CN113528460A - Phage composite powder and preparation method and application thereof - Google Patents
Phage composite powder and preparation method and application thereof Download PDFInfo
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- CN113528460A CN113528460A CN202110795145.5A CN202110795145A CN113528460A CN 113528460 A CN113528460 A CN 113528460A CN 202110795145 A CN202110795145 A CN 202110795145A CN 113528460 A CN113528460 A CN 113528460A
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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Abstract
The invention provides a phage composite powder, comprising 1-4 parts of phage fermentation liquid, 0.02-0.1 part of block polyether, 0.002-0.05 part of hydroxyapatite and 4-16 parts of carrier by weight, wherein the content of phage in the composite powder is more than 3.1 multiplied by 109PFU/g; the application also provides the application of the phage composite powder in preparing feed additives and medicaments for preventing and treating livestock diarrhea; the phage composite powder can obviously improve the weight of broiler chickens, can also prevent bacterial infection of pigs and the like, and has good practical application and market prospect.
Description
Technical Field
The invention belongs to the field of biological agents, and particularly relates to a preparation method and application of phage composite powder.
Background
Bacterial resistance is severe due to early drug abuse, which severely affects food safety and public health. Under the situation of 'resistance reduction and resistance limitation' at the culture end, the bacteriophage is a novel potential 'antibacterial agent' which attracts attention because of the effective bacterium lysis. Phage is expected to replace antibiotics to treat bacterial infection, and can be used for clearing specific harmful flora in animal intestinal tracts and maintaining animal intestinal tract health.
At present, the phage technology is mainly applied in the fields of animal husbandry, environmental protection, agriculture, disease treatment and the like. The composition is mainly used for preventing and treating escherichia coli and salmonella infection in the field of animal husbandry, such as treatment of perihepatitis, pericarditis, air sacculitis, salpingitis, enteritis and the like caused by piglet yellow-white dysentery, chick white dysentery, swine typhoid fever, paratyphoid fever and adult chicken escherichia coli infection.
The existing phage liquid preparation can be reduced by 2-3 orders of magnitude in one month at normal temperature, and is easily polluted by other mixed bacteria in the process of preserving the phage liquid preparation. The powder is beneficial to the preservation of the phage and the prevention of the pollution of infectious microbes, but the survival rate of the phage in the shelf life is difficult to guarantee by the existing powder preparation technology, and a lot of phage die particularly in the environments of dryness, high temperature, acid and alkali and the like, so the preparation technology needs to be optimized. Common microbial preparations such as probiotics and the like are usually prepared into powder by adopting a freeze drying method, although the activity of microorganisms can be kept, the freeze drying cost is high, the energy consumption is high, and the popularization and the application of the microbial preparations on powder of feed and the like are difficult. Joan et al can store 3 months at 4 ℃ after encapsulating salmonella phage in liposome, and can also add drinking water and animal feed (Appl Environ Microbiol,2015,81(14): 4841-4849), but this method gives the preparation steps tedious, difficult, also does not benefit to the industrialized production.
Disclosure of Invention
Aiming at the problems of complicated preparation steps, difficult preservation and low survival rate of the conventional phage preparation, the application provides the phage solid composite powder prepared by the low-cost low-temperature air blast, vacuum drying or low-temperature vacuum spray drying method, so as to improve the survival rate of the phage solid in the preparation and prolong the shelf life of the preparation.
Specifically, the method is realized by the following technical scheme:
the application firstly provides a phage composite powder, which comprises the following components: 1-4 parts of phage fermentation liquid, 0.02-0.1 part of block polyether, 0.002-0.05 part of hydroxyapatite and 4-16 parts of carrier; the phage content in the powder>3.1×109PFU/g。
Further, in the phage composite powder provided by the application, the carrier may be one or more of common carriers such as rice bran, corncob powder, soybean meal, corn flour, straw powder, fish meal, meat and bone powder, skimmed milk powder, maltodextrin, corn starch, whey powder and the like.
