Culture medium for staphylococcus aureus phage fermentation and fermentation method

Abstract

The invention relates to the technical field of microorganisms, in particular to a culture medium for staphylococcus aureus phage fermentation and a fermentation method. According to the culture medium for staphylococcus aureus bacteriophage fermentation, the problems that in large-scale culture of staphylococcus aureus bacteriophages, excessive accumulation of host bacteria biofilm precursor substances is caused by a high-nutrition environment is achieved through cooperation of whey protein hydrolysate, humic acid, chitosan and the like are solved, and the yield of the staphylococcus aureus bacteriophages is increased; the method solves the problems of bacteriophage infection efficiency reduction and amplification resistance, improves the bacteriophage titer, and shortens the fermentation period.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a culture medium and fermentation method for Staphylococcus aureus phage fermentation. Background Technology

[0002] Staphylococcus aureus ( 金黄色葡萄球菌 Staphylococcus aureus is a Gram-positive bacterium belonging to the genus Staphylococcus. It is a common pathogen widely distributed in nature, causing various infectious diseases in humans and animals, such as local abscesses, sepsis, septicemia, endocarditis, osteomyelitis, and fatal pneumonia. It is also one of the main pathogens causing mastitis in dairy cows, often leading to decreased milk production, contamination of dairy products, and compromised dairy product safety, resulting in significant economic losses to livestock production. Antibiotics, such as penicillin, sulfonamides, and quinolones, are commonly used clinically to treat Staphylococcus aureus infections. However, the overuse of antibiotics has led to the emergence of drug-resistant strains and even multidrug-resistant strains, such as methicillin-resistant (MRSA) strains. 耐甲氧西林金黄色葡萄球菌 MRSA strains and vancomycin-resistant strains 万古霉素 耐万古霉素金黄色葡萄球菌 The increasing drug resistance of *Vitamin A* strains, especially *Staphylococcus aureus*, in recent years has brought great challenges to treatment and prevention.

[0003] Bacteriophages are bacterial viruses that complete their lysis or lysogenic cycle by specifically adsorbing onto bacteria. Vicious bacteriophages primarily reproduce progeny through the lysis cycle, while simultaneously lysing the bacteria. Therefore, vicious bacteriophages have a "killing" effect on bacteria and can be used as a treatment for bacterial infections. Bacteriophage therapy is characterized by high specificity, exponential growth, and no effect on other normal flora. Furthermore, it can be used synergistically with antibiotics to achieve the best therapeutic effect, making it a promising antibiotic alternative.

[0004] Staphylococcus aureus, once a common bacterium, has evolved into a representative of MRSA (Metabolic Respiratory Syndrome) superbugs that pose a serious threat to global public health systems. Its increasing resistance to antibiotics has spurred the rise of bacteriophages as an alternative auxiliary strategy for mitigating and inhibiting Staphylococcus aureus. Therefore, achieving low-cost cultivation and efficient fermentation of Staphylococcus aureus bacteriophages is crucial for effectively combating these drug-resistant bacteria and addressing the antibiotic crisis. Summary of the Invention

[0005] In view of this, the present invention proposes a culture medium and fermentation method for Staphylococcus aureus phage fermentation. By utilizing whey protein hydrolysate, humic acid, and chitosan as culture medium components, the invention synergistically solves the problem of excessive accumulation of host bacterial biofilm precursors caused by a high-nutrient environment in large-scale Staphylococcus aureus phage culture, which leads to decreased phage infection efficiency and inhibited amplification. This improves phage titer and shortens the fermentation cycle.

[0006] The technical solution of this invention is implemented as follows: In a first aspect, the present invention provides a culture medium for Staphylococcus aureus phage fermentation, the components of which include whey protein hydrolysate and humic acid.

[0007] Whey protein hydrolysate contains a variety of amino acids and growth factors, such as β-lactoglobulin and immunoglobulins, which can be rapidly absorbed by the host bacteria, promoting host bacterial metabolism and division, and providing sufficient raw materials for phage replication. Humic acid provides carbon source and trace elements, forming a nutritional complement with the nitrogen source of whey protein hydrolysate. At the same time, the vitamins it contains can promote the enzyme activity of the host bacteria, and it can also chelate metal ions, further synergistically promoting the replication efficiency of phage with whey protein hydrolysate.

[0008] Based on the above technical solutions, preferably, the amount of whey protein hydrolysate used is 2.5~4.0 g / L, and the amount of humic acid used is 0.2~0.35 g / L.

