CN113046339B - Filtering aid for phage fermentation broth post-treatment and preparation method and application thereof - Google Patents

Filtering aid for phage fermentation broth post-treatment and preparation method and application thereof Download PDF

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CN113046339B
CN113046339B CN201911380306.3A CN201911380306A CN113046339B CN 113046339 B CN113046339 B CN 113046339B CN 201911380306 A CN201911380306 A CN 201911380306A CN 113046339 B CN113046339 B CN 113046339B
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filter aid
enzyme activity
fermentation broth
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treatment
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CN113046339A (en
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于浩
樊小九
肖逍
丁良
丛郁
徐旭凌
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Phagelux Nanjing Biotechnology Co ltd
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Phagelux Nanjing Biotechnology Co ltd
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    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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Abstract

The invention relates to the field of biology, and particularly provides a filter aid for phage fermentation broth post-treatment, and a preparation method and application thereof. The filter aid of the present invention includes dnase, lipase and xylanase. The bacteriophage fermentation broth post-treatment filter aid has reasonable components, easily obtained materials, low production cost and no harm to human bodies, and is mainly used in the food industry. The invention is simple and easy to operate and has obvious effect. The phage fermentation broth post-treatment filter aid has extremely high application prospect, and is convenient to use and convenient to apply.

Description

Filtering aid for phage fermentation broth post-treatment and preparation method and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a filter aid for phage fermentation broth post-treatment, and a preparation method and application of the filter aid.
Background
With the increasing of the drug resistance of bacteria to antibacterial drugs, the curative effect of some antibacterial drugs is reduced, and superbacteria are even induced, so that the search for novel antibacterial drugs is urgent. The bacteriophage has high antibacterial activity and specificity as a natural bacterial virus, can prevent and treat drug-resistant bacteria and can avoid damaging normal microbial flora. Compared with antibiotic treatment, the side effect is extremely low, the method is more rapid and effective, the natural immunity of the organism is not inhibited or allergic reaction is not caused, and the method has no infectivity and toxicity to human beings or other mammals, and is a living antimicrobial microorganism with great drug development prospect.
The phage has wide application prospect in the field of medicine, and mature fermentation and post-treatment production process is an important link in the application process. The principle of the fermentation production of phage is: after the phage injects the DNA itself into the splitting host bacteria, the host bacteria genetic material is utilized to make self-replication, and finally the splitting host bacteria are split and a large number of progeny phage are released, and the progeny phage continue to repeat the above process, thereby achieving the proliferation speed increased in geometric progression. Purification is the first step after phage propagation and the main purpose is to separate target phage from host bacteria that have not been lysed and the organelles of host bacteria that have been lysed and their fragments by Microfiltration (MF). Bacterial lysates are more concentrated after phage propagation (especially in the case of higher biomass concentrations) due to the release of nucleic acids and other secondary metabolites. The substances with large molecular weight cannot pass through the bacterial filter membrane, so that the filter membrane is easily blocked, the utilization rate of the bacterial filter membrane is influenced, the aftertreatment efficiency of phage products is further reduced, the production cost is increased, and the application and popularization of phage products are severely restricted.
Bacteria such as staphylococcus aureus, escherichia coli, vibrio parahaemolyticus, ralstonia solanaceae, salmonella pullorum, xanthomonas carpet grass and the like are important pathogenic bacteria of human, animal and plant bacterial diseases, and are also important target bacteria of phage products. In the fermentation production process of the laboratory, the condition that the fermentation liquid seriously blocks the bacterial filter membrane in the purification process appears. The only solution adopted at present is to replace a new bacterial filter membrane for filtration. Yi Xin et al, published separation of enterohemorrhagic Escherichia coli O157 phage and its biological properties in sewage treatment systems, university of Chinese academy of sciences, 2015,32 (04): 512-519; zhao Gongli et al, published concentrated isolation of phage and extraction of macroqualified genomic DNA in sewage treatment systems, university of Henan university (Nature science edition), 2016,44 (04): 131-136.
In the above methods, a method of replacing a bacterial filter is adopted, but the cost of the bacterial filter is expensive, and the large-scale use of the bacterial filter causes a great increase in production cost. There is therefore a need to develop a new technique to address filter clogging.
Disclosure of Invention
In view of the above state of the art, the present invention provides a filter aid for the post-treatment of phage fermentation broth, and its preparation and use.
The filter aid for the post-treatment of phage fermentation broth comprises: dnase, lipase and xylanase.
In the present invention, as one of the embodiments, wherein the filter aid comprises: the dosage of the DNase is 1500000-7500000U/100mL, optimally 3000000U/100mL in terms of enzyme activity unit;
in the present invention, as one of the embodiments, wherein the filter aid comprises: the dosage of the lipase is 1000-5000U/100mL, and optimally 4000U/100mL in terms of enzyme activity units;
in the present invention, as one of the embodiments, wherein the filter aid comprises: the xylanase is dosed at 18000-90000U/100mL, optimally 72000U/100mL, in terms of enzyme activity units.
In the present invention, as one of the embodiments, the activity unit of the dnase includes, but is not limited to, 15000U/mg to 30000U/mg; as an exemplary illustration, there may be mentioned 15000U/mg, 17000U/mg, 20000U/mg, 22000U/mg, 25000U/mg, 27000U/mg, 30000U/mg of DNase, or other natural number activity units within the above-mentioned range, as well as other higher number activity units of DNase in the field of the present invention. As one embodiment, the invention preferably has a DNase activity unit of 15000U/mg or 30000U/mg.
In the present invention, as one of the embodiments, the activity unit of the lipase includes, but is not limited to 10000U/g to 25000U/g, and as an exemplary illustration, it may be 10000U/g, 13000U/g, 15000U/g, 17000U/g, 20000U/g, 22000U/g, 25000U/g, or other natural number activity units in the range, but also other higher activity units in the field of the present invention; as one embodiment, the activity unit of the lipase is preferably 10000U/g or 25000U/g.
In the present invention, as one embodiment, the xylanase activity units include, but are not limited to, 180000U/g to 300000U/g; as an exemplary illustration, it may be 180000U/g, 200000U/g, 220000U/g, 250000U/g, 280000U/g, 300000U/g of xylanase, or other natural number activity units within this range, but also other higher activity units of xylanase in the field of the invention; as one embodiment, the viability unit of the xylan is preferably 180000U/g or 300000U/g.
The filter aid comprising DNase, lipase and xylanase of the invention may be used in the treatment of phage broths including, but not limited to, vibrio parahaemolyticus, staphylococcus aureus, ralstonia solanacearum, escherichia coli, salmonella pullorum or Xanthomonas carpet.
In one embodiment, the invention also provides a filter aid which contains DNase, lipase and xylanase, and further comprises cellulase and pectinase.
In the invention, as one embodiment, the dosage of the cellulase in the filter aid is 1500-2500U/100mL, preferably 1500-2000U/100mL, and most preferably 1500U/100mL in terms of enzyme activity units;
in the present invention, as one embodiment, the dosage of pectase in the filter aid is 2500-12500U/100mL, preferably 7500-10000U/100mL, and most preferably 7500U/100mL, in terms of enzyme activity units.
