleraesuls fermentation, bdellovibrio bacteriovorus preparation A with Enterococcus faecalis fermentation not only has stronger lytic effect to the negative intestinal bacteria of gram, and also better than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive staphylococcus epidermidis of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by enterococcus faecalis not only can directly make preparation, also can by further centrifugal, make 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 microbial strains preservation center, Guangdong Province) 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, place 37 ℃ of shaking tables to cultivate 20h, make it be in logarithmic phase, nutrient solution under 10 ℃ of conditions, the centrifugal 25min of 6000rpm, keep the precipitation abandoning supernatant, add potassium phosphate salt damping fluid (concentration is 0.3mol/L, pH7.3) in precipitation, obtaining concentration is 10 17Then the lactobacillus bulgaricus suspension of cfu/mL place 2～4 ℃ of refrigerators to save backup;
The same method, preparation concentration is 10 17The intestinal bacteria suspension of cfu/mL places 2～4 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the intestinal bacteria suspension of step (1) preparation in potassium phosphate salt damping fluid (concentration is 0.3mol/L, pH7.3), adjusting colibacillary concentration is 10 13Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the separation of cultivating out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University from the Bdellovibrio BDJ02(of seawater on January 14th, 2008, deposit number is CCTCC NO:M208012) spot, then this nutrient solution is cultivated 40h at 30 ℃, again at 10 ℃, the centrifugal 25min of 6000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 15 ℃, the centrifugal 20min of 15000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.3mol/L, the potassium phosphate salt damping fluid of pH7.3, the concentration of adjusting Bdellovibrio nectophore is 10 8Pfu/mL obtains Bdellovibrio BDJ02 telotroch concentrated solution, places 2 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
Add sodium-chlor in distilled water, the adding quality of sodium-chlor is 2.8% of distilled water volume, the fermentor tank of packing into, and 121 ℃ of sterilization 20min obtain fermention medium; Add the lactobacillus bulgaricus suspension of step (1) preparation and the Bdellovibrio BDJ02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the lactobacillus bulgaricus in the fermention medium is 10 13Cfu/mL, the Bdellovibrio initial concentration is 10pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 28 ℃; The fermention medium pH value during the fermentation that adds lactobacillus bulgaricus suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.6; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 21%; Add lactobacillus bulgaricus suspension in the culturing process 2 times, add 18h pitch time of lactobacillus bulgaricus suspension at every turn, the amount that at every turn adds lactobacillus bulgaricus suspension is take the concentration of lactobacillus bulgaricus in fermention medium of new adding as 10 13Cfu/mL is as the criterion, and it is 1 * 10 that fermentation culture 40h namely makes concentration 11The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the intestinal bacteria suspension of step (1) preparation and the Bdellovibrio BDJ02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making 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 to the positive streptococcus equisimilis of gram (Streptococcus equinus, strain number: cvcc1925 derive from national veterinary microorganism DSMZ) with to the example that is cracked into of the negative Salmonella typhimurium of gram.Be equipped with in 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, respectively add respectively the bacterial sediment that the cellar culture method obtains streptococcus equisimilis and Salmonella typhimurium, two bottles of every kind of bacterium are regulated the cell concentration consistent (10 of two bottles of damping fluids 11Cfu/mL).In two bottles of buffer systems that contain streptococcus equisimilis, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, in two bottles of buffer systems that contain Salmonella typhimurium, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL bacterium, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, these eight moment of 36h, 42h, by 10 -1～10 -10Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in Figure 5.As can be seen from Figure 5, compare with the bdellovibrio bacteriovorus preparation B with Escherichia coli fermentation, bdellovibrio bacteriovorus preparation A with fermentation using lactobacillus bulgaricus not only has stronger lytic effect to the negative Salmonella typhimurium of gram, and also better than bdellovibrio bacteriovorus preparation B to the positive streptococcus equisimilis streptococcus equisimilis lytic effect of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by lactobacillus bulgaricus not only can directly make preparation, also can by further centrifugal, make simple telotroch, leech liposome preparation.
