CN110592056A - Phage lyase compound powder and its preparation method and application - Google Patents

Abstract

The invention relates to a phage lyase composite powder, the composition and mass percentage of which are phage lyase TSPpgh 0.15-0.2%, phage lyase MMPpgh 0.15-0.2%, trehalose 0.3-0.5%, citric acid 0.1-0.2%, Mannitol 0.2‑0.3%, vitamin C 0.2‑0.3%, diatomaceous earth 98.3‑98.9%; the phage lyase compound powder was tested for antibacterial activity in vitro by double-layer plate method, stored at room temperature for 12 months and the enzyme activity Maintain above 95% (enzyme activity loss within 5%); the compound lyase preparation can be used to replace antibiotics to inhibit the proliferation of harmful pathogenic bacteria such as Escherichia coli, Salmonella, and Staphylococcus aureus in the intestinal tract of animals, and maintain the intestinal microecological balance.

Description

Phage lyase compound powder and its preparation method and application

technical field

The invention belongs to the technical field of enzyme preparations, and in particular relates to a composite powder of phage lyase and its preparation method and application.

Background technique

Phage lyase is a type of cell wall hydrolytic enzyme that can hydrolyze peptidoglycan. After phage infects the host, the double-stranded DNA phage destroys the bacterial cell wall structure through the holin-lyase cleavage system, thereby causing the host to rupture. Lyases generally have two to three domains involved in the catalysis and binding of substrates. As a new type of bactericidal agent, the high affinity of lyase is related to the species-specific cell wall sugar, and the latter is a necessary component for the survival of bacteria, so it is difficult for bacteria to develop resistance to lyase. In nature, almost all All bacteria have corresponding phages, so the development and research of lyase is of great value. There are two ways for phage lyase to lyse bacteria: "lysis from interior" and "lysis from external". When the purified phage lyase is mixed with the host, the lyase degrades the bacteria Cell wall peptidoglycan, which lyses the bacteria to death. The unique antibacterial mechanism of phage lyase makes it possible to replace antibiotics in the treatment of bacterial infections, and can be used to remove specific harmful bacteria in the intestinal tract of animals and maintain intestinal health of animals.

Antibacterial drugs used as feed additives for a long time include sulfonamides, tetracyclines, penicillins, chloramphenicol, aureomycin, kanamycin, gentamicin, etc. (2) cause the immunity of livestock and poultry to decline; (3) cause endogenous infection and secondary infection of livestock and poultry; (4) cause residues in livestock and poultry products.

The abuse of antibiotics has also led to increasingly serious food safety and health problems. The European Union has banned the addition of antibiotics in feed in 2006, and China will also completely ban the addition of antibiotics in feed in 2020; as a new type of fungicide, lyase has The main three characteristics; (1) Specificity. Lyase only lyses its host bacteria and has no killing effect on other strains. As an antibacterial drug, the specificity of lyase can effectively avoid the occurrence of bacterial flora imbalance. (2) Efficiency. Lyase has stronger bactericidal activity than antibiotics. When lysing the same amount of strains, the sterilizing efficiency of lyase is 12-24 times that of antibiotics, and it can eliminate biofilm. (3) Security. In the single-dose toxicity test, the experimental animals did not have any adverse reactions, and no serious inflammatory reactions and other lesions were seen in important organs and tissues, and the lyase was not easy to produce drug resistance. It is expected to replace antibiotics as another way to inhibit bacteria in the intestinal tract of animals.

Phage lyase belongs to protease, and it is easily interfered by gastric acid and bile salt during direct feeding; in addition, a large amount of pepsin and trypsin in the gastrointestinal tract of animals will decompose phage lyase, resulting in loss of its activity; and phage Residues on amino acids of lyases are susceptible to oxidation, resulting in reduced activity.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a composite powder of phage lyase, whose composition and mass percentage are 0.15-0.2% of phage lyase TSPpgh, 0.15-0.2% of phage lyase MMPpgh, and 0.3-0.5% of trehalose , citric acid 0.1-0.2%, mannitol 0.2-0.3%, vitamin C 0.2-0.3%, diatomaceous earth 98.3-98.9%.