[0009] Rapidly proliferating Staphylococcus aureus secretes excessive amounts of biofilm precursors such as extracellular polysaccharides and extracellular DNA (eDNA), forming an invisible barrier layer in the fermentation system. This barrier layer adsorbs bacteriophage capsids, physically blocks contact between bacteriophages and free Staphylococcus aureus, reduces phage passage efficiency, and indirectly weakens the continuity of lysis and amplification. Subsequently, positively charged chitosan is added, which can bind to the negatively charged phage capsid, enhancing its stability. At the same time, it can electrostatically bind to negatively charged biofilm precursors (extracellular polysaccharides and eDNA), disrupting the conditions for them to aggregate and form a complete biofilm, thus dismantling the "invisible barrier layer." This allows Staphylococcus aureus and bacteriophages to fully contact, lyse, and amplify, stabilizing the bacteriophages while increasing the rate of lysis and amplification.

[0010] Based on the above technical solutions, preferably, the culture medium also includes chitosan.

[0011] Based on the above technical solutions, preferably, the amount of chitosan used is 0.6~2.6 g / L.

[0012] Based on the above technical solutions, preferably, the culture medium also includes sodium acetate and red yeast rice pigment.

[0013] Based on the above technical solutions, preferably, the amount of sodium acetate used is 5~9 g / L; more preferably, the amount of sodium acetate used is 9 g / L.

[0014] High concentrations of sodium acetate provide a high osmotic pressure environment, which Staphylococcus aureus can tolerate, while other bacteria have difficulty growing under these conditions. This can prevent the growth of other bacteria, provide a contamination barrier, and reduce the risk of contamination. At the same time, appropriate concentrations of sodium acetate can also enhance the titer of bacteriophages.

[0015] Based on the above technical solutions, preferably, the culture medium also includes corn cob hydrolysate, tryptone, bile salts, and dipotassium hydrogen phosphate.

[0016] Based on the above technical solution, a further preferred embodiment is that the amount of corn cob hydrolysate used is 9 ml / L, the amount of tryptone used is 8~13 g / L, the amount of bile salts used is 1.1 g / L, and the amount of dipotassium hydrogen phosphate used is 2.5 g / L.

[0017] Corn cob powder contains 40%-50% cellulose, which can be converted into glucose through dilute acid hydrolysis. Glucose is the most readily available carbon source for bacteriophage host bacteria, providing an energy basis for host bacteria proliferation and subsequent bacteriophage replication. It also reduces the cost of carbon source by more than 80%. The small amount of xylose remaining in the hydrolysate can also help regulate the metabolism of host bacteria.

[0018] Based on the above technical solutions, preferably, the pH of the culture medium is 6.7~7.3.

[0019] Secondly, a method for fermenting Staphylococcus aureus phage is provided, comprising the culture medium for Staphylococcus aureus phage fermentation as described above.

[0020] Based on the above technical solutions, preferably, the red yeast rice pigment in the culture medium is used as an indicator for determining the fermentation stage.

[0021] The present invention discovered that the culture medium for enrichment is too dark to be observed with the naked eye, which affects the judgment of the key stage of fermentation. Therefore, red yeast rice pigment was added as an indicator to judge the condition of the fermentation broth by the color change of the culture medium.

[0022] Bright red: Corresponds to the early stage / adaptation period of fermentation. At this time, Staphylococcus aureus has not produced a large amount of acid, and the pH of the fermentation broth is ≥7.0. It can also confirm whether the bacteria have recovered normally after inoculation (e.g., OD). 600 ≥0.05); Orange-red (pH=6.5-7.0): This corresponds to the logarithmic growth phase, during which the cells proliferate rapidly and begin to produce a small amount of acid. The pH is approximately between 6.5 and 7.0, indicating that dissolved oxygen (25%-30%) needs to be maintained during subsequent fermentation to avoid excessively high local cell density. Orange-yellow: This corresponds to the stationary phase, when Staphylococcus aureus enters the toxin / enzyme production stage. The pH is ≤6.5, indicating that samples can be taken to detect secondary metabolites (such as coagulase, enterotoxin, etc.) to determine the optimal harvest time.

[0023] The culture medium for Staphylococcus aureus phage fermentation of the present invention has the following advantages over the prior art: 1. By using whey protein hydrolysate, humic acid and chitosan and other culture medium components, the problem of excessive accumulation of host bacterial biofilm precursors caused by high nutrient environment in the large-scale culture of Staphylococcus aureus phage was solved, which led to a decrease in phage infection efficiency and inhibited amplification. This improved phage titer and shortened the fermentation cycle.