Embodiment 6
Respectively with pediococcus acidilactici (bacterium numbering: GIM1.263, derive from microbial strains preservation center, Guangdong Province) and Aeromonas hydrophila (Aeromonas hydrophila, bacterium numbering: GIM1.172, derive from microbial strains preservation center, Guangdong Province) for Host Strains prepares bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B, 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, place 30 ℃ of shaking tables to cultivate 18h, make it be in logarithmic phase, nutrient solution under 5 ℃ of conditions, the centrifugal 15min of 7000rpm, keep the precipitation abandoning supernatant, add sodium phosphate salt damping fluid (concentration is 0.1mol/L, pH7.4) in precipitation, obtaining concentration is 10 22Then the pediococcus acidilactici suspension of cfu/mL place 15 ℃ of refrigerators to save backup;
The same method, preparation concentration is 10 22The Aeromonas hydrophila suspension of cfu/mL places 15 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the Aeromonas hydrophila suspension of step (1) preparation in sodium phosphate salt damping fluid (concentration is 0.1mol/L, pH7.4), the concentration of adjusting Aeromonas hydrophila is 10 15Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the separation of cultivating out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University from the Bdellovibrio BDF03(of fresh water on January 13rd, 2008, deposit number is CCTCC NO:M208010) spot, then this nutrient solution is cultivated 54h at 30 ℃, again at 5 ℃, the centrifugal 20min of 7000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 5 ℃, the centrifugal 25min of 15000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.1mol/L, the sodium phosphate salt damping fluid of pH7.4, the concentration of adjusting Bdellovibrio nectophore is 10 9Pfu/mL obtains Bdellovibrio BDF03 telotroch concentrated solution, places 10 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
With the distilled water fermentor tank of packing into, 121 ℃ of sterilization 20min obtain fermention medium; Add the pediococcus acidilactici suspension of step (1) preparation and the Bdellovibrio BDF03 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the pediococcus acidilactici in the fermention medium is 10 14Cfu/mL, the Bdellovibrio initial concentration is 10pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 30 ℃; The fermention medium pH value during the fermentation that adds pediococcus acidilactici suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.4; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 24%; Add pediococcus acidilactici suspension in the culturing process twice, add 15h pitch time of pediococcus acidilactici suspension at every turn, the amount that at every turn adds pediococcus acidilactici suspension is take the concentration of pediococcus acidilactici in fermention medium of new adding as 10 14It is 5 * 10 that cfu/mL, fermentation culture 45h namely make concentration 8Bdellovibrio bacteriovorus preparation A.
Same method adds the Aeromonas hydrophila suspension of step (1) preparation and the Bdellovibrio BDF03 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 5 * 10 8Bdellovibrio bacteriovorus preparation B.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B have good lytic effect to pathogenic bacterium such as intestinal bacteria, Salmonellas, streptococcus uberises.Respectively with to streptococcus uberis and the colibacillary example that is cracked into.Be equipped with in 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, respectively add respectively the cellar culture method and obtain streptococcus uberis and colibacillary bacterial sediment, 2 bottles of every kind of bacterium are regulated the cell concentration consistent (10 of 4 bottles of damping fluids 10Cfu/mL).In two bottles of buffer systems that contain streptococcus uberis, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, contain in the colibacillary buffer system at two bottles, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, these nine moment of 48h, by 10 -1～10 -10Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in Figure 6.As can be seen from Figure 6, compare with the bdellovibrio bacteriovorus preparation B with the Aeromonas hydrophila fermentation, bdellovibrio bacteriovorus preparation A with the pediococcus acidilactici fermentation not only has stronger lytic effect to the negative intestinal bacteria of gram, and also better than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive streptococcus uberis of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by pediococcus acidilactici not only can directly make preparation, also can by further centrifugal, make 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 microbial strains preservation center, Guangdong Province) and streptococcus suis II respectively, detailed process is as follows:
(1) preparation of streptococcus acidi lactici suspension
Streptococcus acidi lactici is inoculated in nutrient broth medium (peptone 10g, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2) in, place 37 ℃ of shaking tables to cultivate 18h, make it be in logarithmic phase, nutrient solution under 4 ℃ of conditions, the centrifugal 27min of 6000rpm, keep the precipitation abandoning supernatant, add potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5) in precipitation, obtaining concentration is 10 20Then the streptococcus acidi lactici suspension of cfu/mL place 4 ℃ of refrigerators to save backup;
The same method, preparation concentration is 10 20The streptococcus suis II suspension of cfu/mL places 4 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the streptococcus suis II suspension of step (1) preparation in potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), the concentration of adjusting streptococcus suis II is 10 15Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the Bdellovibrio BDSM08(that cultivates separation soil out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on August 7th, 2009, deposit number is CCTCC NO:M209171) spot, then this nutrient solution is cultivated 60h at 30 ℃, again at 4 ℃, the centrifugal 20min of 6000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 4 ℃, the centrifugal 25min of 16000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.2mol/L, the potassium phosphate salt damping fluid of pH7.5, the concentration of adjusting Bdellovibrio nectophore is 10 7Pfu/mL obtains Bdellovibrio BDSM08 telotroch concentrated solution, places 4 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