The preparation method of the above-mentioned phage lyase compound powder is as follows: according to the conventional method, respectively construct the genetically engineered bacteria containing the phage lyase TSPpgh gene and the genetically engineered bacteria containing the phage lyase MMPpgh gene, and construct the successfully genetically engineered bacteria within 35-38 Cultivate at 100-300rmp/min for 10-12h until the biomass OD ₆₀₀ of the genetically engineered bacteria is 30-35, and then induce with lactose. After 5-6h of induction, remove the medium and resuspend in phosphate buffered saline solution , under the pressure of 1000-1100 bar, carry out high-pressure homogenization and crushing, centrifuge to collect the supernatant and filter it with a filter membrane with a pore size of 0.22 μm. The liquid is mixed with trehalose, citric acid, mannitol, vitamin C, and diatomaceous earth, and dried to a water content of 8-10% to obtain a phage lytic enzyme composite powder.

The phage lytic enzyme TSPpgh and the phage lytic enzyme MMPpgh of the present invention have been disclosed in the published invention application documents, and the construction method of the recombinant expression vector is a conventional method, which is used to express the phage lytic enzyme TSPpgh and the phage lytic enzyme MMPpgh. The genetic engineering strain is Escherichia coli BL21, and the transformation method is also a conventional method.

The above two phage lyase genes were respectively cloned into the pET-28a(+) plasmid, and after successful verification, they were transformed into Escherichia coli BL21 to realize the genetic engineering expression of phage lyase TSPpgh and phage lyase MMPpgh.

The two phage lyases were prepared by fermentation, and the seed medium was LB medium, whose components were NaCl10.0g/L, tryptone10.0g/L, yeast powder5.0g/L, pH=7.0-7.2, 121 Sterilize at ℃ for 20 minutes; the components of the fermentation medium are NaCl 10.0g/L, tryptone 10.0g/L, yeast powder 5.0 g/L, glucose 10 g/L, pH=7.0-7.5, sterilized at 115℃ for 20min, During the fermentation process, 2mol/L NaOH was used to adjust pH=7.0.

The induced lactose concentration is 20g/L, 50mL of lactose solution of this concentration is added to each liter of bacterial liquid, the induction temperature is 35-38°C, and the rotation speed is 120-150rmp/min.

The carrier used to adsorb phage lytic enzymes TSPpgh and MMPpgh is food-grade diatomite (Henan Jiekang Environmental Protection Technology Co., Ltd.), which is white and fine powder. It is a biogenic siliceous sedimentary rock, mainly composed of ancient diatoms and The siliceous part of the remains of other tiny organisms such as radiolarians and spicules, etc., the content of diatoms reaches 90%; using diatomaceous earth as the adsorbent of phage lyase can effectively increase the load of phage lyase, and can be combined with Phage lyase plays a synergistic role. It can be evenly dispersed when added to the feed, and it can be bonded and mixed with the feed particles, which is not easy to separate and precipitate; after the livestock and poultry eat, it can promote digestion, and can absorb and excrete the bacteria in the gastrointestinal tract of livestock and poultry, enhancing Animal physique plays a role in strengthening the tendons and invigorating the stomach.

Trehalose, mannitol, and vitamin C, as a class of substances that improve the body's immunity and anti-aging, can be used as enzyme protection agents to improve the antioxidant capacity and thermal stability of proteases, and to increase the shelf life of enzyme preparations. Citric acid can Effectively enhance the antibacterial activity of lyase.

Another object of the present invention is to apply the above-mentioned phage lyase compound powder as a feed additive for livestock and poultry.

The phage lyase compound powder provided by the invention can effectively inhibit the value-added of Escherichia coli, Salmonella and Staphylococcus aureus, can replace antibiotic additives in feed, and is used to remove harmful bacteria in the intestinal tract of livestock and poultry; it can specifically kill bacteria within the lysate spectrum bacteria without disturbing other intestinal flora, maintaining the intestinal micro-ecological balance of animals, and enhancing the ability of animals to resist infection by external pathogenic bacteria.