[0024] 2. Compared with other culture media, the main fermentation endpoint can be preliminarily determined by visual inspection of the color. It also has the advantages of low cost, low susceptibility to contamination by other microorganisms, and simple preparation method suitable for industrial production. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a morphological diagram of the Staphylococcus aureus bacteriophage GRNSAP04 of the present invention; Figure 2 This is a morphological diagram of the Staphylococcus aureus bacteriophage GRNSAP30 of the present invention. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] The tryptone used in this invention was purchased from Angel Yeast (product number FP318); sodium acetate was purchased from Sinopharm (product number 10018718); humic acid was purchased from Sinopharm (product number 69013960); chitosan was purchased from Aladdin (product number C105802-100g); dipotassium hydrogen phosphate was purchased from Sinopharm (product number 10017518); bile salts were purchased from Yuanye Biotechnology (product number S31320); red yeast rice pigment was purchased from Beijing Beifangwei (product number BWJ426620161G); and whey protein hydrolysate was purchased from Sinopharm Wokai (product number 69014483).

[0029] The preparation method of corn cob hydrolysate in this invention is as follows: Add 1g of corn cob powder and 10mL of 1wt% dilute sulfuric acid to a beaker, control the solid-liquid ratio at 1:10, heat in an 80℃ water bath for 2 hours, stirring 2-3 times during the process, to obtain corn cob hydrolysate.

[0030] The two Staphylococcus aureus bacteriophages used in this invention are: bacteriophage numbered GRNSAP30, host bacterium GRNJP01, accession number CCTCC NO: M 2024537; and bacteriophage numbered GRNSAP04, host bacterium GRNJP15, accession number CCTCC NO: M 2024536.

[0031] Example 1 Optimization of sodium acetate content.

[0032] Weigh 9 ml of corn cob hydrolysate, 10 g of tryptone, 1.1 g of bile salts, 2.5 g of dipotassium hydrogen phosphate, and 0.01 g of red yeast rice pigment as the basal culture medium. Add 5 g, 7 g, 9 g, and 11 g of sodium acetate respectively, then add to 1000 ml of solvent and mix well. Adjust the pH to 6.7, sterilize at 121℃ for 20 minutes, and set up a control group without sodium acetate. Inoculate Staphylococcus aureus phage host bacteria GRNJP01 and GRNJP015, and phages GRNSAP30 and GRNSAP04 at an MOI of 0.01, respectively, and culture at 37℃, 160 rpm, and 0.20 vvm aeration for 8 hours. After being transferred to a fermentation tank, determine the potency of the fermentation broth using the methods described below.

[0033] The methods for determining potency are as follows: 1. Prepare a sufficient number of TSA (or LB solid) plates in advance.

[0034] 2. Pick a single colony or scrape a slant from the host bacteria plate and transfer it to a certain volume of TSB (or LB) medium. Incubate overnight at a suitable temperature in an incubator to obtain a pure bacterial solution.

[0035] 3. Take the phage sample to be tested, serially dilute it 10 times to a certain concentration, take 1 ml of the appropriate concentration of the sample to be tested into a 10 ml centrifuge tube, add 300 μl of host bacterial solution to each tube and let it stand for 10-15 min.

[0036] 4. Add 5-10 ml of TSB semi-solid medium (or LB semi-solid medium, i.e., 0.7 g agar powder added to 100 ml of medium to prepare and sterilize) to a centrifuge tube at a temperature not higher than 45℃. Immediately pour the mixture onto a TSA (or LB solid) medium plate, shake to mix, and then let it stand to solidify.

[0037] 5. After incubating the plates overnight in a 37°C incubator, remove them and count the plaques on the plates to calculate the bacterial phage concentration. The results are shown in Table 1.

[0038] Table 1

[0039] As shown in Table 1, without the addition of sodium acetate, GRNSAP04 fermentation failed due to contamination by other microorganisms in the actual experiment. Adding a high amount of sodium acetate helps to avoid contamination by other microorganisms. Within a certain range, 5~9 g / L also has a certain promoting effect on the titer of bacteriophages. However, the titer of the fermentation broth begins to be affected when the amount added exceeds 9 g / L.

[0040] Example 2: Optimization of whey protein hydrolysate dosage.