Add sodium-chlor in the pH value is 7.2 DNB liquid nutrient medium, the adding quality of sodium-chlor is 1.5% of DNB liquid nutrient medium volume, the fermentor tank of packing into, and 121 ℃ of sterilization 20min obtain fermention medium; Add the streptococcus acidi lactici suspension of step (1) preparation and the Bdellovibrio BDSM08 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the streptococcus acidi lactici in the fermention medium is 10 13Cfu/mL, Bdellovibrio initial concentration are 10 2Pfu/mL ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 30 ℃; The fermention medium pH value during the fermentation that adds streptococcus acidi lactici suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.5; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 27%; Add streptococcus acidi lactici suspension in the culturing process 2 times, add 14h pitch time of streptococcus acidi lactici suspension at every turn, the amount that at every turn adds streptococcus acidi lactici suspension is take the concentration of streptococcus acidi lactici in fermention medium of new adding as 10 13Cfu/mL is as the criterion, and it is 5 * 10 that fermentation culture 42h namely makes concentration 9The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the streptococcus suis II suspension of step (1) preparation and the Bdellovibrio BDSM08 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 5 * 10 9The bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all have lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, staphylococcus epidermidiss.Respectively with the example that is cracked into to Salmonella choleraesuls and staphylococcus epidermidis.Be equipped with in 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, respectively add respectively the bacterial sediment that the cellar culture method obtains Salmonella choleraesuls and staphylococcus epidermidis, 2 bottles of every kind of bacterium are regulated the cell concentration consistent (10 of 4 bottles of damping fluids 10Cfu/mL).In two bottles of buffer systems that contain Salmonella choleraesuls, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, in two bottles of buffer systems that contain staphylococcus epidermidis, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, these nine moment of 48h, by 10 -1～10 -10Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in Figure 7.As can be seen from Figure 7, compare with the bdellovibrio bacteriovorus preparation B with the streptococcus suis II fermentation, bdellovibrio bacteriovorus preparation A with the streptococcus acidi lactici fermentation not only has stronger lytic effect to the negative Salmonella choleraesuls of gram, and also better than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive staphylococcus epidermidis of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by streptococcus acidi lactici not only can directly make preparation, also can by further centrifugal, make 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 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, place 32 ℃ of shaking tables to cultivate 24h, make it be in logarithmic phase, nutrient solution under 4 ℃ of conditions, the centrifugal 20min of 5000rpm, keep the precipitation abandoning supernatant, add potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5) in precipitation, obtaining concentration is 10 18Then the faecium suspension of cfu/mL place 4 ℃ of refrigerators to save backup;
The same method, preparation concentration is 10 18The intestinal bacteria suspension of cfu/mL places 4 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the intestinal bacteria suspension of step (1) preparation in potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), adjusting colibacillary concentration is 10 15Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the separation of cultivating out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University from the Bdellovibrio BDJ02(of seawater on January 14th, 2008, deposit number is CCTCC NO:M208012) spot, then this nutrient solution is cultivated 60h at 30 ℃, again at 4 ℃, the centrifugal 20min of 6000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 4 ℃, the centrifugal 25min of 16000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.2mol/L, the potassium phosphate salt damping fluid of pH7.5, the concentration of adjusting Bdellovibrio nectophore is 10 6Pfu/mL obtains Bdellovibrio BDJ02 telotroch concentrated solution, places 4 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
Add sodium-chlor in the pH value is 7.2 DNB liquid nutrient medium, the adding quality of sodium-chlor is 2.7% of DNB liquid nutrient medium volume, the fermentor tank of packing into, and 121 ℃ of sterilization 20min obtain fermention medium; Add the faecium suspension of step (1) preparation and the Bdellovibrio BDJ02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the faecium in the fermention medium is 10 13Cfu/mL, Bdellovibrio initial concentration are 10 3Pfu/mL ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 30 ℃; The fermention medium pH value during the fermentation that adds faecium suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.3; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 26%; Add faecium suspension in the culturing process twice, add 14h pitch time of faecium suspension at every turn, the amount that at every turn adds faecium suspension is take the concentration of faecium in fermention medium of new adding as 10 13Cfu/mL is as the criterion, and it is 1 * 10 that fermentation culture 42h namely makes concentration 9The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the intestinal bacteria suspension of step (1) preparation and the Bdellovibrio BDJ02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 1 * 10 9The bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all have lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, listeria monocytogenes.Respectively with the example that is cracked into to listeria monocytogenes and Salmonella typhimurium.Be equipped with in 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, respectively add respectively the bacterial sediment that the cellar culture method obtains listeria monocytogenes and Salmonella typhimurium, 2 bottles of every kind of bacterium are regulated the cell concentration consistent (10 of 4 bottles of damping fluids 11Cfu/mL).In two bottles of buffer systems that contain listeria monocytogenes, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, in two bottles of buffer systems that contain Salmonella typhimurium, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, these eight moment of 36h, 42h, by 10 -1～10 -11Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in Figure 8.As can be seen from Figure 8, compare with the bdellovibrio bacteriovorus preparation B with Escherichia coli fermentation, bdellovibrio bacteriovorus preparation A with the faecium fermentation not only has stronger lytic effect to the negative Salmonella typhimurium of gram, and also better than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive listeria monocytogenes of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by faecium not only can directly make preparation, also can by further centrifugal, make 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 microbial strains preservation center, Guangdong Province) and intestinal bacteria respectively, detailed process is as follows:
(1) preparation of Bacillus licheniformis suspension
Bacillus licheniformis is inoculated in (peptone 10g in the nutrient broth medium, extractum carnis powder 3g, sodium-chlor 5g, pH7.4 ± 0.2), place 33 ℃ of shaking tables to cultivate 18h, make it be in logarithmic phase, nutrient solution under 4 ℃ of conditions, the centrifugal 25min of 5000rpm, keep the precipitation abandoning supernatant, add potassium-sodium phosphates salt buffer (concentration is 0.2mol/L, pH7.6) in precipitation, obtaining concentration is 10 19Then the Bacillus licheniformis suspension of cfu/mL place 4 ℃ of refrigerators to save backup;
The same method, preparation concentration is 10 19The intestinal bacteria suspension of cfu/mL places 4 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the intestinal bacteria suspension of step (1) preparation in potassium-sodium phosphates salt buffer (concentration is 0.2mol/L, pH7.6), adjusting colibacillary concentration is 10 12Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the separation of cultivating out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University from the Bdellovibrio BDM01(of seawater on April 28th, 2008, deposit number is CCTCC NO:M208066) spot, then this nutrient solution is cultivated 48h at 30 ℃, again at 4 ℃, the centrifugal 30min of 5000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 4 ℃, the centrifugal 30min of 13000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.2mol/L, the potassium-sodium phosphates salt buffer of pH7.6, the concentration of adjusting Bdellovibrio nectophore is 10 8Pfu/mL obtains Bdellovibrio BDM01 telotroch concentrated solution, places 4 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
Add sodium-chlor in the DNB liquid nutrient medium, the adding quality of sodium-chlor is 3.0% of DNB liquid nutrient medium volume, the fermentor tank of packing into, and 121 ℃ of sterilization 20min obtain fermention medium; Add the Bacillus licheniformis suspension of step (1) preparation and the Bdellovibrio BDM01 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the Bacillus licheniformis in the fermention medium is 10 12Cfu/mL, Bdellovibrio initial concentration are 10 2Pfu/mL ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 30 ℃; The fermention medium pH value during the fermentation that adds Bacillus licheniformis suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.2; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 28%; Add Bacillus licheniformis suspension in the culturing process twice, add 15h pitch time of Bacillus licheniformis suspension at every turn, the amount that at every turn adds Bacillus licheniformis suspension is take the concentration of Bacillus licheniformis in fermention medium of new adding as 10 12Cfu/mL is as the criterion, and it is 1 * 10 that fermentation culture 48h namely makes concentration 12The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the intestinal bacteria suspension of step (1) preparation and the Bdellovibrio BDM01 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 1 * 10 12The bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all have good lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, streptococcus aureuses.Respectively with to the negative Aeromonas hydrophila of gram with to the example that is cracked into of the positive streptococcus equisimilis of gram (Streptococcus equinus, strain number: cvcc1925 derive from national veterinary microorganism DSMZ).Be equipped with 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, add respectively the bacterial sediment that the cellar culture method obtains Aeromonas hydrophila and streptococcus equisimilis, every kind of bacterium has two bottles.Regulate the cell concentration consistent (10 of 4 bottles of damping fluids 10Cfu/mL).In two bottles of buffer systems that contain Aeromonas hydrophila, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, in two bottles of buffer systems that contain streptococcus equisimilis, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, these nine moment of 48h, by 10 -1～10 -10Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in Figure 9.As can be seen from Figure 9, compare with the bdellovibrio bacteriovorus preparation B that obtains with Escherichia coli fermentation, bdellovibrio bacteriovorus preparation A with the lichen bacillus ferments not only has stronger lytic effect to the negative Aeromonas hydrophila of gram, and also good than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive streptococcus equisimilis of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by Bacillus licheniformis not only can directly make preparation, also can by further centrifugal, make 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 microbial strains preservation center, Guangdong Province) 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, place 32 ℃ of shaking tables to cultivate 24h, make it be in logarithmic phase, nutrient solution under 4 ℃ of conditions, the centrifugal 20min of 5000rpm, keep the precipitation abandoning supernatant, add potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5) in precipitation, obtaining concentration is 10 17Then the Lactobacterium acidophilum suspension of cfu/mL place 8 ℃ of refrigerators to save backup;
The same method, preparation concentration is 10 17The intestinal bacteria suspension of cfu/mL places 8 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the intestinal bacteria suspension of step (1) preparation in potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), adjusting colibacillary concentration is 10 15Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the separation of cultivating out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University from the Bdellovibrio BDS02(of soil on August 7th, 2009, deposit number is CCTCC NO:M209170) spot, then this nutrient solution is cultivated 60h at 30 ℃, again at 4 ℃, the centrifugal 20min of 6000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 15 ℃, the centrifugal 20min of 13000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.2mol/L, the potassium phosphate salt damping fluid of pH7.5, the concentration of adjusting Bdellovibrio nectophore is 10 7Pfu/mL obtains Bdellovibrio BDS02 telotroch concentrated solution, places 6 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
