阅读说明：本技术 噬菌体裂解酶复合粉剂及其制备方法和应用 (Phage lyase composite powder and preparation method and application thereof ) 是由 林连兵 张关令 熊燕 嵇歆彧 蔡赛波 邓先余 王峰 张棋麟 郭军 于 2019-09-19 设计创作，主要内容包括：本发明涉及一种噬菌体裂解酶复合粉剂,其组成物及质量百分比为噬菌体裂解酶TSPpgh 0.15-0.2%、噬菌体裂解酶MMPpgh 0.15-0.2%、海藻糖0.3-0.5%、柠檬酸0.1-0.2%、甘露醇0.2-0.3%、维生素C 0.2-0.3%、硅藻土98.3-98.9%；该噬菌体裂解酶复合粉剂采用双层平板法进行体外抑菌活性检测,在常温下储存12个月且酶活保持在95%以上(酶活损失在5%以内)；该复合裂解酶制剂可用于替代抗生素抑制动物肠道内大肠杆菌、沙门氏菌、金黄色葡萄球菌等有害病原菌的增殖,维持肠道微生态平衡。(The invention relates to a phage lyase composite powder, which comprises 0.15-0.2% of phage lyase TSPpgh, 0.15-0.2% of phage lyase MMPpgh, 0.3-0.5% of trehalose, 0.1-0.2% of citric acid, 0.2-0.3% of mannitol, 0.2-0.3% of vitamin C and 98.3-98.9% of diatomite by mass percent; the phage lyase composite powder is used for in-vitro antibacterial activity detection by adopting a double-layer plate method, and is stored for 12 months at normal temperature, and the enzyme activity is kept above 95% (the enzyme activity loss is within 5%); the compound lyase preparation can be used for replacing antibiotics to inhibit proliferation of harmful pathogenic bacteria such as escherichia coli, salmonella, staphylococcus aureus and the like in animal intestinal tracts and maintain intestinal tract microecological balance.)

1. A phage lyase composite powder is characterized in that: the composition and the mass percentage are 0.15 to 0.2 percent of bacteriophage lytic enzyme TSPpgh, 0.15 to 0.2 percent of bacteriophage lytic enzyme MMPpgh, 0.3 to 0.5 percent of trehalose, 0.1 to 0.2 percent of citric acid, 0.2 to 0.3 percent of mannitol, 0.2 to 0.3 percent of vitamin C and 98.3 to 98.9 percent of diatomite.

2. The method for preparing a phage lytic enzyme complex powder of claim 1, comprising: respectively constructing a genetically engineered bacterium containing a phage lyase TSPpgh gene and a genetically engineered bacterium containing a phage lyase MMPpgh gene, and fermenting and culturing the successfully constructed genetically engineered bacterium at 35-38 ℃ and 300rmp/min for 10-12h until the biomass OD of the genetically engineered bacterium is reached₆₀₀And =30-35, then using lactose for induction, removing the culture medium after the induction time is 5-6h, resuspending the culture medium by phosphate buffer solution, carrying out high-pressure homogenization and crushing under the pressure of 1000-1100 bar, centrifugally collecting the supernatant, filtering the supernatant by using a filter membrane with the pore diameter of 0.22 mu m, mixing the supernatant containing the lyase TSPpgh and the supernatant containing the lyase MMPpgh with trehalose, citric acid, mannitol, vitamin C and diatomite uniformly under the aseptic condition, and drying the mixture until the water content is 8-10% to obtain the phage lyase composite powder.

3. The use of the phage lyase complex powder of claim 1 as a feed additive for livestock and poultry, characterized in that: the feed additive can improve the immunity of livestock and poultry and increase the capability of the livestock and poultry for resisting the infection of exogenous pathogenic bacteria.

Technical Field

The invention belongs to the technical field of enzyme preparations, and particularly relates to phage lyase composite powder as well as a preparation method and application thereof.

Background

The phage lyase is a cell wall hydrolase, can hydrolyze peptidoglycan, and after the phage infects a host, the double-stranded DNA phage destroys a bacterial cell wall structure through a cave protein-lyase cracking system, so that host bacteria are cracked. Lyases generally have two to three domains involved in catalysis and binding of substrates. As a novel bactericidal preparation, the high affinity of the lyase is related to the species-specific cell wall glycosyl, and the latter is an essential component for the survival of bacteria, so that the bacteria are difficult to generate resistance to the lyase, and in nature, almost all bacteria have corresponding phages, so that the development and research of the lyase have important value. The phage lyase lyses bacteria in two ways, namely "self-internal lysis" (lysine from internal) and "self-external lysis" (lysine from external), and when the purified phage lyase is cultured in combination with a host, the lyase degrades bacterial cell wall peptidoglycan, thereby killing the bacteria by lysis. The unique bacteriostatic mechanism of the phage lyase makes the phage lyase hopeful to replace antibiotics to treat bacterial infection, and can be used for clearing specific harmful flora in animal intestinal tracts and maintaining the health of the animal intestinal tracts.