The two phage lyases of the present invention are obtained from the phages of Thermus TC16 and Thermus subhibens TG17 isolated from Tengchong Rehai, wherein the phage lyase TSPpgh has antibacterial activity in the range of 37°C-65°C, and the phage Lyase MMPpgh has antibacterial activity in the range of 37°C-50°C.

The phage lytic enzyme composite powder of the present invention can be stored at normal temperature for a long time, and the experiment proves that the phage lytic enzyme composite powder is stored at normal temperature, and after 12 months of use, the enzyme activity still remains above 95% (enzyme activity loss is within 5%) ).

The phage lysing enzyme composite powder of the present invention can effectively reduce the feed-to-weight ratio of white-feather broilers, and can promote the daily weight gain of white-feather broilers while removing pathogenic microorganisms in the cecum of white-feather broilers mainly including Escherichia coli and Salmonella, and can effectively improve the bursa index of white-feather broilers , cecal index, etc. Experiments have proved that the compound phage lytic enzyme preparation can effectively improve the immunity of white-feathered broilers and increase the ability to resist the infection of exogenous pathogenic bacteria.

The extensive use of antibiotics has led to the emergence of drug-resistant bacteria, which is a serious threat to human health; phage lyase is a special type of bacterial cell wall hydrolytic enzyme, which can specifically kill bacteria without causing damage to other beneficial bacteria. It can effectively control the infection and multiplication of pathogenic bacteria and is not easy to produce drug resistance; phage lyase preparation is expected to replace antibiotics and become a new product against bacterial diseases.

Detailed ways

The present invention will be described in further detail below by the examples, but the scope of protection of the present invention is not limited to said content; In the following examples, the seed culture medium adopts LB medium, and its components are NaCl 10.0g/L, tryptone 10.0 g/L, yeast powder 5.0g/L, pH=7.0-7.2, sterilized at 121°C for 20min; the components of the fermentation medium were NaCl 10.0g/L, tryptone 10.0g/L, yeast powder 5.0 g/L, Glucose 10 g/L, pH=7.0-7.5, sterilized at 115°C for 20 minutes, and 2mol/L NaOH was used to adjust pH=7.0 during fermentation.

Phosphate buffered saline (PBS): K ₂ PHO ₄ 0.27g, NaHPO ₄ 1.42g, NaCl 8g, KCl 0.2g, add deionized water and stir to dissolve, adjust pH to 7.4, dilute to 1L, sterilize at 121°C for 20min.

Example 1: The composition and mass percentage of the phage lyase compound powder are phage lyase TSPpgh 0.15%, phage lyase MMPpgh 0.2%, trehalose 0.5%, citric acid 0.1%, mannitol 0.2%, vitamin C0.2 %, diatomaceous earth 98.65%.

The preparation method of the above-mentioned phage lyase compound powder is as follows: the phage lyase TSPpgh gene and the phage lyase MMPpgh gene were respectively cloned into the pET-28a(+) plasmid, and after successful verification, they were transformed into E. coli BL21 to obtain The genetically engineered bacteria with the TSPpgh gene and the genetically engineered bacteria containing the phage lytic enzyme MMPpgh gene were inoculated into 300mL seed medium respectively, cultivated at 37°C to OD ₆₀₀ =2.0, and then inoculated at 2% inoculum to In the fermentation medium (20L automatic fermenter (Shanghai Bailun Biotechnology Co., Ltd.)), cultivate at 37°C and 200rmp/min for 10h until the biomass OD ₆₀₀ of the genetically engineered bacteria is 35;