Be the fermentor tank of packing in 7.2 the DNB liquid nutrient medium with the pH value, 121 ℃ of sterilization 20min obtain fermention medium; Add the Lactobacterium acidophilum suspension of step (1) preparation and the Bdellovibrio BDS02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the Lactobacterium acidophilum in the fermention medium is 10 10Cfu/mL, the Bdellovibrio initial concentration is 10pfu/mL, ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 30 ℃; The fermention medium pH value during the fermentation that adds Lactobacterium acidophilum suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.5; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 20%; Add Lactobacterium acidophilum suspension in the culturing process twice, add 14h pitch time of Lactobacterium acidophilum suspension at every turn, the amount that at every turn adds Lactobacterium acidophilum suspension is take the concentration of Lactobacterium acidophilum in fermention medium of new adding as 10 13Cfu/mL is as the criterion, and it is 1 * 10 that fermentation culture 42h namely makes concentration 8The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the intestinal bacteria suspension of step (1) preparation and the Bdellovibrio BDS02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 1 * 10 8The bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all have lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, streptococcus aureuses.Respectively with the example that is cracked into to streptococcus aureus and Salmonella choleraesuls.Be equipped with in 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, respectively add respectively the bacterial sediment that the cellar culture method obtains streptococcus aureus and Salmonella choleraesuls, 2 bottles of every kind of bacterium are regulated the cell concentration consistent (10 of 4 bottles of damping fluids 11Cfu/mL).In two bottles of buffer systems that contain streptococcus aureus, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, in two bottles of buffer systems that contain Salmonella choleraesuls, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, these eight moment of 36h, 42h, by 10 -1～10 -11Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in figure 10.As can be seen from Figure 10, compare with the bdellovibrio bacteriovorus preparation B with Escherichia coli fermentation, bdellovibrio bacteriovorus preparation A with the Lactobacterium acidophilum fermentation not only has stronger lytic effect to the negative Salmonella choleraesuls of gram, and also better than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive streptococcus aureus of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by Lactobacterium acidophilum not only can directly make preparation, also can by further centrifugal, make 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 microbial strains preservation center, Guangdong Province) 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, place 30 ℃ of shaking tables to cultivate 18h, make it be in logarithmic phase, nutrient solution under 5 ℃ of conditions, the centrifugal 15min of 8000rpm, keep the precipitation abandoning supernatant, add sodium phosphate salt damping fluid (concentration is 0.1mol/L, pH7.4) in precipitation, obtaining concentration is 10 22Then the lactobacterium casei suspension of cfu/mL place 15 ℃ of refrigerators to save backup;
The same method, preparation concentration is 10 22The Aeromonas hydrophila suspension of cfu/mL places 15 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the Aeromonas hydrophila suspension of step (1) preparation in sodium phosphate salt damping fluid (concentration is 0.1mol/L, pH7.4), the concentration of adjusting Aeromonas hydrophila is 10 15Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the separation of cultivating out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University from the Bdellovibrio BDF02(of fresh water on January 13rd, 2008, deposit number is CCTCC NO:M208009) spot, then this nutrient solution is cultivated 54h at 30 ℃, again at 5 ℃, the centrifugal 20min of 7000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 5 ℃, the centrifugal 15min of 20000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.1mol/L, the sodium phosphate salt damping fluid of pH7.4, the concentration of adjusting Bdellovibrio nectophore is 10 7Pfu/mL obtains Bdellovibrio BDF02 telotroch concentrated solution, places 2 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
With the distilled water fermentor tank of packing into, 121 ℃ of sterilization 20min obtain fermention medium; Add the lactobacterium casei suspension of step (1) preparation and the Bdellovibrio BDF02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the lactobacterium casei in the fermention medium is 10 14Cfu/mL, Bdellovibrio initial concentration are 10 3Pfu/mL ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 29 ℃; The fermention medium pH value during the fermentation that adds lactobacterium casei suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.2; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 30%; Add lactobacterium casei suspension in the culturing process twice, add 15h pitch time of lactobacterium casei suspension at every turn, the amount that at every turn adds lactobacterium casei suspension is take the concentration of lactobacterium casei in fermention medium of new adding as 10 14Cfu/mL is as the criterion, and it is 1 * 10 that fermentation culture 45h namely makes concentration 9The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the Aeromonas hydrophila suspension of step (1) preparation and the Bdellovibrio BDF02 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 1 * 10 9The bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B have good lytic effect to pathogenic bacterium such as intestinal bacteria, Salmonellas, streptococcus equisimilises.Respectively with to streptococcus equisimilis and the colibacillary example that is cracked into.Be equipped with in 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, respectively add respectively the cellar culture method and obtain streptococcus equisimilis and colibacillary bacterial sediment, 2 bottles of every kind of bacterium are regulated the cell concentration consistent (10 of 4 bottles of damping fluids 12Cfu/mL).In two bottles of buffer systems that contain streptococcus equisimilis, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, contain in the colibacillary buffer system at two bottles, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, these eight moment of 36h, 42h, by 10 -1～10 -12Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in figure 11.As can be seen from Figure 11, compare with the bdellovibrio bacteriovorus preparation B with the Aeromonas hydrophila fermentation, bdellovibrio bacteriovorus preparation A with the lactobacterium casei fermentation not only has stronger lytic effect to the negative intestinal bacteria of gram, and also better than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive streptococcus equisimilis of gram.In addition, the bdellovibrio bacteriovorus preparation A that the Cheesecake Bacterium lacticum makes not only can directly make preparation, also can by further centrifugal, make 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, place 37 ℃ of shaking tables to cultivate 18h, make it be in logarithmic phase, nutrient solution under 4 ℃ of conditions, the centrifugal 20min of 6000rpm, keep the precipitation abandoning supernatant, add potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5) in precipitation, obtaining concentration is 10 20Then the lactobacillus lactis suspension of cfu/mL place 4 ℃ of refrigerators to save backup;