In the present invention, as one embodiment, the filter aid comprises the following components in the following amounts: in terms of the unit of enzyme activity,
dnase: 1500000-7500000U/100mL, preferably 3000000-6000000U/100mL, and most preferably 3000000U/100mL;
lipase: 1000-5000U/100mL, preferably 4000-5000U/100mL, and most preferably 4000U/100mL;
xylanase: 18000-90000U/100mL, preferably 36000-72000U/100mL, and most preferably 72000U/100mL;
cellulase: 1500-2500U/100mL, preferably 1500-2000U/100mL, and most preferably 1500U/100mL; and
pectase: 2500-12500U/100mL, preferably 7500-10000U/100mL, and most preferably 7500U/100mL.
In the present invention, as one of the embodiments, wherein the activity unit of dnase in the filter aid is 15000U/mg or 30000U/mg;
in the present invention, as one of the embodiments, wherein the activity unit of lipase in the filter aid is 10000U/g or 25000U/g;
in the present invention, as one of the embodiments, wherein the xylanase activity unit in the filter aid is 180000U/g or 300000U/g;
in the present invention, as one of the embodiments, wherein the activity unit of the cellulase in the filter aid is 5000U/g;
in the present invention, as one of the embodiments, wherein the activity unit of the pectase in the filter aid is 25000U/g.
As one embodiment, the filter aid comprising DNase, lipase, xylanase, cellulase and pectinase of the invention is preferably used for post-treatment of a phage fermentation broth of Vibrio parahaemolyticus.
As one embodiment, the invention also provides a filter aid which contains DNase, lipase and xylanase and further comprises cellulase and phospholipase.
In the present invention, as one embodiment, the dosage of the cellulase in the filter aid is 500-2500U/100mL, preferably 1000-2500U/100mL, and most preferably 1500U/100mL;
In the present invention, as one embodiment, the dosage of phospholipase in the filter aid is 20000 to 50000U/100mL, preferably 40000 to 50000U/100mL, and most preferably 40000U/100mL.
In the present invention, as one of the embodiments, the filter aid comprises the following components in terms of enzyme activity units:
dnase: 1500000-7500000U/100mL, preferably 3000000-7500000U/100mL, most preferably 4500000/100mL;
lipase: 1000-5000U/100mL, preferably 2000-5000U/100mL, and most preferably 4000U/100mL;
xylanase: 18000-90000U/100mL, preferably 48000-90000U/100mL, more preferably 54000-90000U/100mL, and most preferably 72000U/100mL;
cellulase: 500-2500U/100mL, preferably 1000-2500U/100mL, and most preferably 1500U/100mL; and
phospholipase: 20000-50000U/100mL, preferably 40000-50000U/100mL, most preferably 40000U/100mL.
The present invention, as one embodiment, the filter aid of the present invention comprises:
the activity unit of the DNase is 15000U/mg or 30000U/mg;
the activity unit of the lipase is 10000U/g or 25000U/g;
the activity unit of the xylanase is 180000U/g or 300000U/g;
the activity unit of the cellulase is 5000U/g;
The activity unit of the phospholipase is 100000U/g.
As one embodiment, the filter aid comprising dnase, lipase, xylanase, cellulase and phospholipase is preferably used for post-treatment of staphylococcus aureus phage fermentation broth.
In one embodiment, the invention also provides a filter aid which contains DNase, lipase and xylanase and further comprises phospholipase and pectinase.
In the present invention, as one of the embodiments, the dosage of the phospholipase in the filter aid is 10000-50000U/100mL, preferably 10000-40000U/100mL, and most preferably 20000U/100mL, in terms of enzyme activity units; the dosage of the pectase is 2500-12500U/100mL, preferably 5000-12500U/100mL, and most preferably 10000U/100mL.
In the present invention, as one of the embodiments, the filter aid comprises the following components in terms of enzyme activity units:
dnase: 1500000-7500000U/100mL, preferably 3000000-7500000U/100mL, and most preferably 3000000U/100mL;
lipase: 1000-5000U/100mL, preferably 2000-4000U/100mL, and most preferably 3000U/100mL;
xylanase: 36000-90000U/100mL, preferably 48000-90000U/100mL, further 54000-90000U/100mL, optimally 72000U/100mL;
Phospholipase: 10000-50000U/100mL, preferably 10000-40000U/100mL, and most preferably 20000U/100mL; and
pectase: 2500-12500U/100mL, preferably 5000-12500U/100mL, and most preferably 10000U/100mL.
As one embodiment, the filter aid of the present invention comprises:
the activity unit of the DNase is 15000U/mg or 30000U/mg;
the activity unit of the lipase is 10000U/g or 25000U/g;
the activity unit of the xylanase is 180000U/g or 300000U/g;
the activity unit of the phospholipase is 100000U/g;
the activity unit of the pectase is 25000U/g.
In the present invention, as one embodiment, a filter aid comprising dnase, lipase, xylanase, phospholipase and pectinase is preferably used for post-treatment of a bacteriophage fermentation broth of solanaceae.
As one embodiment, the invention also provides a filter aid which contains DNase, lipase and xylanase and further comprises cellulase, phospholipase and pectinase.
As one embodiment, the filter aid comprises, in terms of enzyme activity units:
the dosage of the cellulase is 500-2500U/100mL, preferably 500-2000U/100mL, more preferably 1500-2000U/100mL, and most preferably 1500U/100mL;
The dosage of the phospholipase is 10000-50000U/100mL, preferably 20000-50000U/100mL, and optimally 40000U/100mL;
the dosage of the pectase is 2500-12500U/100mL, preferably 5000-10000U/100mL, and most preferably 7500U/100mL.
As one embodiment, wherein the filter aid comprises the following components in terms of enzyme activity units:
dnase: 1500000-7500000U/100mL, preferably 3000000-7500000/100mL, most preferably 3000000/100mL;
lipase: 2000-5000U/100mL, preferably 3000-5000U/100mL, and most preferably 4000U/100mL;
xylanase: 36000-90000U/100mL, preferably 54000-90000U/100mL, most preferably 54000U/100mL;
cellulase: 500-2500U/100mL, preferably 500-2000U/100mL, preferably 1500-2000U/100mL, and most preferably 1500U/100mL;
phospholipase: 10000-50000U/100mL, preferably 20000-50000U/100mL, optimally 40000U/100mL; and
pectase: 2500-12500U/100mL, preferably 5000-10000U/100mL, and most preferably 7500U/100mL.
As one embodiment, the filter aid of the present invention comprises:
the activity unit of the DNase is 15000U/mg or 30000U/mg;
the activity unit of the lipase is 10000U/g or 25000U/g;
the activity unit of the xylanase is 180000U/g or 300000U/g;
The activity unit of the cellulase is 5000U/g;
the activity unit of the phospholipase is 100000U/g;
the activity unit of the pectase is 25000U/g.
The filter aid containing DNase, lipase, xylanase, cellulase, phospholipase and pectinase is preferably used for the post-treatment of coliphage fermentation broth.
As one embodiment, the filter aid of the present invention has a pH of 7.2; the adjustment can be performed using buffers commonly used in the art, including but not limited to, PBS, borate or citrate buffer solutions, preferably PBS buffer solutions.
As one embodiment, the filter aid for the post-treatment of phage fermentation broth comprises the following components in terms of enzyme activity units:
dnase: 3000000U/100mL;
lipase: 4000U/100mL;
xylanase: 72000U/100mL; and
10 x PBS buffer (ph=7.2): 10mL/100mL.