Then use lactose to induce, the induced lactose concentration is 20g/L, add 50mL lactose solution per liter of bacterial liquid, the induction temperature of the host expressing phage lyase TSPpgh is 37°C, the rotation speed is 150rmp/min, and the induction time is 5h; The host induction temperature of the lyase MMPpgh is 37°C, the rotation speed is 120rmp/min, and the induction time is 5h; the culture medium is removed by centrifugation, resuspended in phosphate buffered saline solution, and the host concentration for high-pressure homogenization is OD ₆₀₀ =15, under the pressure of 1000 bar, carry out high-pressure homogenate crushing, after centrifugation at 13000r/min, collect the supernatant, and filter it with a filter membrane with a pore size of 0.22 μm. Under sterile conditions, the supernatant containing lyase TSPpgh, containing The supernatant of the lyase MMPpgh was mixed with trehalose, citric acid, mannitol, vitamin C, and diatomaceous earth, and dried by boiling, with the inlet air temperature at 37°C and the outlet air temperature at 30°C, and dried until the water content was 8%, to obtain phage lytic enzyme compound powder.

Example 2: The composition and mass percentage of the phage lyase compound powder in this example are phage lyase TSPpgh 0.2%, phage lyase MMPpgh 0.15%, trehalose 0.3%, citric acid 0.2%, mannitol 0.3%, vitamin C 0.3%, diatomaceous earth 98.55%.

The preparation method of the above-mentioned phage lyase compound powder is as follows: the phage lyase TSPpgh gene and the phage lyase MMPpgh gene were respectively cloned into the pET-28a(+) plasmid, and after successful verification, they were transformed into E. coli BL21 to obtain The genetically engineered bacteria with the TSPpgh gene and the genetically engineered bacteria containing the phage lyase MMPpgh gene were inoculated into 300mL seed medium respectively, cultivated at 37°C to OD ₆₀₀ =1.5, and then inoculated at 1.5% inoculum to In the fermentation medium (20L automatic fermenter (Shanghai Bailun Biotechnology Co., Ltd.)), cultivate at 37°C and 150rmp/min for 12h until the biomass OD ₆₀₀ of the genetically engineered bacteria is 30;

Then use lactose to induce, the induced lactose concentration is 20g/L, add 50mL lactose solution per liter of bacterial liquid, the induction temperature of the host expressing phage lyase TSPpgh is 37°C, the rotation speed is 120rmp/min, and the induction time is 6h; The host induction temperature of the lyase MMPpgh is 37°C, the rotation speed is 150rmp/min, and the induction time is 6h; the medium is removed by centrifugation, resuspended in phosphate buffered saline solution, and the host concentration for high-pressure homogenization is OD ₆₀₀ =20, under the pressure of 1100 bar, the high-pressure homogenate was crushed, and after centrifugation at 13000r/min, the supernatant was collected and filtered with a filter membrane with a pore size of 0.22 μm. Under sterile conditions, the supernatant containing the lyase TSPpgh, containing The supernatant of the lyase MMPpgh was mixed with trehalose, citric acid, mannitol, vitamin C, and diatomaceous earth, and dried by boiling, with the inlet air temperature at 37°C and the outlet air temperature at 30°C, and dried until the water content was 9%, to obtain phage lytic enzyme compound powder.

Example 3: In vitro antibacterial activity detection of phage lytic enzyme composite powder

1. Take 1 g of the phage lytic enzyme composite powder prepared in the above examples in a 4 mL EP tube, and take 1 g of sterilized diatomaceous earth in a 4 mL EP tube as a control;

2. Add 3 mL PBS to the experimental group and the control group respectively;

3. Add 200 μL of the bacteria solution to be tested diluted to 10 ^-5 into the experimental group and the control group;

4. After fully shaking, incubate in a 37°C water bath for 30 minutes, shaking every 5 minutes;

5. After incubation, take 300 μL of double-layer medium for detection, and incubate at 37°C for 12 hours for colony counting;

The components of the lower solid medium are: NaCl 10.0g/L, tryptone 10.0g/L, yeast powder 5.0 g/L, agar powder 15.0 g/L;

The upper semi-solid medium components are: NaCl 10.0g/L, tryptone 10.0g/L, yeast powder: 5.0 g/L, agar powder: 5.6g/L;

Escherichia coli used for detection, strain number: CMC (B) 44102; Salmonella paratyphi, strain number: CMCC (B) 50094; Staphylococcus aureus, strain number: ATCC6538, provided by Kunming University of Science and Technology Provided by Faculty of Science and Technology; _OD600 = 1.2-1.5 when used.