Further, in the phage composite powder provided by the present application, the phage fermentation liquid is prepared as follows: adding bacteriophage and corresponding host bacteria into LB liquidCulturing the culture medium at 37 ℃ for 6-18 hours, centrifuging (10000 rpm, 10 minutes), performing filtration sterilization (0.22 mu M polyethersulfone), and removing the culture medium by ultrafiltration (100KD ultrafiltration membrane), wherein the titer of phage in the fermentation broth is not less than 1011CFU/mL；
LB liquid medium formulation (1L): 5g of yeast extract, 10g of tryptone and 10g of sodium chloride, and the balance is distilled water.
Further, in the phage composite powder provided by the application, the block polyether comprises at least one of polyether F-68 (poloxamer 188) or block polyether F-127 (poloxamer 407).
Further, in the phage composite powder provided by the present application, hydroxyapatite, preferably nano-hydroxyapatite or micro-hydroxyapatite, is used.
The term "bacteriophage and its corresponding host bacterium" as used herein refers to a bacteriophage and its host as conventional in the art, such as bacteriophage PA13076 of host bacterium Salmonella CMCC13076, bacteriophage JS-15 of host bacterium Escherichia coli K88, bacteriophage JS116 of host bacterium Salmonella pullorum s96116, and the like.
Secondly, the application also provides a preparation method of the phage composite powder, which comprises the following specific steps:
1) the titer is more than or equal to 1011Adding block polyether into CFU/mL phage fermentation liquid, coating for 1-6 hours at 37 ℃ and 180rpm to obtain coated phage; the coating of phage by block polyether is facilitated at 37 ℃;
2) then adding hydroxyapatite into the coated phage, stirring uniformly, oscillating at 37 ℃ and 180rpm for 1-2 hours, then adding a carrier, and stirring uniformly to obtain a powder precursor for later use;
or uniformly mixing the hydroxyapatite with the carrier aqueous solution to obtain a mixed solution; dissolving the coated phage prepared in the step 1) into the mixed solution to obtain a powder solution for later use;
3) drying the powder precursor or the powder solution obtained in the step 2) to constant weight to obtain the phage composite powder.
Further, the above step 3) "drying" means: drying the powder precursor to constant weight by air drying or vacuum drying at 10-70 deg.C; or, the powder solution is dried to constant weight in a spray drying mode; the spray drying parameters are preferably: the needle-through time interval is 96s at 120 ℃, and the peristaltic speed is 15 rpm.
Thirdly, the application provides the application of the phage composite powder in serving as a feed additive.
Fourthly, the application also provides application of the phage composite powder in preparation of livestock diarrhea prevention and treatment medicines or raw materials thereof.
Compared with the conventional phage preparation, the phage composite powder provided by the invention has high phage content (phage content)>3.1×109PFU/g) and improves the gastric acid resistance of the bacteriophage and the stability of long-term storage at normal temperature. The block polyether can coat the phage to form a protective film on the surface of the phage, so that the stability of the phage in the processes of drying, storing and using is enhanced. The hydroxyapatite can release hydroxide under the acidic (such as gastric acid) condition, neutralize hydrogen ions around the phage and avoid the inactivation of the phage; the stability of the phage can be improved and the powdering rate can be improved by loading the coated phage on the carrier.
Drawings
FIG. 1 photograph of Salmonella enteritidis phage complex rice bran.
FIG. 2 photograph of Salmonella enteritidis bacteriophage complexed corn starch.
FIG. 3 photograph of Salmonella enteritidis phage complex maltodextrin.
FIG. 4 photograph of coliphage complex corn flour.
FIG. 5 is a schematic diagram showing the protective effect of different coating agents on phage.
FIG. 6 is a schematic diagram showing the protective effect of different addition amounts of block polyether PF-68 on bacteriophage.
FIG. 7 is a schematic diagram of the detection result of improving the gastric acid resistance of the phage composite powder by nano-hydroxyapatite.