As one embodiment, the phage fermentation broth includes, but is not limited to, phage fermentation broth of vibrio parahaemolyticus, staphylococcus aureus, ralstonia solanacearum, escherichia coli, salmonella pullorum, or xanthomonas carpet grass.
As one embodiment, the filter aid for post-treatment of a phage fermentation broth of vibrio parahaemolyticus of the present invention comprises the following components in terms of enzyme activity units:
Dnase: 3000000U/100mL;
cellulase: 1500U/100mL;
pectase: 7500U/100mL;
lipase: 4000U/100mL;
xylanase: 72000U/100mL; and
10 x PBS buffer (ph=7.2): 10mL/100mL.
As one embodiment, the filter aid for post-treatment of staphylococcus aureus phage fermentation broth of the invention comprises the following components in terms of enzyme activity unit:
dnase: 4500000U/100mL;
cellulase: 1500U/100mL;
phospholipase: 40000U/100mL;
lipase: 4000U/100mL;
xylanase: 72000U/100mL; and
10 x PBS buffer (ph=7.2): 10mL/100mL.
As one embodiment, the invention is used for post-treating the filter aid of the fermentation liquor of the solanaceae Ralstonia phage, and comprises the following components in terms of enzyme activity units:
dnase: 3000000U/100mL;
phospholipase: 20000U/100mL;
pectase: 10000U/100mL;
lipase: 3000U/100mL;
xylanase: 72000U/100mL; and
10 x PBS buffer (ph=7.2): 10mL/100mL.
As one embodiment, the filter aid for the post-treatment of coliphage fermentation broth of the present invention comprises the following components in terms of enzyme activity units:
Dnase: 3000000U/100mL;
cellulase: 1500U/100mL;
phospholipase: 40000U/100mL;
pectase: 7500U/100mL;
lipase: 4000U/100mL;
xylanase: 54000U/100mL; and
10 x PBS buffer (ph=7.2): 10mL/100mL.
The phage fermentation broth purifying filter aid has reasonable components.
Wherein various organic matter digestive enzymes can act with fermentation products other than phage, decomposing the fermentation products, making them into substances smaller than 0.22 μm in diameter. For example: the DNase can cut off the phosphodiester bond to hydrolyze the phosphodiester bond on the main chain of the sugar-phosphate, so that a large amount of deoxyribonucleic acid substances released by host bacteria after phage cleavage in the fermentation process can be effectively degraded, active nucleic acid is deactivated, the viscosity is reduced, and the active nucleic acid can smoothly pass through a filter membrane; the cellulase can effectively degrade cellulose synthesized by bacteria in the fermentation process; phospholipase acts on each ester bond in phospholipid molecule specifically, and can degrade phospholipid component in cell membrane structure remained after host bacteria rupture in fermentation process effectively; the phage fermentation broth has complex components and contains substrates for pectase, and the substrates are converted into water-soluble pectin under the action of pectase, then catalyzed to remove methyl ester groups and then degraded to produce galacturonic acid. Lipases catalyze hydrolysis, alcoholysis, esterification, transesterification of triacylglycerides and other water-insoluble esters and reverse synthesis of esters, and in addition, exhibit other enzymatic activities such as phospholipase, lysophospholipase, cholesterol esterase, acyl peptide hydrolase, and the like. The lipase can effectively degrade phospholipid components and other lipid substances in cell membrane structures remained after the rupture of host bacteria in the fermentation process. The xylanase can digest non-starch saccharides in the fermentation broth.
Further, a filter aid is post-treated against phage fermentation broth, wherein the water is ddH 2 O。ddH 2 And the quality of the filtering and sterilizing filter aid for the post-treatment of the phage fermentation broth can be ensured to be stable and controllable.
Furthermore, aiming at the phage fermentation broth post-treatment filter aid, the buffer solution preparation is beneficial to the protection of enzyme activity and the preservation of the filter aid.
The invention also provides a preparation method of the phage fermentation broth filter aid, which comprises the following steps:
1) Accurately weighing 10×pbs buffer (ph=7.2) with a measuring cylinder, and placing in a volumetric flask;
2) Accurately weighing the enzyme solids used in the filter aid, and using a proper amount of sterile ddH 2 Adding O into the volumetric flask of the step 1) after dissolving, and fixing the volume by using sterile water;
3) Passing the obtained solution through a 0.22 μm aseptic filter for aseptic treatment;
4) The obtained solution is packaged in a sterile centrifuge tube and stored in a refrigerator at 4 ℃ for standby.
Wherein the enzyme solid comprises:
dnase: activity unit 15000U/mg or 30000U/mg;
cellulase: the activity unit is 5000U/g;
phospholipase: activity unit 10 0000U/g;
pectase: activity unit 25000U/g;
lipase: activity units 10000U/g or 25000U/g; or (b)
Xylanase: activity units 180000U/g or 300000U/g.
As one embodiment, the invention provides a method for preparing a post-treatment filter aid for phage fermentation broth comprising vibrio parahaemolyticus, staphylococcus aureus, ralstonia solanacearum, escherichia coli, salmonella pullorum or xanthomonas carpet oxalis: accurately weighing 0.2g of DNase, 0.4g of lipase and 0.4g of xylanase, and adding a proper amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then sub-packaged in a 50mL sterile centrifuge tube, and stored at 4℃for use.
As one embodiment, the preparation method of the filter aid for post-treatment of the phage fermentation broth of vibrio parahaemolyticus comprises the following steps: accurately weighing 0.2g of DNase, 0.3g of cellulase, 0.3g of pectase, 0.4g of lipase and 0.4g of xylanase, and adding a proper amount of ddH 2 After stirring until O is sufficiently dissolved, it is added to 10mL of 10×pbs buffer (ph=7.2), and then ddH is used 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then packaged in sterile centrifuge tubes, and stored at 4℃for further use.
As one embodiment, the preparation method of the filter aid for post-treatment of staphylococcus aureus phage fermentation broth comprises the following steps: accurately weighing 0.3g of DNase, 0.3g of cellulase, 0.4g of phospholipase, 0.4g of lipase and 0.4g of xylanase, and adding a proper amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then packaged in sterile centrifuge tubes, and stored at 4℃for further use.
As one embodiment, the preparation method of the filter aid for post-treatment of the fermentation liquor of the Lei's phage of the Solanaceae comprises the following steps: accurately weighing 0.2g of DNase and 0.2g of phospholipaseAdding appropriate amount of ddH into pectase 0.4g, lipase 0.3g and xylanase 0.4g 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then packaged in sterile centrifuge tubes, and stored at 4℃for further use.
As one embodiment, the preparation method of the filter aid for the post-treatment of the coliphage fermentation broth comprises the following steps: accurately weighing 0.2g of DNase, 0.3g of cellulase, 0.4g of phospholipase, 0.3g of pectase, 0.4g of lipase and 0.3g of xylanase, and adding a proper amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then packaged in sterile centrifuge tubes, and stored at 4℃for further use.
The preparation and preservation method of the phage fermentation broth purification filter aid has the advantages of simple and feasible process, high efficiency, low application cost, simple operation, easy realization and convenient application.