[0041] Weigh 9 ml of corn cob hydrolysate, 10 g of tryptone, 1.1 g of bile salts, 2.5 g of dipotassium hydrogen phosphate, 0.01 g of red yeast rice pigment, and 7 g of sodium acetate as the basal culture medium. Add 1.5 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g, 4.5 g, and 5.0 g of whey protein hydrolysate, respectively, and then add them to 1000 ml of solvent and mix well. Adjust the pH to 6.7, sterilize at 121℃ for 20 minutes, and set up a control group without whey protein hydrolysate. Inoculate Staphylococcus aureus phage host bacteria GRNJP01 and GRNJP015, and phages GRNSAP30 and GRNSAP04 at an MOI of 0.01, respectively, and culture at 37℃, 160 rpm, and 0.20 vvm aeration for 8-10 hours. After being transferred to a fermentation tank, determine the potency of the fermentation broth using the method in Example 1.

[0042] The titer results of the two bacteriophages in this embodiment are shown in Table 2.

[0043] Table 2

[0044] Table 2 shows that the dosage of whey protein hydrolysate is 2.5~4.0 g / L, which has a good effect on improving phage titer, and the optimal dosage is 3.5 g / L.

[0045] Example 3: Optimize the dosage of humic acid.

[0046] Based on Example 2, 9 ml of corn cob hydrolysate, 10 g of tryptone, 1.1 g of bile salts, 2.5 g of dipotassium hydrogen phosphate, 0.01 g of red yeast rice pigment, 7 g of sodium acetate, and 3.5 g of whey protein hydrolysate were weighed as the basal culture medium. 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, and 0.5 g of humic acid were added respectively, and then mixed evenly in 1000 ml of solvent. The pH was adjusted to 6.7, and the mixture was sterilized at 121°C for 20 minutes. A control group without humic acid was also set up. Staphylococcus aureus phage host bacteria GRNJP01 and GRNJP015, and phages GRNSAP30 and GRNSAP04 were inoculated at an MOI of 0.01 and cultured at 37°C, 160 rpm, and 0.20 vvm aeration for 8-10 hours. After being placed in a fermentation tank, the titer of the fermentation broth was determined using the method in Example 1. The results are shown in Table 3.

[0047] Table 3

[0048] According to Table 3, the preferred content of humic acid is 0.2~0.35g / L.

[0049] Example 4: Optimization of chitosan dosage.

[0050] Based on Example 3, 9 ml of corn cob hydrolysate, 10 g of tryptone, 1.1 g of bile salts, 2.5 g of dipotassium hydrogen phosphate, 0.01 g of red yeast rice pigment, 7 g of sodium acetate, 3.5 g of whey protein hydrolysate, and 0.35 g of humic acid were weighed as the basal culture medium. Chitosan was added at concentrations of 0.6 g, 1.0 g, 1.4 g, 1.8 g, 2.2 g, 2.6 g, 3.0 g, and 3.4 g, respectively. The mixture was then added to 1000 ml of solvent and mixed thoroughly. The pH was adjusted to 6.7, and the mixture was sterilized at 121°C for 20 minutes. A control group without chitosan was also included. Staphylococcus aureus phage host bacteria GRNJP01 and GRNJP015, and phages GRNSAP30 and GRNSAP04 were inoculated at an MOI of 0.01 and cultured at 37°C, 160 rpm, and 0.20 vvm aeration for 8-10 hours. After being placed in a fermentation tank, the titer of the fermentation broth was determined using the method in Example 1. The results are shown in Table 4.

[0051] Table 4

[0052] Table 4 shows that the titer increases with the gradual addition of chitosan from 0.6 g / L to 2.6 g / L. However, the titer is affected when the chitosan concentration exceeds 2.6 g / L, and decreases further with additions from 2.6 g / L. The increased positive charge of chitosan exceeds the negative charge of the phages, hindering further phage lysis. The blank control shows that adding chitosan has a positive effect on titer improvement, which is significant for increasing titer and large-scale production. Therefore, adding chitosan improves the titer of Staphylococcus aureus phage fermentation. When using the medium of this invention with 2.6 g / L chitosan, it binds to the negatively charged phage capsid, disrupting the conditions for aggregation and formation of a complete biofilm, thus better breaking down the "invisible barrier layer." RNSAP30 and GRNSAP04 show higher titers.

[0053] Example 5: An optimized culture medium for culturing Staphylococcus aureus bacteriophages.