The same method, preparation concentration is 10 20The streptococcus suis II suspension of cfu/mL places 4 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the streptococcus suis II suspension of step (1) preparation in potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), the concentration of adjusting streptococcus suis II is 10 15Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque occurs on the double-deck agar plate of Host Strains to be contained.) the Bdellovibrio BDS01(that cultivates separation soil out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on August 7th, 2009, deposit number is CCTCC NO:M209169) spot, then this nutrient solution is cultivated 20h at 40 ℃, again at 4 ℃, the centrifugal 20min of 6000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 4 ℃, the centrifugal 40min of 12000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.2mol/L, the potassium phosphate salt damping fluid of pH7.5, the concentration of adjusting Bdellovibrio nectophore is 10 8Pfu/mL obtains Bdellovibrio BDS01 telotroch concentrated solution, places 4 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
Add sodium-chlor in the pH value is 7.2 DNB liquid nutrient medium, the adding quality of sodium-chlor is 0.5% of DNB liquid nutrient medium volume, the fermentor tank of packing into, and 121 ℃ of sterilization 20min obtain fermention medium; Add the lactobacillus lactis suspension of step (1) preparation and the Bdellovibrio BDS01 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the lactobacillus lactis in the fermention medium is 10 13Cfu/mL, Bdellovibrio initial concentration are 10 2Pfu/mL ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 30 ℃; The fermention medium pH value during the fermentation that adds lactobacillus lactis suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.2～7.6; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 23%; Add lactobacillus lactis suspension in the culturing process 1 time, add 18h pitch time of lactobacillus lactis suspension at every turn, the amount that at every turn adds lactobacillus lactis suspension is take the concentration of lactobacillus lactis in fermention medium of new adding as 10 13Cfu/mL is as the criterion, and it is 5 * 10 that fermentation culture 42h namely makes concentration 9The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the streptococcus suis II suspension of step (1) preparation and the Bdellovibrio BDS01 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 5 * 10 9The bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all have lytic effect to pathogenic bacterium such as Aeromonas, Salmonellas, staphylococcus epidermidiss.Respectively with the example that is cracked into to Aeromonas hydrophila and staphylococcus epidermidis.Be equipped with in 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, respectively add respectively the bacterial sediment that the cellar culture method obtains Aeromonas hydrophila and staphylococcus epidermidis, 2 bottles of every kind of bacterium are regulated the cell concentration consistent (10 of 4 bottles of damping fluids 11Cfu/mL).In two bottles of buffer systems that contain Aeromonas hydrophila, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, in two bottles of buffer systems that contain staphylococcus epidermidis, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, these nine moment of 48h, by 10 -1～10 -11Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in figure 12.As can be seen from Figure 12, compare with the bdellovibrio bacteriovorus preparation B with the streptococcus suis II fermentation, bdellovibrio bacteriovorus preparation A with the lactobacillus lactis fermentation not only has stronger lytic effect to the negative Aeromonas hydrophila of gram, and also better than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive staphylococcus epidermidis of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by lactobacillus lactis not only can directly make preparation, also can by further centrifugal, make 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 microbial strains preservation center, Guangdong Province) 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, place 30 ℃ of shaking tables to cultivate 24h, make it be in logarithmic phase, nutrient solution under 8 ℃ of conditions, the centrifugal 20min of 6000rpm, keep the precipitation abandoning supernatant, add sodium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.4) in precipitation, obtaining concentration is 10 20Then the plant lactobacillus suspension of cfu/mL place 4 ℃ of refrigerators to save backup;
The same method, the concentration of preparation are 10 20Cfu/mL Salmonella choleraesuls suspension places 4 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the Salmonella choleraesuls suspension of step (1) preparation in sodium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.4), the concentration of adjusting Salmonella choleraesuls is 10 12Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the separation of cultivating out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University from the Bdellovibrio BDF01(of fresh water on January 13rd, 2008, deposit number is CCTCC NO:M208008) spot, then this nutrient solution is cultivated 20h at 30 ℃, again at 2 ℃, the centrifugal 15min of 8000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 4 ℃, the centrifugal 22min of 15000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.2mol/L, the sodium phosphate salt damping fluid of pH7.4, the concentration of adjusting Bdellovibrio nectophore is 10 6Pfu/mL obtains Bdellovibrio BDF01 telotroch concentrated solution, places 15 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