The invention also provides an application method of the phage fermentation broth filter aid, and the phage fermentation broth filter aid is applied:
1. mixing the filter aid with the final volume ratio of 10% in phage fermentation broth, uniformly stirring, and carrying out interaction for 2-10 hours at room temperature;
2. the solution was filtered using a 0.22 μm sterile filter. Phage solutions were obtained that removed the host bacteria and most of the secondary metabolites.
As one embodiment, the filter aid for post-treatment of phage fermentation broth comprising vibrio parahaemolyticus, staphylococcus aureus, ralstonia solanacearum, escherichia coli, salmonella pullorum or xanthomonas carpet, the optimal treatment time for phage fermentation broth of vibrio parahaemolyticus is 12 hours; or the optimal treatment time for staphylococcus aureus phage fermentation broth is 10 hours; or the optimal treatment time for the phage fermentation broth of the Ralstonia solanaceae is 12 hours; the optimal treatment time for the coliphage fermentation broth is 10 hours; or the optimal treatment time for the salmonella pullorum phage fermentation broth is 10 hours; or the optimal treatment time for the xanthomonas carpet grass phage fermentation broth is 8 hours.
As one embodiment, the optimal treatment time for the vibrio parahaemolyticus phage fermentation broth for the post-treatment filter aid is 6 hours.
As one embodiment, the optimal treatment time for the staphylococcus aureus phage fermentation broth for the post-treatment filter aid is 8 hours.
As one embodiment, the optimal treatment time of the filter aid for post-treatment of the fermentation broth of the Leucopia utilis bacteriophages of Solanaceae is 8 hours.
As one embodiment, the optimal treatment time for the coliphage fermentation broth for the filter aid for the coliphage fermentation broth is 4 hours.
The phage fermentation broth filter aid is convenient to use and can be used for a plurality of different phages. The adaptability is good, the method can be carried out under the room temperature condition, and the method has extremely high application prospect.
Compared with the prior art, the invention has the following advantages: the bacteriophage fermentation broth post-filtration auxiliary agent disclosed by the invention has the advantages of reasonable components, wide raw material sources, easiness in obtaining, low cost, capability of maintaining extremely high survival rate and activity of bacteriophage and good adaptability. The preparation and storage methods and the methods of the phage purification filter aid are reasonable, the process is simple and feasible, the efficiency is high, the application cost is low, the operation is simple, and the phage purification filter aid is easy to realize. Has extremely high application prospect, low requirement on preservation temperature, convenient use, and convenient application, and can be used for preservation under various conditions.
Detailed Description
The following examples and experimental examples serve to further illustrate the invention but do not limit the effective scope of the invention in any way.
In the following examples of the present invention,
the formula of the LB liquid medium is as follows: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water.
The formula of the LB solid medium is as follows: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 15g of agar and 1000mL of distilled water.
0.22 μm sterile filter (Millpore)
Sterile centrifuge tube (Eppendorf)
Dnase I enzyme (activity unit: 15000U/mg, available from SIGMA Co.);
cellulase (vitality unit: 5000U/g, purchased from the summer-top industry group);
phospholipase (activity unit: 100000U/g, available from Zhengzhou Wanbo chemical Co., ltd.);
pectase (activity unit: 25000U/g, purchased from Xiasheng industries);
lipase (activity units: 10000U/g, purchased from the Xiasheng Utility group);
xylanase (activity unit: 180000U/g, purchased from Xiasheng industries);
10 x PBS buffer (ph=7.2) (available from ThomerFisher);
ddH 2 O。
the phage strains described in examples 7 to 9 below were all obtained from the patents disclosed, and the phage strains used in examples 10 to 12 were as follows:
the preservation unit of the coliphage CL1 (Escherichia coli phage CL 1) is China center for type culture collection, and the address is Chinese university of Wuhan, mail code 430072; the preservation date is 2018, 12 and 28; the preservation number is CCTCC NO: m2018936.
The preservation unit of the salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG P1) is China center for type culture collection, and the address is China university of Wuhan, and post code 430072; the preservation date is 2018, 11 and 09; the preservation number is CCTCC NO: m2018765.
The storage unit of the xanthomonas carpet grass phage YHC5 (Xanthomonas axonopodis phage YHC 5) is China center for type culture collection, and the address is Chinese university of Wuhan, and mail code 430072; the preservation date is 2018, 08 and 30; the preservation number is CCTCC NO: m2018579.
EXAMPLE 1 preparation of formulations of six enzymes at different concentrations
Accurately weighing 0.1g, 0.2g, 0.3g, 0.4g and 0.5g of DNase (activity unit: 15000U/mg), and adding appropriate amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then sub-packaged in a 50mL sterile centrifuge tube, and stored at 4℃for use.
Accurately weighing 0.1g, 0.2g, 0.3g, 0.4g and 0.5g of cellulase (activity unit: 5000U/g), and adding appropriate amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then sub-packaged in a 50mL sterile centrifuge tube, and stored at 4℃for use.
Accurately weighing 0.1g, 0.2g, 0.3g, 0.4g and 0.5g of phospholipase (activity unit: 10 0000U/g), and adding appropriate amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then sub-packaged in a 50mL sterile centrifuge tube, and stored at 4℃for use.
Accurately weighing pectase (activity unit: 2 5000U/g) 0.1g, 0.2g, 0.3g, 0.4g and 0.5g, and adding appropriate amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then sub-packaged in a 50mL sterile centrifuge tube, and stored at 4℃for use.
Accurately weighing lipase (activity unit: 10000U/g) 0.1g, 0.2g, 0.3g, 0.4g and 0.5g, and adding appropriate amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10×pbs buffer (ph=respectively 7.2 Before adding ddH 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then sub-packaged in a 50mL sterile centrifuge tube, and stored at 4℃for use.
Accurately weighing xylanase (activity unit: 180000U/g) 0.1g, 0.2g, 0.3g, 0.4g and 0.5g, and adding appropriate amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then sub-packaged in a 50mL sterile centrifuge tube, and stored at 4℃for use.
To 10mL of 10 x PBS buffer (ph=7.2) was added ddH 2 O constant volume to 100mL; the resulting 100mL solution was sterilized by a 0.22 μm sterile filter and then packaged in 50mL sterile centrifuge tubes as a blank and stored at 4℃for further use.
Example 2 preparation of phage fermentation broth Filter aid
Accurately weighing 0.2g of DNase, 0.4g of lipase and 0.4g of xylanase, and adding a proper amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then sub-packaged in a 50mL sterile centrifuge tube, and stored at 4℃for use.
Example 3 preparation of Vibrio parahaemolyticus phage fermentation broth Filter aid
Accurately weighing 0.2g of DNase, 0.3g of cellulase, 0.3g of pectase, 0.4g of lipase and 0.4g of xylanase, and adding a proper amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then sub-packaged in a 50mL sterile centrifuge tube, and stored at 4℃for use.
Example 4 preparation of Filter aid for post-treatment of Staphylococcus aureus phage fermentation broth
Accurately weighing 0.3g of DNase and fibers0.3g of luciferase, 0.4g of phospholipase, 0.4g of lipase and 0.4g of xylanase, and a proper amount of ddH is added respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then sub-packaged in a 50mL sterile centrifuge tube, and stored at 4℃for use.