FIG. 8 is a schematic diagram of the detection of the normal temperature storage stability of the phage composite powder.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples reagent sources:
segmented polyether F-68 and segmented polyether F-127 were purchased from Biotechnology engineering (Shanghai) Ltd; nano-hydroxyapatite was purchased from shanghai bailing waffle chemical technologies ltd;
the phages used in the following examples are all phages common in the art and deposited in the applicant's laboratory. Salmonella phage PA13076, Salmonella pullorum phage JS116 see the literature "Pao hong duo et al, isolation and identification of Salmonella lytic phage and their biological properties food science, 2015,36(05), 131";
enterobacter phage JS-15 is described in "royal et al, identification of isolation and biological properties of escherichia coli K88 phage, north china agro-paper, 2012,27(4), 163";
shigella phages vB _ SflM _004, vB _ SdyM _006 and vB _ SssoS _008 are described in "Wanan et al, biology of New Lytic Bacteriophages Infecting Shigella spp. in freeshwater Environment, Front. Microbiol.,2021,12,619323.
Example 1: preparation of salmonella bacteriophage PA13076 composite rice bran
The preparation method of the phage composite powder in the embodiment comprises the following steps:
the phage titer is 2 multiplied by 10 to 1 part of the fermentation liquor of the phylogenetic phage PA13076 by weight part10(the preparation method is detailed in wrapping red flower, etc., the separation and identification of salmonella lytic phage and the biological characteristics thereof. food science 2015,36(05),131.), 0.04 part of block polyether F-68 is added, and the mixture is wrapped for 6 hours at 37 ℃ and 180 rpm; then theAdding 0.02 part of nano hydroxyapatite into the coated phage, oscillating at 37 ℃ and 180rpm for 1 hour; then 6 parts of rice bran are added, evenly stirred and finally dried by air blowing at 40 ℃ to constant weight, thus obtaining the phage composite rice bran (figure 1).
4g of phage complex rice bran obtained in this example was dissolved in 30mL of PBS buffer solution, shaken at 37 ℃ and 180rpm for 2 hours, filtered and sterilized, and the titer of phage in rice bran was measured to obtain a phage titer of 3.1X 109PFU/g。
In the specific implementation, the drying is carried out to constant weight in a blowing or vacuum mode within the temperature range of 10-70 ℃.
Example 2: preparation of salmonella bacteriophage PA13076 composite corn starch
The preparation method of the phage composite powder in the embodiment comprises the following steps:
adding 0.03 part of block polyether F-127 to 3 parts of Salmonella phage PA13076 fermentation broth (same as in example 1), and coating at 37 ℃ and 180rpm for 2 hours; then dissolving the coated phage into an aqueous solution of 0.3 part of corn starch and 0.03 part of hydroxyapatite, and uniformly stirring; spray-drying to constant weight under conditions of atomization temperature of 120 deg.C, needle-through time interval of 96s, and peristaltic speed of 15rpm to obtain bacteriophage composite corn starch (figure 2).
1g of bacteriophage corn starch obtained in the example is respectively dissolved in 10mL of PBS buffer solution, the temperature is 37 ℃, the rpm is 180, the oscillation is carried out for 2 hours, the filtration sterilization is carried out, the bacteriophage titer in the corn starch is measured, and the bacteriophage titer is measured to be 7.0 multiplied by 1010PFU/g。
Example 3: preparation of salmonella phage PA13076 composite maltodextrin
The preparation method of the phage composite powder in the embodiment comprises the following steps:
adding 0.06 part of block polyether F-68 into 3 parts of salmonella phage PA13076 fermentation liquor, and coating for 3 hours at 37 ℃ and 180 rpm; then dissolving the coated phage into 0.9 part of maltodextrin and 0.01 part of hydroxyapatite aqueous solution, and uniformly stirring; and finally, spray drying at the atomization temperature of 115 ℃, the needle-through time interval of 96s and the peristaltic speed of 15rpm to obtain the phage composite maltodextrin (figure 3).