Sodium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.6), the fermentor tank of packing into, 121 ℃ of sterilization 20min obtain fermention medium; Add the plant lactobacillus suspension of step (1) preparation and the Bdellovibrio BDF01 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the plant lactobacillus in the fermention medium is 10 10Cfu/mL, Bdellovibrio initial concentration are 10 2Pfu/mL ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 30 ℃; The fermention medium pH value during the fermentation that adds plant lactobacillus suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.4; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 30%; Add plant lactobacillus suspension in the culturing process twice, add 18h pitch time of plant lactobacillus suspension at every turn, the amount that at every turn adds plant lactobacillus suspension is take the concentration of plant lactobacillus in fermention medium of new adding as 10 10Cfu/mL is as the criterion, and it is 5 * 10 that fermentation culture 48h namely makes concentration 10The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the Salmonella choleraesuls suspension of step (1) preparation and the Bdellovibrio BDF01 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 5 * 10 10The bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all have lytic effect to pathogenic bacterium such as intestinal bacteria, Salmonellas, streptococcus agalactiaes.Respectively with the example that is cracked into to streptococcus agalactiae and Salmonella typhimurium.Be equipped with in 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, respectively add respectively the bacterial sediment that the cellar culture method obtains streptococcus agalactiae and Salmonella typhimurium, two bottles of every kind of bacterium are regulated the cell concentration consistent (10 of 4 bottles of damping fluids 10Cfu/mL).In two bottles of buffer systems that contain streptococcus agalactiae, add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, in two bottles of buffer systems that contain Salmonella typhimurium, add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, these 98 moment of 36h, 42h, by 10 -1～10 -10Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in figure 13.As can be seen from Figure 13, compare with the bdellovibrio bacteriovorus preparation B with the Salmonella choleraesuls fermentation, bdellovibrio bacteriovorus preparation A with the plant lactobacillus fermentation not only has stronger lytic effect to the negative Salmonella typhimurium of gram, and also better than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive streptococcus agalactiae of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by plant lactobacillus not only can directly make preparation, also can by further centrifugal, make 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 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, place 32 ℃ of shaking tables to cultivate 24h, make it be in logarithmic phase, nutrient solution under 4 ℃ of conditions, the centrifugal 20min of 5000rpm, keep the precipitation abandoning supernatant, add potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5) in precipitation, obtaining concentration is 10 18Then the Pediococcus pentosaceus suspension of cfu/mL place 8 ℃ of refrigerators to save backup;
The same method, preparation concentration is 10 18The intestinal bacteria suspension of cfu/mL places 8 ℃ of refrigerators to save backup;
(2) preparation of Bdellovibrio nectophore concentrated solution:
Add the intestinal bacteria suspension of step (1) preparation in potassium phosphate salt damping fluid (concentration is 0.2mol/L, pH7.5), adjusting colibacillary concentration is 10 15Cfu/mL, access (is 200910042274.6 according to application number with ordinary method again, name is called the patent application of " a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof that prevents and treats mastadenitis of cow ", be specially the double-layer plate method that adopts Stolp and Petzhold: get 0.5mL Bdellovibrio nectophore concentrated solution and 0.5mL intestinal bacteria suspension and mix, 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) mixes, be poured into dull and stereotyped (the nutrient broth 0.8g of DNB lower floor, tyrosine acid hydrolysis thing 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ° of C constant incubators to cultivate double-layer plate, plaque appears on the double-deck agar plate of Host Strains to be contained) and the separation of cultivating out is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University from the Bdellovibrio BDJ01(of seawater on January 14th, 2008, deposit number is CCTCC NO:M208011) spot, then this nutrient solution is cultivated 60h at 30 ℃, again at 8 ℃, the centrifugal 30min of 5000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 4 ℃, the centrifugal 25min of 16000rpm, keep the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, in precipitation, add 0.2mol/L, the potassium phosphate salt damping fluid of pH7.5, the concentration of adjusting Bdellovibrio nectophore is 10 9Pfu/mL obtains Bdellovibrio BDJ01 telotroch concentrated solution, places 4 ℃ of Refrigerator stores for subsequent use;
(3) preparation of bdellovibrio bacteriovorus preparation
Add sodium-chlor in the pH value is 7.2 DNB liquid nutrient medium, the adding quality of sodium-chlor is 2.5% of DNB liquid nutrient medium volume, the fermentor tank of packing into, and 121 ℃ of sterilization 20min obtain fermention medium; Add the Pediococcus pentosaceus suspension of step (1) preparation and the Bdellovibrio BDJ01 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, the initial concentration of the Pediococcus pentosaceus in the fermention medium is 10 13Cfu/mL, Bdellovibrio initial concentration are 10 3Pfu/mL ferments; Each Parameter Conditions that ferments is as follows: temperature is controlled at 30 ℃; The fermention medium pH value during the fermentation that adds Pediococcus pentosaceus suspension and Bdellovibrio nectophore concentrated solution is controlled at 7.6; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 25%; Add Pediococcus pentosaceus suspension in the culturing process 3 times, add 12h pitch time of Pediococcus pentosaceus suspension at every turn, the amount that at every turn adds Pediococcus pentosaceus suspension is take the concentration of Pediococcus pentosaceus in fermention medium of new adding as 10 13Cfu/mL is as the criterion, and it is 1 * 10 that fermentation culture 48h namely makes concentration 9The bdellovibrio bacteriovorus preparation A of pfu/mL.
Same method adds the intestinal bacteria suspension of step (1) preparation and the Bdellovibrio BDJ01 telotroch concentrated solution of step (2) preparation in 30 ℃ fermention medium, making concentration is 1 * 10 9The bdellovibrio bacteriovorus preparation B of pfu/mL.