Antibacterial drugs used as feed additives for a long time include sulfonamides, tetracyclines, penicillins, chloramphenicol, chlortetracycline, kanamycin, gentamicin and the like, and the harm is mainly expressed in that (1) bacteria generate drug resistance; (2) the immunity of the livestock and poultry body is reduced; (3) causing endogenous infection and secondary infection of livestock and poultry; (4) causing residues in the livestock and poultry products.

Food safety and health problems caused by the abuse of antibiotics are becoming more serious, the addition of antibiotics to the feed is prohibited in 2006 in the European Union, and the addition of antibiotics to the feed is also prohibited comprehensively in 2020 in China; the lyase is used as a novel bactericide and has 3 main characteristics; (1) specificity. The lyase only cleaves host bacteria and has no killing effect on other strains, and the specificity of the lyase can effectively avoid flora imbalance as an antibacterial drug. (2) High efficiency. Compared with antibiotics, lyase has stronger bactericidal activity. The same amount of strain is cracked, the sterilizing efficiency of the lyase is 12-24 times that of the antibiotic, and the biofilm can be eliminated. (3) And (4) safety. In a single-dose toxicity experiment, the experimental animals have no adverse reaction, important organs and tissues have no serious inflammatory reaction and other pathological changes, and the lyase is not easy to generate drug resistance. Is expected to replace antibiotics to become another way for inhibiting bacteria of animal intestinal tracts.

The phage lyase belongs to proteases and is easily interfered by gastric acid and bile salt in the direct feeding process; in addition, the large amount of pepsin and trypsin in animal gastrointestinal tract can decompose bacteriophage lyase, resulting in loss of its activity; and residues on the amino acids of the phage lyase are easily oxidized, resulting in reduced activity.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides phage lyase composite powder, which comprises 0.15-0.2% of phage lyase TSPpgh, 0.15-0.2% of phage lyase MMPpgh, 0.3-0.5% of trehalose, 0.1-0.2% of citric acid, 0.2-0.3% of mannitol, 0.2-0.3% of vitamin C and 98.3-98.9% of diatomite by mass percent.

The preparation method of the phage lyase composite powder comprises the following steps: respectively constructing a genetically engineered bacterium containing a phage lyase TSPpgh gene and a genetically engineered bacterium containing a phage lyase MMPpgh gene according to a conventional method, and culturing the successfully constructed genetically engineered bacterium at 35-38 ℃ and 100-300rmp/min for 10-12h until the biomass OD (OD) of the genetically engineered bacterium₆₀₀=30-35, then induction with lactose, after an induction time of 5-6h, removal of the medium, resuspension with phosphate buffered saline, high-pressure homogenization disruption at a pressure of 1000-1100 bar, centrifugation to collect the supernatant and filtration with a filter having a pore size of 0.22 μm, sterile filtration of the supernatant containing the lyase TSPpgh, of the supernatant containing the lyase TSPpghMixing the supernatant of lyase MMPpgh with trehalose, citric acid, mannitol, vitamin C and diatomite, and drying to water content of 8-10% to obtain phage lyase composite powder.

The phage lyase TSPpgh and the phage lyase MMPpgh are disclosed in published patent application documents, the construction method of the recombinant expression vector is a conventional method, the genetic engineering strain for expressing the phage lyase TSPpgh and the phage lyase MMPpgh is Escherichia coli BL21, and the transformation method is also a conventional method.

The two phage lyase genes are respectively cloned on pET-28a (+) plasmids, and are transformed into escherichia coli BL21 after successful verification, so that the genetic engineering expression of phage lyase TSPpgh and phage lyase MMPpgh is realized.

The two phage lyases are prepared by fermentation, an LB culture medium is adopted as a seed culture medium and comprises the components of 10.0g/L of NaCl, 10.0g/L of tryptone and 5.0g/L, pH =7.0-7.2 of yeast powder, and the phage lyases are sterilized at 121 ℃ for 20 min; the components of the fermentation medium comprise NaCl 10.0g/L, tryptone 10.0g/L, yeast powder 5.0g/L, glucose 10 g/L, pH =7.0-7.5, sterilization is carried out at 115 ℃ for 20min, and the pH =7.0 is adjusted by 2mol/L NaOH in the fermentation process.