1g of phage complex maltodextrin obtained in this example was dissolved in 10mL of PBS buffer solution at 37 ℃ and 180rpm for 2 hours with shaking, and the phage titer in maltodextrin was measured by filtration sterilization to be 9.0X 1010PFU/g。
Example 4: preparation of Escherichia coli phage JS-15 composite corn powder
The preparation method of the phage composite powder comprises the following steps:
adding 2 parts of Escherichia coli phage JS-15 fermentation liquor with titer of 4.2 × 1010PFU/mL (details of a fermentation liquid preparation method are shown in Wanan et al, separation and identification of Escherichia coli K88 bacteriophage and biological characteristics thereof, North China agricultural science, 2012,27(4),163.) 0.1 part of block polyether F-127 is added, coating is carried out for 3 hours at 37 ℃ and 180rpm, then 0.02 part of nano-hydroxyapatite is added to the coated bacteriophage, corn flour is added after oscillation is carried out for 2 hours at 37 ℃ and 180rpm, and blast drying is carried out at 40 ℃ until constant weight is achieved, so that the bacteriophage composite corn flour (shown in figure 4) is obtained.
Dissolving 1g of composite phage corn flour obtained in the embodiment in 10mL of PBS buffer solution, respectively, oscillating for 2 hours at 37 ℃ and 180rpm, filtering and sterilizing, measuring the titer of phage in corn flour, and measuring the titer of phage to be 8.1 × 109PFU/g。
Example 5: detection of protective effect of different coating agents on phage
To 1 part of Salmonella phage PA13076 fermentation broth (phage titer 2X 10)10) PBS, 5 parts of glycerol, 0.05 part of maltooligosaccharide, 0.15 part of sorbitol and 0.1 part of segmented polyether F-68 were added to the above aqueous solutions, respectively, and the mixture was coated at 37 ℃ and 180rpm for 2 hours. Then, 0.02 part of nano-hydroxyapatite is respectively added into the coated phage, rice bran is added after oscillation is carried out for 1 hour at 37 ℃ and 180rpm, the mixture is uniformly stirred, and vacuum drying is carried out at 37 ℃ until the weight is constant, thus obtaining the phage composite powder.
4g of phage rice bran is dissolved in 30mL of PBS buffer solution, the mixture is shaken at 37 ℃ and 180rpm for 2 hours, and the titer of phage in the rice bran is measured after filtration sterilization. The results showed that F-68 had the best protective effect on the phage, and was 2.2 times and 1.9 times that of the blank control (PBS group), respectively, in the case of glycerol. Isomaltooligosaccharides and sorbitol had lower titers than the PBS group, about 1/2 and 1/5, respectively (figure 5).
Example 6: protective effect of addition amount of block polyether F-68 on bacteriophage
0 part, 0.02 part, 0.04 part, 0.06 part, 0.08 part and 0.1 part of block polyether F-68 are respectively added into 6 parts of salmonella phage PA13076 fermentation liquor, and the mixture is coated for 2 hours at 37 ℃ and 180 revolutions.
Then, the coated phage is added into a mixture of 0.005 part of hydroxyapatite and 4 parts of rice bran, and the mixture is stirred uniformly. Vacuum drying at 40 deg.C to constant weight.
4g of phage rice bran is respectively dissolved in 30mL of PBS buffer solution, the mixture is shaken at 37 ℃ and 180rpm for 2 hours, and the phage titer in the rice bran is measured after filtration sterilization. The results show that the higher the phage titer with increasing concentration of the block polyether, indicating that the block polyether can increase the stability of the phage (FIG. 6).
Example 7: detection of gastric acid resistance of phage composite powder by nano-hydroxyapatite
Adding 3 parts of salmonella phage PA13076 fermentation liquor into block polyether F-68, coating for 2 hours at 37 ℃ and 180rpm, dividing into 5 equal parts, and respectively adding 0.001 part, 0.002 part, 0.005 part, 0.010 part, 0.020 part and 0.050 part of nano hydroxyapatite. Meanwhile, the control group is not added with nano hydroxyapatite.
Then, 5 parts of rice bran is added into the coated phage, and the mixture is stirred uniformly. Vacuum drying at 40 deg.C to constant weight to obtain phage composite powder.
4g of phage rice bran were dissolved in 30mL of artificial simulated gastric juice (1.0 g of sodium chloride and 1.6g of pepsin were dissolved to 500mL by adding 3.5mL of hydrochloric acid and water, and the pH of the solution was 1.2). The titer was determined by shaking at 37 ℃ and 180rpm for 2 hours, filter sterilization (FIG. 7). The result shows that the phage titer is higher with the increase of the content of the hydroxyapatite, when the content is more than 0.01 part, the phage titer is highest, and the increase of the amount of the hydroxyapatite is almost unchanged, which indicates that the hydroxyapatite can improve the gastric acid resistance of the phage.