This bdellovibrio bacteriovorus preparation A and bdellovibrio bacteriovorus preparation B all have lytic effect to pathogenic bacterium such as streptococcus uberis, Salmonellas, streptococcus suis II.Respectively with the example that is cracked into to streptococcus uberis and streptococcus suis II.Be equipped with in 4 bottles of potassium-sodium phosphates salt buffers that 50mL respectively is housed, respectively add respectively the bacterial sediment that the cellar culture method obtains streptococcus uberis and streptococcus suis II, 2 bottles of every kind of bacterium are regulated the cell concentration consistent (10 of 4 bottles of damping fluids 10Cfu/mL).In two bottles of buffer systems that contain streptococcus uberis, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL; Equally, in two bottles of buffer systems that contain streptococcus suis II, respectively add respectively bdellovibrio bacteriovorus preparation A1mL and bdellovibrio bacteriovorus preparation B1mL, in 30 ℃, cultivate on the shaking table of 200rpm.Take a sample respectively in 0h, 6h, 12h, 18h, 24h, 30h, these eight moment of 36h, 42h, by 10 -1～10 -10Extension rate dilute, the diluent of respectively getting again 100 μ L carries out respectively the flat board coating, cultivate 24h under 30 ℃ of conditions after, calculate colony number.Experimental result as shown in figure 14.As can be seen from Figure 14, compare with the bdellovibrio bacteriovorus preparation B with Escherichia coli fermentation, bdellovibrio bacteriovorus preparation A with the Pediococcus pentosaceus fermentation not only has stronger lytic effect to the negative streptococcus suis II of gram, and also better than bdellovibrio bacteriovorus preparation B to the lytic effect of the positive streptococcus uberis of gram.In addition, the bdellovibrio bacteriovorus preparation A that is made by Pediococcus pentosaceus not only can directly make preparation, also can by further centrifugal, make simple telotroch, leech liposome preparation.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (5)

1. the fermentation process of a bdellovibrio bacteriovorus preparation is characterized in that may further comprise the steps:
(1) preparation of host bacteria suspension:
Host Strains is cultured to logarithmic phase, collects thalline, adjust concentration with phosphate buffered saline buffer, obtaining concentration is 10 17～10 22Then the Host Strains suspension of cfu/mL place 2～15 ℃ to save backup; Described Host Strains is subtilis (Bacillus subtilis) GIM1.136, lactobacillus bulgaricus (Lactobacillus bulgaricus) GIM1.80 or faecium (Enterococcus faecium) CGMCC1.2136;
(2) preparation of Bdellovibrio nectophore concentrated solution:
The Host Strains suspension that adds step (1) preparation in phosphate buffered saline buffer, the concentration of adjusting Host Strains is 10 10～10 15Cfu/mL, access again the Bdellovibrio spot, then this nutrient solution is cultivated 20～60h at 20～40 ℃, again at 2～15 ℃, the centrifugal 15～35min of 5000～8000rpm, keep supernatant liquor and discard precipitation, then with supernatant liquor at 2～15 ℃, the centrifugal 15～40min of 12000～20000rpm keeps the precipitation abandoning supernatant, be precipitated as Bdellovibrio nectophore, the concentration that adds phosphate buffered saline buffer adjustment Bdellovibrio nectophore in precipitation is 10 6～10 9Pfu/mL obtains the Bdellovibrio nectophore concentrated solution, places 2～15 ℃ to save backup; Described Bdellovibrio is BDJ02, and BDJ02 is preserved in Chinese Typical Representative culture collection center in the Wuhan, China city Wuhan University on January 14th, 2008, and deposit number is CCTCC NO:M208012;
(3) preparation of bdellovibrio bacteriovorus preparation:
With the solvent sterilization, obtain fermention medium; Add the Host Strains suspension of step (1) preparation and the Bdellovibrio nectophore concentrated solution of step (2) preparation in fermention medium, making the Host Strains initial concentration is 10 10～10 15Cfu/mL, Bdellovibrio nectophore initial concentration are 10 1～10 3Pfu/mL ferments; Leavening temperature is controlled at 28～30 ℃, and the pH value is controlled at 7.2～7.6; The interlock of mixing speed and dissolved oxygen is set, makes dissolved oxygen level be controlled at 20%～30%; Add Host Strains suspension in the fermenting process 1～3 time, add 12～18h pitch time of Host Strains suspension at every turn, the amount that at every turn adds Host Strains suspension is take the concentration of Host Strains in fermention medium of new adding as 10 10～10 15Cfu/mL is as the criterion; Fermentation culture 36～48h namely 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, it is characterized in that: the mode of the described collection thalline of step (1) is for to obtain by centrifugation, and centrifugal condition is 2～15 ℃, the centrifugal 15～35min of 5000～8000rpm; The concentration of described Host Strains suspension is 10 17～10 22Cfu/mL; The concentration of step (1)～(3) each described phosphate buffered saline buffer is 0.1～0.3mol/L, and pH is 7.2～7.6; The concentration of the described Bdellovibrio nectophore concentrated solution of step (2) is 10 8～10 12Pfu/mL.
3. the fermentation process of a kind of bdellovibrio bacteriovorus preparation according to claim 1 is characterized in that: the condition of the described sterilization of step (3) is 121 ℃ of sterilization 20min; The concentration of described bdellovibrio bacteriovorus preparation is 10 8～10 12Pfu/mL; Described DNB liquid nutrient medium is that nutrient broth 0.8g, caseinic acid hydrolyzate 0.5g and yeast extract 0.1g are dissolved in the 1000mL distilled water, regulates pH value to 7.2～7.6.
4. a bdellovibrio bacteriovorus preparation is prepared by each described method of claim 1～3.
5. the application of bdellovibrio bacteriovorus preparation according to claim 4 in preparation control Salmonella typhimurium, streptococcus aureus, streptococcus equisimilis and listeria monocytogenes medicine.
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