Example 5 preparation of a Filter aid for post-treatment of a phage fermentation broth of Ralstonia solanaceae
Accurately weighing 0.2g of DNase, 0.2g of phospholipase, 0.4g of pectase, 0.3g of lipase and 0.4g of xylanase, and adding a proper amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then sub-packaged in a 50mL sterile centrifuge tube, and stored at 4℃for use.
Example 6 preparation of E.coli phage fermentation broth post-treatment Filter aid
Accurately weighing 0.2g of DNase, 0.3g of cellulase, 0.4g of phospholipase, 0.3g of pectase, 0.4g of lipase and 0.3g of xylanase, and adding a proper amount of ddH respectively 2 After O was stirred until it was sufficiently dissolved, it was added to 10mL of 10 x PBS buffer (ph=7.2), respectively, and then ddH was added 2 O constant volume to 100mL; the 100mL of the obtained solution was sterilized by a 0.22 μm sterile filter, and then sub-packaged in a 50mL sterile centrifuge tube, and stored at 4℃for use.
EXAMPLE 7 preparation of Vibrio parahaemolyticus phage fermentation broth
And (3) respectively inoculating the preserved vibrio parahaemolyticus single colonies into 43 bottles of conical flasks containing 100mL of LB culture medium, and when the temperature is 37 ℃ and the shaking culture is carried out at 240rpm until the OD value is 0.2, respectively adding 100 mu L of 1000PFU/mL vibrio parahaemolyticus phage VP46 (Vibrio parahaemolyticus phage VP, preservation number CCTCC NO: M2016190) into the conical flasks, and carrying out the shaking culture at 240rpm for 18 hours to obtain vibrio parahaemolyticus phage fermentation liquor.
EXAMPLE 8 preparation of Staphylococcus aureus phage fermentation broth
And (3) respectively inoculating the preserved staphylococcus aureus single colonies into 43 conical flasks containing 100mL of LB culture medium, and when the temperature is 37 ℃ and the shaking culture is carried out at 240rpm until the OD value is 0.2, adding 100 mu L of 1000PFU/mL staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J P2 and the preservation number is CCTCC NO: M2016185) into the conical flasks, and shaking culture is carried out at 240rpm for 18 hours at 37 ℃ to obtain staphylococcus aureus phage fermentation liquor.
Example 9 preparation of a fermentation broth of a Ralstonia solanaceae bacteriophage
Inoculating preserved single bacterial colonies of Solanaceae Ralstonia respectively into 43 conical flasks containing 100mL of LB culture medium, and adding 1000PFU/mL of Solanaceae Ralstonia phage GP1 (Ralstonia solanacearum phage GP 1) when the OD value is 0.2 by shake culture at 28 ℃ and 240rpm, wherein the preservation number CCTCC NO: m2016633) 100. Mu.L, 28 ℃,240rpm, and shake culturing for 18 hours to obtain a fermentation broth of the Lei-Barbamate bacteria.
Example 10 preparation of E.coli phage fermentation broth
The stored E.coli single colonies were inoculated into 43 conical flasks containing 100mL of LB medium, and when the culture was performed at 37℃and 240rpm with shaking until the OD was 0.2, 1000PFU/mL of E.coli phage CL1 (Escherichia coli phage CL1, accession number: CCTCC NO: M2018936) was added thereto, 100. Mu.L was performed, and the culture was performed at 37℃and 240rpm with shaking for 18 hours, to thereby obtain E.coli phage fermentation broth.
Example 11 preparation of Salmonella pullorum phage fermentation broth
And (3) respectively inoculating the preserved single colonies of the salmonella pullorum into 7 conical flasks containing 100mL of LB culture medium, and when the culture is carried out at 28 ℃ and 240rpm in a shaking way until the OD value is 0.2, adding 100 mu L of 1000PFU/mL salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG P1, preservation number is CCTCC NO: M2018765) into the conical flasks, and carrying out the shake culture at 28 ℃ and 240rpm for 18 hours to obtain the salmonella pullorum phage fermentation broth.
EXAMPLE 12 preparation of Xanthomonas carpet phage fermentation broth
The preserved single bacterial colony of the xanthomonas carpet grass is respectively inoculated into 7 conical flasks containing 100mL LB culture medium, when the temperature is 37 ℃, and the shaking culture is carried out at 240rpm until the OD value is 0.2, 100 mu L of 1000PFU/mL xanthomonas carpet grass phage YHC5 (Xanthomonas axonopodis phage YHC, preservation number is CCTCC NO: M2018579) is added into the conical flasks, and the shaking culture is carried out at 37 ℃, and the shaking culture is carried out at 240rpm for 18 hours, thus obtaining the xanthomonas carpet grass phage fermentation broth.
Experimental example 1 Effect of six enzymes on Vibrio parahaemolyticus phage fermentation broth
The concentration gradients of 5 enzyme preparations such as Dnase I enzyme, cellulase, phospholipase, pectase, lipase and xylanase prepared in example 1 were selected for single factor experiments.
31 parts of 100mL of Vibrio parahaemolyticus phage fermentation broth are prepared according to the method in example 7, 10mL of Dnase I enzyme, cellulase, phospholipase, pectase, lipase and xylanase preparation are added into 30 bottles respectively, 10mL of sterile PBS buffer solution is added into 31 st bottle as a control, the mixture is uniformly mixed, the mixture is kept stand for 2 hours under the room temperature condition (25-28 ℃), and then filtered by a 0.22 mu m sterile filter membrane, the filtration quantity is measured, and the component effect of each filter aid is observed.
The filtration results are shown in Table 1, and in the DNase treatment of different dosages, the addition of 0.2g and 0.3g treatment groups increased the filtration dosage of the vibrio parahaemolyticus phage fermentation broth by 12.5%, so that the minimum dosage of 0.2g was selected as the optimal dosage;
in the treatment of cellulase with different dosages, the treatment groups of 0.3g and 0.4g improve the filtering dosage of the phage fermentation broth of the vibrio parahaemolyticus by 12.5%, so that the minimum dosage of 0.3g is selected as the optimal dosage;
in the treatment of phospholipase with different dosages, the filtering dosage of the phage fermentation broth of the vibrio parahaemolyticus is not improved by each treatment group, so that the phospholipase has no effect on the phage fermentation broth of the vibrio parahaemolyticus;
in the treatment of pectase with different dosages, the treatment group of 0.3g improves the filtering dosage of the phage fermentation liquor of the vibrio parahaemolyticus by 22.5%, so 0.3g is selected as the optimal dosage;
In the lipase treatment with different dosages, the treatment group of 0.4g improves the filtering dosage of the phage fermentation liquor of the vibrio parahaemolyticus by 30 percent, so 0.4g is selected as the optimal dosage;
among the different doses of xylanase treatment, the treatment group of 0.4g improves the filtering dose of the phage fermentation broth of the vibrio parahaemolyticus by 22.5%, so 0.4g is selected as the optimal dose;
in summary, DNase, cellulase, pectinase, lipase and xylanase are essential components of the bacteriophage fermentation broth filter aid, and the optimal dosages of the DNase, the cellulase, the pectinase, the lipase and the xylanase are respectively 0.2g,0.3g, 0.4g and 0.4g; phospholipase is not necessary for purification of the phage fermentation broth of Vibrio parahaemolyticus.
