Combined fermentation process of bacteriophage and probiotics
By performing the combined fermentation of phages and probiotics in the same fermentation medium and adding trace elements, the problem of low yield in the prior art is solved, and more efficient microecological preparation production and flexibility in product form is achieved.
Patent Information
- Application Number
- CN202311540050.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-20
- Publication Date
- 2024-12-17
AI Technical Summary
The existing phages and probiotics are not co-fermented in the same fermentation medium, and cannot effectively utilize the nutrients in the medium, resulting in low yields of microecological preparations.
The combined fermentation of phages and probiotics is carried out in the same fermentation medium. By staggering fermentation and adding trace elements FeCl3, vitamin B12 and biotin, the fermentation effect is enhanced, and water agents and powders are prepared by separation and extraction to improve product applicability.
It improves the yield of microecological preparations, enhances the fermentation effect, and provides flexible product forms for ease of use and storage.
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of combined fermentation of bacteriophage and probiotics, in particular to a combined fermentation process of bacteriophage and probiotics. Background Art
[0002] The abuse of antibiotics has become a global problem. With the emergence of more and more drug-resistant pathogens, researchers have been urged to accelerate the development of alternative antibiotics. Phages and probiotics, as green and new alternative antibiotics, have attracted much attention in recent years.
[0003] Bacteriophages are viruses that can infect and kill bacteria. They were discovered by Twort in 1915, and F'elix d'Herelle first described the phage phenomenon and plaques. Bacteriophages are widely distributed in nature, and most of the early phage research conducted in the early 20th century focused on phage therapy for the treatment of bacterial infections. Bacteriophages are also an important component of the microecology. They exist in the human body and animal intestines, soil environments, and water environments. Bacteriophages are widely distributed in these microecologies and play an important role in regulating the functions of microecologies. The interaction between phages and host bacteria is crucial to the ecology and evolution of microbial communities. Their co-evolution with host bacteria can maintain bacterial phenotypic and genetic diversity, and increase their evolution and differentiation rates.
[0004] Probiotics are a general term for microorganisms that are beneficial to the human body, among which lactic acid bacteria account for the largest proportion. Lactic acid bacteria can ferment carbohydrates to produce a large amount of lactic acid. They are extremely widely distributed in nature. At least 18 genera and more than 200 species have been discovered so far. The well-known lactic acid bacteria are mainly concentrated in the genera of Lactobacillus, Bifidobacterium and Streptococcus. Except for a very small number, most lactic acid bacteria are necessary for the human body and can colonize in the human intestine. They have multiple probiotic functions such as inhibiting the growth of harmful bacteria, improving the intestinal microenvironment, and enhancing the immune system.
[0005] Bacterial diseases are a type of disease in humans or animals caused by pathogenic bacteria. This type of disease and viral diseases are currently one of the main diseases that threaten the health and development of humans and animals. Conventional prevention and treatment methods mainly include vaccination and the use of antibiotics. However, due to a series of problems such as antibiotic restriction and ban caused by the increasingly serious antibiotic resistance, new prevention and treatment methods are urgently needed. As a virus that invades bacteria, bacteriophages have a series of advantages such as specificity, rapid sterilization, and no destruction of the balance of the bacterial flora, and are increasingly attracting attention. Existing bacteriophages are prepared from the corresponding host bacteria through a series of strict infection processes. The existing methods have the following disadvantages: bacteriophages and probiotics are not co-fermented in the same fermentation medium, and the nutrients in the medium cannot be effectively utilized to increase the yield of microecological preparations. Summary of the invention
[0006] The present invention provides a combined fermentation process of bacteriophage and probiotics, which solves the problems raised by the above background technology.