The formula for calculating the antibacterial rate is as follows:

Bacterial inhibition rate (K ₁ ) = the number of colonies in the control group - the number of colonies in the experimental group/the number of colonies in the control group × 100%;

The experimental results showed that the phage lytic enzyme composite powder had an inhibitory rate of 66.23% against Escherichia coli (CMC(B)44102) and 78.3% against Salmonella paratyphi (CMCC(B)50094). The antibacterial rate of Staphylococcus aureus (ATCC6538) was 85.2%.

Embodiment 4: experiment as feed additive

Select 315 0-day-old white-feathered broilers and randomly divide them into 3 groups, namely blank control group, positive control group, and phage lysing enzyme experimental group, with 3 replicates in each group; Group adds aureomycin in basal ration by 50ppm/kg, and phage lyase experimental group adds the phage lyase compound powder described in embodiment 1 in basal ration by 50ppm/kg; Experimental period is 28 days, and experimental process The feed intake and body weight were recorded every day, and weighed before the early feeding on the 28th day. In each group, 3 animals were randomly selected for dissection and weighed the immune organs; the average daily feed intake (ADFI) was finally calculated, Average daily gain (ADG), feed-to-weight ratio (F/G) and immune organ index (mg/g), the calculation formula is as follows:

Average daily feed intake (ADFI) = feed intake/experimental days

Average daily gain (ADG) = (final weight - initial weight) / number of days of experiment

Feed-to-weight ratio (F/G) = average daily feed intake/average daily gain

Immune organ index (mg/g) = Immune organ weight/body weight

The experimental results are as follows:

Average Daily Feed Intake (ADFI) Average Daily Gain (ADG) Material to weight ratio (F/G) Bursa index (mg/g) Spleen index (mg/g) Blank control group 25.404 10.344 0.347 1.169 1.164 positive control group 28.826 16.131 0.256 1.272 1.852 Phage Lyase Experiment Group 28.331 14.656 0.280 1.091 0.894

The experimental results show that the ratio of material to weight (F/G): blank control group > phage lyase experimental group > positive control group, and the difference between the positive control group and the phage lyase experimental group in the ratio of material to weight (F/G) is not large, indicating that The phage lyase compound powder of the present invention is used as a feed additive, which can effectively reduce the feed-to-weight ratio (F/G) of broilers, which is close to the effect of aureomycin.

Immune organ index: positive control group > blank control group > phage lysing enzyme experimental group, and the immune organ index difference between the phage lysing enzyme experimental group and the blank control group is not large, indicating that the phage lysing enzyme compound powder of the present invention does not have immunity to broiler chickens level is affected.

Embodiment 5: Storage activity tracking experiment of phage lyase compound powder

Define the activity of the phage lytic enzyme composite powder just prepared to be 100%, and store samples for 7 days, 15 days, 30 days, 90 days, 180 days, and 360 days respectively for antibacterial activity detection, and the detection method is with reference to Example 3, and Calculate the enzyme activity rate of phage lyase compound powder by the following formula.

Bacterial inhibition rate (Kn) = number of colonies in the control group - number of colonies in the experimental group/number of colonies in the control group × 100%, where n is 0d, 7d, 15d, 30d, 90d, 180d, 360d;

Enzyme activity rate=inhibition rate (Kn)/inhibition rate (K _0d )×100%;

The results are shown in the table below:

time 0d 7d 15d 30d 90d 180d 360d Enzyme activity 100% 98% 98% 97.5% 97% 96% 95.5%

The results of tracking and detection of enzyme activity show that the loss of enzyme activity is within 5% when stored at room temperature for 360 days, which will not affect the characteristics of the finished product.

Claims (3)

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.