Example 8: normal temperature storage stability of phage composite powder
The salmonella phage PA13076 composite powder (example 1) and the liquid phage preparation (the fermentation liquid of the phage in example 1) are placed at 25 ℃ for 7 days, 15 days, 30 days, 60 days and 90 days, the content of the phage in each gram of the phage composite powder and the liquid phage preparation is respectively determined, the content of the phage is only reduced by 0.5 order of magnitude in 1 month after the phage composite powder is stored, and the content of the phage is only reduced by about 1 order of magnitude in three months after the phage composite powder is stored. The phage content of the liquid phage preparation was reduced by only about 2 orders of magnitude for 1 month of storage, and by only about 4 orders of magnitude for three months of storage (fig. 8). The phage composite powder has better stability at normal temperature than the phage liquid preparation.
Example 9: preparation of phage cocktail composite powder
Preparing liquid phage cocktail: shigella phages vB _ SflM _004, vB _ SdyM _006 and vB _ SssoS _008 (Royal et al, biological of New Lytic Bacteriophages Infecting Shigella spp. in Freshwater Environment, Front. Microbiol.,2021,12,619323.), Salmonella phage PA13076 and pullorum Salmonella phage JS116 (Pakistan et al, phage cocktail JS-SP1 research on the cracking kinetics and sterilization effect of mixed infection of salmonella epidemic strains, Chinese antibiotics, 2017,42(10),858.) were mixed in equal volumes to obtain phage mixture.
The preparation method of the phage composite powder of the embodiment is that 1 part of phage mixture (phage titer is 2 multiplied by 10)11) 0.08 part of block polyether F-68 was added thereto, and the mixture was coated at 37 ℃ and 180rpm for 2 hours. And then dissolving the coated phage into an aqueous solution of 0.1 part of corn starch and 0.02 part of hydroxyapatite, and uniformly stirring. And (3) carrying out low-temperature spray drying at the atomizing temperature of 110 ℃, the needle feeding time interval of 96s and the peristaltic speed of 15rpm to obtain the phage composite powder.
1g of bacteriophage corn starch obtained in the present example was dissolved in 10mL of PBS buffer solution at 37 ℃ and 180rpm, shaken for 2 hours, filtered and sterilized, and the titer of the bacteriophage in the corn starch was measured to determine phage phagocytosisThe titer of the thallus is 7.0 multiplied by 1010PFU/g。
Example 10: influence of phage cocktail composite powder on growth performance of broiler chickens
A blank control group, a liquid phage group and a phage composite powder group (5000 in each group) are arranged, the preparation method of the liquid phage and the phage composite powder is detailed in example 9, three groups are fed with phage additives and are simultaneously fed with conventional feed, 37-day-old young foot-blackcock is weighed (test day 1), broilers are weighed on test days 7, 16, 22 and 26 (marketing), and the detection results are shown in table 1.
TABLE 1 bacteriophage composite powder for raising growth performance of meat chicken
As is clear from table 1, the average body weight of the three groups did not change significantly on days 7 and 16. The average body weight of the phage composite powder group was heavier than the liquid phage group on days 22 and 26, and the body weight of the liquid phage group was heavier than that of the control group. Compared with the control group, the weight of the phage composite powder is increased by 6.53% at 22 days, and the weight is increased by 9.7% at 26 days. The embodiment proves that the phage composite powder can be used as a feed additive in the field of animal breeding to promote the growth of animals.
Example 11: escherichia coli phage JS-15 composite powder for preventing diarrhea caused by pig Escherichia coli
15 weaned piglets of 25 days old are treated with 2mL of 1X 10 feed5Oral challenge experiments are carried out, and when all piglets are in diarrhea state, the piglets are divided into 3 groups of blank control groups, bacteriophage liquid preparation treatment groups and bacteriophage composite powder treatment groups (5 groups in each group).