[0054] Weigh out 9 ml of corn cob hydrolysate, 8 g of tryptone, 1.1 g of bile salts, 2.5 g of dipotassium hydrogen phosphate, 0.01 g of red yeast rice pigment, 5 g of sodium acetate, 2.5 g of whey protein hydrolysate, 0.2 g of humic acid, and 0.6 g of chitosan to prepare the culture medium. Add these to 1000 ml of solvent and mix thoroughly. Adjust the pH to 6.7 and sterilize at 121°C for 20 minutes. Inoculate Staphylococcus aureus phage host bacteria GRNJP01 and GRNJP015, and phages GRNSAP30 and GRNSAP04 respectively at an MOI of 0.01 and culture at 37°C, 160 rpm, and 0.20 vvm aeration for 8 hours.

[0055] Example 6: An optimized culture medium for culturing Staphylococcus aureus bacteriophages.

[0056] Weigh out 9 ml of corn cob hydrolysate, 10 g of tryptone, 1.1 g of bile salts, 2.5 g of dipotassium hydrogen phosphate, 0.01 g of red yeast rice pigment, 7 g of sodium acetate, 4.0 g of whey protein hydrolysate, 0.30 g of humic acid, and 1.8 g of chitosan to prepare the culture medium. Add these to 1000 ml of solvent and mix thoroughly. Adjust the pH to 7.0 and sterilize at 121°C for 20 minutes. Inoculate Staphylococcus aureus phage host bacteria GRNJP01 and GRNJP015, and phages GRNSAP30 and GRNSAP04 respectively at an MOI of 0.01 and culture at 37°C, 160 rpm, and 0.20 vvm aeration for 9 hours.

[0057] Example 7: An optimized culture medium for culturing Staphylococcus aureus bacteriophages.

[0058] Weigh out 9 ml of corn cob hydrolysate, 13 g of tryptone, 1.1 g of bile salts, 2.5 g of dipotassium hydrogen phosphate, 0.01 g of red yeast rice pigment, 9 g of sodium acetate, 3.5 g of whey protein hydrolysate, 0.35 g of humic acid, and 2.6 g of chitosan to prepare the culture medium. Add these to 1000 ml of solvent and mix thoroughly. Adjust the pH to 7.3 and sterilize at 121°C for 20 minutes. Inoculate Staphylococcus aureus phage host bacteria GRNJP01 and GRNJP015, and phages GRNSAP30 and GRNSAP04 respectively at an MOI of 0.01 and culture at 37°C, 160 rpm, and 0.20 vvm aeration for 10 hours.

[0059] Comparative Example 1: The difference between this comparative example and Example 4 is that the culture medium was replaced with commercially available TSB medium, while other culture conditions remained the same.

[0060] The titers of the phages cultured in Examples 5-7 and Comparative Example 1 were determined, and the fermentation times were compared. The results are shown in Table 5.

[0061] Table 5

[0062] The culture medium of the present invention has significantly improved the titer of the bacteriophages obtained from fermentation, and the fermentation time is also shorter.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A culture medium for Staphylococcus aureus phage fermentation, characterized in that: The culture medium consists of whey protein hydrolysate and humic acid.

2. The culture medium for Staphylococcus aureus phage fermentation as described in claim 1, characterized in that: The amount of whey protein hydrolysate used is 2.5~4.0 g / L, and the amount of humic acid used is 0.2~0.35 g / L.

3. The culture medium for Staphylococcus aureus phage fermentation as described in claim 1, characterized in that: The culture medium also includes chitosan.

4. The culture medium for Staphylococcus aureus phage fermentation as described in claim 3, characterized in that: The amount of chitosan used is 0.6~2.6 g / L.

5. The culture medium for Staphylococcus aureus phage fermentation as described in claim 3, characterized in that: The culture medium also includes sodium acetate and red yeast rice pigment.

6. The culture medium for Staphylococcus aureus phage fermentation as described in claim 5, characterized in that: The amount of sodium acetate used is 5~9 g / L.

7. The culture medium for Staphylococcus aureus phage fermentation as described in claim 5, characterized in that: The culture medium also includes corn cob hydrolysate, tryptone, bile salts, and dipotassium hydrogen phosphate.

8. A method for fermenting Staphylococcus aureus bacteriophages, characterized in that: The culture medium for Staphylococcus aureus phage fermentation as described in any one of claims 1 to 7.

9. The method for Staphylococcus aureus phage fermentation as described in claim 8, characterized in that: The red yeast rice pigment in the culture medium serves as an indicator for determining the fermentation stage.

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