The induced lactose concentration is 20g/L, 50mL of lactose solution with the concentration is added into each liter of bacterial liquid, the induction temperature is 35-38 ℃, and the rotating speed is 120-150 rmp/min.

The carrier for adsorbing the phage lyase TSPpgh and MMPpgh is food-grade diatomite (Henan Jiekang environmental protection science and technology Limited), is white fine powder in character, is a biogenic siliceous sedimentary rock, mainly comprises ancient diatoms and siliceous parts of other tiny organisms such as actinomycetes, spongia spicata and the like, and the content of the diatoms reaches 90 percent; the diatomite is used as an adsorbent of the phage lyase, the loading capacity of the phage lyase can be effectively increased, the diatomite and the phage lyase can play a synergistic effect, and the diatomite can be uniformly dispersed when being added into feed and is bonded and mixed with feed particles, so that the diatomite is not easy to separate out; after eating, the livestock and poultry can promote digestion, adsorb bacteria in gastrointestinal tracts of the livestock and poultry and then discharge the bacteria out of bodies, enhance the physique of the animals and play a role in strengthening the muscles and the stomach.

Trehalose, mannitol and vitamin C are used as substances for improving the immunity of the organism and resisting aging, the trehalose, the mannitol and the vitamin C are used as enzyme protective agents, the antioxidant capacity and the heat stability of protease can be improved, the storage life of an enzyme preparation product can be prolonged, and citric acid can effectively enhance the bacteriostatic activity of lyase.

The invention also aims to apply the phage lyase composite powder to being used as a feed additive for livestock and poultry.

The phage lyase composite powder provided by the invention can effectively inhibit the proliferation of escherichia coli, salmonella and staphylococcus aureus, can replace an antibiotic additive in feed, and is used for removing harmful bacteria in intestinal tracts of livestock and poultry; can specifically kill bacteria in the lysis spectrum without interfering other flora in the intestinal tract, maintain the micro-ecological balance of the animal intestinal tract and enhance the capability of the animal to resist the infection of external pathogenic bacteria.

The two phage lyase are obtained from phages of Thermus TC16 and Thermus subsp TG17 separated from Tengchong hot sea, wherein the phage lyase TSPpgh has bacteriostatic activity within the range of 37-65 ℃, and the phage lyase MMPpgh has bacteriostatic activity within the range of 37-50 ℃.

The phage lyase composite powder can be stored for a long time at normal temperature, and experiments prove that the activity of the used phage lyase composite powder is still kept above 95 percent (the loss of the activity of the enzyme is within 5 percent) after 12 months of storage at normal temperature.

The phage lyase composite powder can effectively reduce the feed-weight ratio of white feather broilers, promote the daily weight gain of the white feather broilers while removing the blindgut pathogenic microorganisms of the white feather broilers, mainly comprising escherichia coli and salmonella, and effectively improve the bursa of Fabricius index, the cecal index and the like of the white feather broilers.

The use of antibiotics in large quantities has caused the emergence of drug-resistant bacteria and seriously threatened the health of human beings; the phage lyase is a special bacterial cell wall hydrolase, can specifically kill bacteria, cannot damage other beneficial flora, can effectively control the infection and proliferation of pathogenic bacteria, and is not easy to generate drug resistance; the phage lyase preparation is expected to replace antibiotics and become a new product for resisting bacterial diseases.

Detailed Description

The present invention is further illustrated by the following examples, without limiting the scope of the invention thereto; in the following examples, LB medium was used as the seed medium, and the components were NaCl 10.0g/L, tryptone 10.0g/L, yeast powder 5.0g/L, pH =7.0-7.2, sterilized at 121 ℃ for 20 min; the components of the fermentation medium comprise NaCl 10.0g/L, tryptone 10.0g/L, yeast powder 5.0g/L, glucose 10 g/L, pH =7.0-7.5, sterilization is carried out at 115 ℃ for 20min, and the pH =7.0 is adjusted by 2mol/L NaOH in the fermentation process.

Phosphate Buffered Saline (PBS): k₂PHO₄ 0.27g、NaHPO₄1.42g, NaCl 8g and KCl 0.2g, adding deionized water, stirring for dissolving, adjusting pH to 7.4, diluting to 1L, and sterilizing at 121 deg.C for 20 min.