TABLE 1 results of the effect of different concentrations of six enzymes on the filtration amount of Vibrio parahaemolyticus phage fermentation broth
Figure BDA0002341988040000181
Figure BDA0002341988040000191
Experimental example 2 Effect of six enzymes on Staphylococcus aureus phage fermentation broth
5 concentration gradients of each of the 6 enzyme preparations prepared in example 1, namely, dnase I enzyme, cellulase, phospholipase, pectinase, lipase and xylanase, were selected for single factor experiments.
31 parts of 100mL of Staphylococcus aureus phage fermentation broth were prepared as in example 8, 10mL of Dnase I enzyme, cellulase, phospholipase, pectinase, lipase and xylanase preparation were added to 30 bottles of the fermentation broth, 10mL of sterile PBS buffer was added as a control to 31 bottles of the fermentation broth, the fermentation broth was uniformly mixed, and the fermentation broth was allowed to stand at room temperature (25 ℃ C. To 28 ℃ C.) for 2 hours, and then filtered through a 0.22 μm sterile filter membrane, the filtration amount was measured, and the effects of the components of each filter aid were observed.
The filtration results are shown in Table 2, and the addition of 0.3g, 0.4g and 0.5g of treatment groups in the DNase treatment of different doses resulted in an increase in the filtration dose of the Staphylococcus aureus phage broth of 37.14%, so that the minimum dose of 0.3g was selected as the optimal dose;
of the different doses of cellulase treatment, treatment groups of 0.3g, 0.4g and 0.5g all increased the filter dose of staphylococcus aureus phage fermentation broth by 28.57%, so the minimum dose of 0.3g was selected as the optimal dose;
of the different doses of phospholipase treatment, treatment groups of 0.4g and 0.5g each increased the filter dose of staphylococcus aureus phage fermentation broth by 17.14%, so the minimum dose of 0.4g was selected as the optimal dose;
in the treatment of pectinase with different dosages, the treatment group of 0.4g only improves the filtering dosage of the staphylococcus aureus phage fermentation broth by 2.86%, so that the pectinase has no effect on the staphylococcus aureus phage fermentation broth;
of the different doses of lipase treatment, the treatment group of 0.4g increased the filter dose of staphylococcus aureus phage broth by 28.57%, thus selecting 0.4g as the optimal dose;
of the different doses of xylanase treatment, the treatment group of 0.4g increased the filter dose of staphylococcus aureus phage broth by 22.86%, thus selecting 0.4g as the optimal dose;
In sum, DNase, cellulase, phospholipase, lipase and xylanase are essential components of the phage fermentation broth filter aid, and the optimal dosages of the DNase, the cellulase, the phospholipase, the lipase and the xylanase are respectively 0.3g,0.4g and 0.4g; pectase is not necessary for purification of the staphylococcus aureus phage broth.
TABLE 2 results of the effect of different concentrations of six enzymes on the filtration amount of Staphylococcus aureus phage fermentation broth
Figure BDA0002341988040000201
Figure BDA0002341988040000211
Experimental example 3 Effect detection of six enzymes on Solanaceae Ralstonia phage fermentation broth
5 concentration gradients of each of the 6 enzyme preparations prepared in example 1, namely, dnase I enzyme, cellulase, phospholipase, pectinase, lipase and xylanase, were selected for single factor experiments.
31 parts of a fermentation broth of a bacteriophage of Solanaceae, 100mL of which were prepared as in example 9, 10mL of a Dnase I enzyme, a cellulase, a phospholipase, a pectinase, a lipase and a xylanase preparation were added to 30 bottles of the fermentation broth, 10mL of a sterile PBS buffer was added as a control to 31 bottles of the fermentation broth, the fermentation broth was uniformly mixed, and the fermentation broth was allowed to stand at room temperature (25 ℃ C. -28 ℃ C.) for 2 hours, and then filtered with a 0.22 μm sterile filter membrane, and the filtration amount was measured to observe the effects of the components of each filter aid.
The filtration results are shown in Table 3, and in the DNase treatment with different dosages, the addition of 0.2g and 0.3g treatment groups increased the filtration dosage of the fermentation broth of the Solanaceae Ralstonia by 35.71%, so that the minimum dosage of 0.2g was selected as the optimal dosage;
In the treatment of the cellulase with different dosages, the treatment groups of 0.1g, 0.2g, 0.3g, 0.4g and 0.5g do not increase the filtration dosage of the bacteriophage fermentation broth of the Solanaceae, but rather the filtration dosage is reduced, so that the cellulase has no effect on the bacteriophage fermentation broth of the Solanaceae;
in the phospholipase treatment with different dosages, the treatment group with 0.2g improves the filtration dosage of the phage fermentation broth of the Ralstonia solanaceae by 60.71%, so that the dosage of 0.2g is selected as the optimal dosage;
in the treatment of pectinase with different dosages, the treatment group of 0.4g improves the filtering dosage of the bacteriophage fermentation broth of the Ralstonia solanaceae by 50%, so that the dosage of 0.4g is selected as the optimal dosage;
in the lipase treatment with different dosages, the treatment group of 0.3g improves the filtration dosage of the phage fermentation broth of the Ralstonia solanaceae by 42.86%, so that 0.3g is selected as the optimal dosage;
in the xylanase treatment with different dosages, the treatment group with 0.4g improves the filtration dosage of the phage fermentation broth of the Ralstonia solanaceae by 60.71%, so that 0.4g is selected as the optimal dosage;
in summary, DNase, phospholipase, pectinase, lipase and xylanase are essential components of the phage fermentation broth filter aid, and the optimal dosages of the DNase, the phospholipase, the pectinase, the lipase and the xylanase are respectively 0.2g,0.4g,0.3g and 0.4g; and the cellulase is not necessary for purifying the phage fermentation broth of Ralstonia solanaceae.
TABLE 3 results of the effect of different concentrations of six enzymes on the filtration amount of the fermentation broth of Lei's bacteriophage of Solanaceae
Treatment of DNAzymes Cellulase enzymes Phospholipase enzyme Pectase enzyme Lipase enzyme Xylanase enzyme Filtration capacity (mL) Increase ratio
1 0.1g - - - - - 31 10.71％
2 0.2g - - - - - 38 35.71％
3 0.3g - - - - - 38 35.71％
4 0.4g - - - - - 37 32.14％
5 0.5g - - - - - 36 28.57％
6 - 0.1g - - - - 27 -3.57％
7 - 0.2g - - - - 25 -10.71％
8 - 0.3g - - - - 22 -21.43％
9 - 0.4g - - - - 19 -32.14％
10 - 0.5g - - - - 20 -28.57％
11 - - 0.1g - - - 34 21.43％
12 - - 0.2g - - - 45 60.71％
13 - - 0.3g - - - 35 25％
14 - - 0.4g - - - 35 25％
15 - - 0.5g - - - 32 14.29％
16 - - - 0.1g - - 30 7.14％
17 - - - 0.2g - - 34 21.43％
18 - - - 0.3g - - 38 35.71％
19 - - - 0.4g - - 42 50％
20 - - - 0.5g - - 35 25％
21 - - - - 0.1g - 33 17.86％
22 - - - - 0.2g - 36 28.57％
23 - - - - 0.3g - 40 42.86％
24 - - - - 0.4g - 36 28.57％
25 - - - - 0.5g - 32 14.29％
26 - - - - - 0.1g 27 -3.57％
27 - - - - - 0.2g 31 10.71％
28 - - - - - 0.3g 38 35.71％
29 - - - - - 0.4g 45 60.71％
30 - - - - - 0.5g 42 50％
31 - - - - - - 28 0
Experimental example 4 Effect detection of six enzymes on coliphage fermentation broth
5 concentration gradients of each of the 6 enzyme preparations prepared in example 1, namely, dnase I enzyme, cellulase, phospholipase, pectinase, lipase and xylanase, were selected for single factor experiments.