The piglets of the blank control group were fed with normal feed, and the treatment group was orally fed with feed supplemented with 0.01 part of phage JS-15 liquid preparation (royal et al, isolation and identification of Escherichia coli K88 phage and biological properties thereof, North China agro-Proc, 2012,27(4),163.) and 0.01 part of phage JS-15 composite powder (example 4), respectively.
The status of the piglets was recorded every 5 hours of observation and the amount of E.coli K88 in the faeces was determined. The results are shown in Table 2:
TABLE 2 piglet status
The blank control group had severe diarrhea, watery stool, poor spirit and dull sensation in piglets, and the content of K88 in feces was 4.1X 105CFU/g; phage liquid preparation for treating few diarrhea, normal feces and 1.5X 10 content of K88 in feces3CFU/g; the phage composite powder treatment group does not find diarrhea phenomenon, the feces are normal, the mental state is good, and the content of K88 in the feces is 120 CFU/g.
This example demonstrates that the phage composite powder can be used to prepare veterinary drugs or raw materials thereof to prevent diarrhea in animals caused by E.coli.
Although embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. The phage composite powder is characterized by comprising the following components: 1-4 parts of phage fermentation liquid, 0.02-0.1 part of block polyether, 0.002-0.05 part of hydroxyapatite and 4-16 parts of carrier; the content of phage in the composite powder is more than 3.1 multiplied by 109 PFU/g。
2. The phage composite powder of claim 1, wherein the carrier comprises at least one of rice bran, wheat bran, corn cob meal, soybean meal, corn flour, straw powder, fish meal, meat and bone meal, skim milk powder, maltodextrin, corn starch, whey powder.
3. The phage complexing powder of claim 1, wherein said phage fermentation broth is obtained by: adding bacteriophage and corresponding host bacteria into LB liquid culture medium at the same time, 37oC culturing for 6-18 hours, centrifuging, filtering for sterilization, and removing the culture medium by ultrafiltration to obtain the phage in the fermentation liquor with titer not less than 1011 CFU/mL。
4. The phage composite powder of claim 1, wherein said block polyether is at least one of polyether F-68 or polyether F-127.
5. A method for preparing the phage display composite powder of any one of claims 1 to 4, comprising the following steps:
the titer is more than or equal to 1011 Adding block polyether into the CFU/mL phage fermentation liquid, and coating for 1-6 hours to obtain the coated phage;
adding hydroxyapatite into the coated phage, carrying out oscillation reaction for 1-2 hours, and then adding a carrier to obtain a powder precursor for later use;
or uniformly mixing the hydroxyapatite with the carrier aqueous solution to obtain a mixed solution; dissolving the coated phage prepared in the step 1) into the mixed solution to obtain a powder solution for later use;
drying the powder precursor or the powder solution obtained in the step 2) to constant weight to obtain the phage composite powder.
6. The method for preparing a phage display composite powder according to claim 5, wherein the drying in step 3) is: drying the powder precursor to constant weight by air drying or vacuum drying at 10-70 deg.C; alternatively, the powder solution is dried to constant weight by spray drying.
7. Use of a phage display composite powder according to any of claims 1 to 4 as a feed additive.
8. Use of a bacteriophage complex powder according to any one of claims 1 to 4 for the preparation of a veterinary medicament or a raw material thereof.
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| RU2232808C1 (en) * | 2002-10-11 | 2004-07-20 | Закрытое акционерное общество "ПИ - ПРО - ЛАЙФ" | Biopreparation based upon bacteriophages for preventing and treating salmonellosis in animals |
| US20090093041A1 (en) * | 2007-10-01 | 2009-04-09 | Walbeck Alan K | Methods for drying bacteriophage and bacteriophage containing compositions, the resulting dry compositions, and methods of use |
| US20110293671A1 (en) * | 2008-11-28 | 2011-12-01 | Finlay Warren H | Bacteriophage compositions for treatment of bacterial infections |
| CN110982792A (en) * | 2020-01-02 | 2020-04-10 | 瑞科盟(青岛)生物工程有限公司 | Method for storing freeze-dried powder capsules of bacteriophage |
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