31 portions of 100mL of coliphage fermentation broth were prepared as in example 10, 10mL of Dnase I enzyme, cellulase, phospholipase, pectinase, lipase and xylanase preparation were added to 30 bottles of the broth, 10mL of sterile PBS buffer was used as a control, the mixture was mixed uniformly, and the mixture was allowed to stand at room temperature (25 ℃ C. To 28 ℃ C.) for 2 hours, and then filtered through a 0.22 μm sterile filter membrane, the filtration amount was measured, and the effects of the components of each filter aid were observed.
As shown in Table 3, the addition of 0.2g, 0.3g and 0.4g of treatment groups in the DNase treatment of different doses resulted in an increase in the filtered E.coli phage broth dosage of 42.22%, so that the minimum dosage of 0.2g was selected as the optimal dosage;
Of the cellulase treatments of different dosages, the treatment group of 0.3g improved the filter dosage of the coliphage fermentation broth by 28.89%, so that the dosage of 0.3g was selected as the optimal dosage;
of the different doses of phospholipase treatment, the treatment group of 0.4g improved the filter dose of the coliphage broth by 28.89%, so the dose of 0.4g was selected as the optimal dose;
in the treatment of pectinase with different dosages, the treatment group of 0.3g improves the filtering dosage of the coliphage fermentation broth by 31.11%, so that the dosage of 0.3g is selected as the optimal dosage;
among the different doses of lipase treatment, the treatment group of 0.4g improves the filter dose of the coliphage fermentation broth by 22.22%, so 0.4g is selected as the optimal dose;
of the different doses of xylanase treatment, the treatment group of 0.3g improves the filter dose of the coliphage fermentation broth by 31.11%, so 0.3g is selected as the optimal dose;
in summary, DNase, cellulase, phospholipase, pectinase, lipase and xylanase are essential components of the phage fermentation broth filter aid, and the optimal dosages are 0.2g,0.3g,0.4g and 0.3g respectively.
TABLE 4 results of the effect of different concentrations of six enzymes on the filtration amount of E.coli phage fermentation broth
Figure BDA0002341988040000231
Figure BDA0002341988040000241
Experimental example 5 test of phage fermentation broth post-treatment filter aid on phage fermentation broth treatment time
Vibrio parahaemolyticus, staphylococcus aureus, solanaceae Ralstonia and Escherichia coli phage filter aid were formulated as described in example 3, example 4, example 5 and example 6, respectively.
5 parts of each of 100mL phage fermentation broths prepared in example 7, example 8, example 9 and example 10 was taken, and 10mL of the corresponding filter aid was added to each 100mL, and the mixture was allowed to stand at room temperature (25℃to 28 ℃) for 2 hours, 4 hours, 6 hours, 8 hours and 10 hours, and then filtered through a 0.22 μm sterile filter membrane to determine the filtration amount.
As shown in Table 5, 100mL of the Vibrio parahaemolyticus phage fermentation broth after adding 10mL of the filter aid, after 6 hours of treatment, can be completely passed through a 0.22 μm filter membrane, thereby indicating that 6 hours is the optimal treatment time;
100mL of staphylococcus aureus phage fermentation broth can be completely filtered through a 0.22 mu m filter membrane after being treated for 8 hours after 10mL of filter aid is added, thereby indicating that 8 hours is the optimal treatment time;
100mL of the fermentation broth of the Lei-Barbamate of Solanaceae can completely pass through a 0.22 mu m filter membrane after being treated for 8 hours after adding 10mL of filter aid, thereby indicating that 8 hours is the optimal treatment time;
After adding 10mL of filter aid to 100mL of coliphage fermentation broth, the whole broth can pass through a 0.22 μm filter after 4h treatment, thus indicating that 4h is the optimal treatment time.
TABLE 5 post-treatment of phage fermentation broth filtration adjuvant treatment for different times of filtration
Figure BDA0002341988040000251
Experimental example 6 test of phage fermentation broth post-treatment filter aid on phage fermentation broth treatment time
Phage filter aids were formulated as described in example 2.
The 100mL phage fermentation broths prepared in example 7, example 8, example 9, example 10, example 11 and example 12 were each taken in 7 parts, 100mL portions were each added with 10mL phage filtration aid, and the mixture was allowed to stand at room temperature (25℃to 28 ℃) for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours and 14 hours, and then filtered with a 0.22 μm sterile filter membrane, and the filtration amount was measured.
As shown in Table 6, 100mL of the Vibrio parahaemolyticus phage fermentation broth after being treated for 12 hours with 10mL of the filter aid can be completely passed through the 0.22 μm filter membrane, thereby indicating that 12 hours is the optimal treatment time;
100mL of staphylococcus aureus phage fermentation broth can be completely filtered through a 0.22 mu m filter membrane after 10mL of filter aid is added and treated for 10 hours, thereby indicating that 10 hours is the optimal treatment time;
100mL of the fermentation broth of the Lei-Barbamate of Solanaceae can completely pass through a 0.22 mu m filter membrane after being treated for 12 hours after adding 10mL of filter aid, thereby indicating that 12 hours is the optimal treatment time;
after 10mL of filter aid is added into 100mL of coliphage fermentation broth, the coliphage fermentation broth can be completely filtered through a 0.22 mu m filter membrane after being treated for 10 hours, thereby indicating that 10 hours is the optimal treatment time;
100mL of the pullorum disease salmonella phage fermentation broth can be completely filtered through a 0.22 mu m filter membrane after 10mL of filter aid is added and treated for 10 hours, so that the 10 hours are the optimal treatment time;
100mL of the Xanthomonas carpet grass phage broth after adding 10mL of filter aid, after 8h of treatment, can be completely passed through a 0.22 μm filter, thus indicating 8h as the optimal treatment time.
TABLE 6 post-treatment of phage fermentation broth filtration adjuvant treatment for different times of filtration
Figure BDA0002341988040000261
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Claims (33)

1. A vibrio parahaemolyticus phage fermentation broth post-treatment filter aid, characterized in that the filter aid consists of: calculated in enzyme activity units:
dnase: 3000000-6000000U/100mL;
lipase: 4000-5000U/100mL;
xylanase: 36000-72000U/100mL;
cellulase: 1500-2000U/100mL;
pectase: 7500-10000UU/100mL;
PBS buffer solution; and
and (3) water.
2. The filter aid according to claim 1, wherein the filter aid comprises: the dosage of the DNase is 3000000U/100mL in terms of enzyme activity unit.
3. The filter aid according to claim 1, wherein the filter aid comprises: the dosage of the lipase is 4000U/100mL in terms of enzyme activity units.
4. The filter aid according to claim 1, wherein the filter aid comprises: the xylanase was dosed 72000U/100mL in enzyme activity units.
5. The filter aid according to claim 1, wherein the filter aid comprises: the dosage of the cellulase is 1500U/100mL in terms of enzyme activity units.
6. The filter aid according to claim 1, wherein the filter aid comprises: the dosage of the pectase is 7500U/100mL in terms of enzyme activity units.
7. The filter aid according to claim 1, wherein the filter aid consists of the following doses of components: in terms of the unit of enzyme activity,
dnase: 3000000U/100mL;
cellulase: 1500U/100mL;
pectase: 7500U/100mL;
Lipase: 4000U/100mL;
xylanase: 72000U/100mL;
10 x PBS buffer, ph=7.2: 10mL/100mL; and
and a proper amount of water.
8. A staphylococcus aureus phage fermentation broth post-treatment filter aid, characterized in that the filter aid consists of the following components: in terms of the unit of enzyme activity,
dnase: 3000000-7500000U/100mL;
lipase: 2000-5000U/100mL;
xylanase: 54000-90000U/100mL;
cellulase: 1000-2500U/100mL;
phospholipase: 40000-50000U/100mL;
PBS buffer solution; and
and (3) water.
9. The filter aid according to claim 8, wherein the filter aid comprises: the dosage of the DNase is 4500000/100mL in terms of enzyme activity units.
10. The filter aid according to claim 8, wherein the filter aid comprises: the dosage of the lipase is 4000U/100mL in terms of enzyme activity units.
11. The filter aid according to claim 8, wherein the filter aid comprises: the xylanase was dosed 72000U/100mL in enzyme activity units.
12. The filter aid according to claim 8, wherein the filter aid comprises: the dosage of the cellulase is 1500U/100mL in terms of enzyme activity units.
13. The filter aid according to claim 8, wherein the filter aid comprises: the dosage of the phospholipase is 40000U/100mL in terms of enzyme activity units.
14. The filter aid according to claim 8, wherein the filter aid consists of the following doses of components: in terms of the unit of enzyme activity,
dnase: 4500000U/100mL;
cellulase: 1500U/100mL;
phospholipase: 40000U/100mL;
lipase: 4000U/100mL;
xylanase: 72000U/100mL;
10 x PBS buffer, ph=7.2: 10mL/100mL; and
and a proper amount of water.
15. A filter aid for post-treatment of a bacteriophage fermentation broth of ralstonia solanacearum, characterized in that the filter aid consists of the following components: in terms of the unit of enzyme activity,
dnase: 3000000-7500000U/100mL;
lipase: 2000-4000U/100mL;
xylanase: 54000-90000U/100mL;
phospholipase: 10000-40000U/100mL;
pectase: 5000-12500U/100mL;
PBS buffer solution; and
and (3) water.
16. The filter aid according to claim 15, wherein the filter aid comprises: the dosage of the DNase is 3000000U/100mL in terms of enzyme activity unit.
17. The filter aid according to claim 15, wherein the filter aid comprises: the dosage of the lipase is 3000U/100mL in terms of enzyme activity units.
18. The filter aid according to claim 15, wherein the filter aid comprises: the xylanase was dosed 72000U/100mL in enzyme activity units.
19. The filter aid according to claim 15, wherein the filter aid comprises: the dosage of the phospholipase is 20000U/100mL in terms of enzyme activity units.
20. The filter aid according to claim 15, wherein the filter aid comprises: the dosage of the pectase is 10000U/100mL calculated by enzyme activity unit.
21. The filter aid according to claim 15, wherein the filter aid consists of the following doses of components: in terms of the unit of enzyme activity,
dnase: 3000000U/100mL;
phospholipase: 20000U/100mL;
pectase: 10000U/100mL;
lipase: 3000U/100mL;
xylanase: 72000U/100mL;
10 x PBS buffer, ph=7.2: 10mL/100mL; and
and a proper amount of water.
22. An escherichia coli bacteriophage fermentation broth post-treatment filter aid, wherein the filter aid comprises the following components: in terms of the unit of enzyme activity,
dnase: 3000000-7500000U/100mL;
lipase: 3000-5000U/100mL;
Xylanase: 54000-90000U/100mL;
cellulase: 1500-2000U/100mL;
phospholipase: 20000-50000U/100mL;
pectase: 5000-10000U/100mL;
PBS buffer solution; and
and (3) water.
23. The filter aid according to claim 22, wherein the filter aid comprises: the dosage of the DNase is 3000000/100mL in terms of enzyme activity unit.
24. The filter aid according to claim 22, wherein the filter aid comprises: the dosage of the lipase is 4000U/100mL in terms of enzyme activity units.
25. The filter aid according to claim 22, wherein the filter aid comprises: the xylanase was dosed at 54000U/100mL in enzyme activity units.
26. The filter aid according to claim 22, wherein the filter aid comprises: the dosage of the cellulase is 1500U/100mL in terms of enzyme activity units.
27. The filter aid according to claim 22, wherein the filter aid comprises: the dosage of the phospholipase is 40000U/100mL in terms of enzyme activity units.
28. The filter aid according to claim 22, wherein the filter aid comprises: the dosage of the pectase is 7500U/100mL in terms of enzyme activity units.
29. The filter aid according to claim 22, wherein the filter aid consists of: in terms of the unit of enzyme activity,
dnase: 3000000U/100mL;
cellulase: 1500U/100mL;
phospholipase: 40000U/100mL;
pectase: 7500U/100mL;
lipase: 4000U/100mL;
xylanase: 54000U/100mL;
10 x PBS buffer, ph=7.2: 10mL/100mL; and
and a proper amount of water.
30. The filter aid according to any one of claims 1, 8, 15, 22, wherein the filter aid has a pH of 7.2; the solution for adjusting the pH value of the filter aid is PBS buffer solution.
31. A method of preparing a filter aid according to any of claims 1-30, comprising:
(1) Accurately weighing 10 x PBS buffer solution with ph=7.2 with a measuring cylinder, and placing in a volumetric flask;
(2) Accurately weighing the enzyme used in the filter aid, and using a proper amount of sterile ddH 2 Adding O into the volumetric flask described in (1) after dissolving, and fixing the volume with sterile water;
(3) Passing the obtained solution through a 0.22 μm aseptic filter for aseptic treatment;
(4) The obtained solution is packaged in a sterile centrifuge tube and stored in a refrigerator at 4 ℃ for standby.
32. A method of using the filter aid according to any of claims 1-30, wherein the method comprises:
(1) Mixing the filter aid with the final volume ratio of 10% in phage fermentation broth, uniformly stirring, and carrying out interaction for 2-10 hours at room temperature;
(2) The solution was filtered using a 0.22 μm sterile filter to obtain a phage solution from which the host bacteria and most of the secondary metabolites were removed.
33. The application method according to claim 32, characterized in that the application method comprises:
the treatment time of the filtering auxiliary agent for the post-treatment of the vibrio parahaemolyticus phage fermentation liquor is 6 hours; or (b)
The treatment time of the filter aid for the post-treatment of the staphylococcus aureus phage fermentation broth is 8 hours; or (b)
The treatment time of the filter aid for the post-treatment of the bacteriophage fermentation broth of the Ralstonia solanaceae is 8 hours; or (b)
The treatment time of the filter aid for the post-treatment of the coliphage fermentation broth is 4 hours.
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