[0007] The present invention provides the following technical solution: a combined fermentation process of bacteriophage and probiotics, comprising the following steps:
[0008] Step S1: inoculating the activated host bacteria into TSB culture medium at an inoculation ratio of 1% to 20%, culturing temperature of 25 to 45° C., rotating speed of 20 to 240 rpm, and culturing time of 4 to 60 hours to obtain host bacteria seed solution; inoculating the activated probiotic bacteria into seed culture medium at an inoculation ratio of 1% to 20%, culturing temperature of 25 to 45° C., rotating speed of 20 to 240 rpm, and culturing time of 4 to 60 hours to obtain probiotic seed solution;
[0009] Step S2: inoculating probiotics, bacteriophages and phage host bacteria at the same time, mixing them and co-fermenting them; wherein the probiotics and / or their metabolites can kill the phage host bacteria, and the phages and probiotics are fermented at different times;
[0010] Step S3: After the fermentation culture is completed, centrifugal separation is performed, the supernatant is filtered and the phage product is collected, and the precipitate is spray-dried or freeze-dried to obtain the probiotic product;
[0011] Step S4: Aseptic packaging: Aseptically packaging the fermentation sterilization clear liquid in step 3 to obtain an aqueous solution of a mixture of bacteriophages and probiotic metabolites;
[0012] Step S5: adding a protective agent and / or a carrier in an amount of 1 to 15 times the amount of the bacteria in step 3, emulsifying and mixing, drying, crushing and sieving, and aseptically packaging to obtain a powder of a mixture of bacteriophages and probiotics.
[0013] Preferably, step S1 specifically includes that the probiotic is Bacillus subtilis BLCC1-0155, whose deposit number is CCTCCNO: M2015278;
[0014] The phages selected were Salmonella typhimurium phage φSa-1 and Aeromonas hydrophila phage BLCC16-001;
[0015] Among them, the deposit number of Salmonella typhimurium phage φSa-1 is CCTCCNO: M2017217;
[0016] The deposit number of Aeromonas hydrophila phage BLCC16-001 is CCTCCNO: M2020456.
[0017] Preferably, step S2 specifically includes inoculating the phage and host bacteria seed liquid into the fermentation medium, fermenting for 4 to 60 hours to obtain the phage fermentation liquid, sterilizing the fermentation liquid with a 0.1 μm to 0.22 μm membrane, inoculating the probiotic seed liquid into the sterile phage fermentation liquid, fermenting for 6 to 120 hours, and obtaining the phage and probiotic fermentation liquid.
[0018] Preferably, step S3 specifically includes: the fermentation liquid in step 3 is separated by one or more separation processes including flocculation, centrifugation or membrane filtration to obtain fermentation sterilization clear liquid and bacterial cells.
[0019] Preferably, step S2 specifically comprises: inoculating the phage and host bacteria seed solution into the fermentation medium, and adding trace elements FeCl3, vitamin B12 and biotin, with concentrations of 0.01 g / L, 0.002 g / L and 0.001 g / L respectively, fermenting for 4-30 hours, and obtaining the phage fermentation solution.
[0020] The present invention has the following beneficial effects:
[0021] 1. The combined fermentation process of bacteriophage and probiotics improves efficiency through combined fermentation: by co-fermenting bacteriophage and probiotics in the same fermentation medium, the nutrients in the medium can be more effectively utilized and the output of microecological preparations can be increased.
[0022] 2. The combined fermentation process of bacteriophage and probiotics, the addition of trace elements and vitamins: adding trace elements FeCl3, vitamin B12 and biotin to the fermentation medium helps to provide auxiliary factors required for microbial growth, thereby enhancing the fermentation effect.
[0023] 3. The combined fermentation process of bacteriophage and probiotics, flexible preparation of aqueous solution and powder: After separation and extraction, aqueous solution and powder can be prepared separately, improving the applicability and convenience of the product. Aqueous solution is suitable for direct use, while powder is easier to store and transport. DETAILED DESCRIPTION
[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0025] Embodiment 1
[0026] A combined fermentation process of bacteriophage and probiotics comprises the following steps:
[0027] Step S1: inoculating the activated host bacteria into TSB culture medium at an inoculation ratio of 1% to 20%, culturing temperature of 25 to 45° C., rotating speed of 20 to 240 rpm, and culturing time of 4 to 60 hours to obtain host bacteria seed solution; inoculating the activated probiotic bacteria into seed culture medium at an inoculation ratio of 1% to 20%, culturing temperature of 25 to 45° C., rotating speed of 20 to 240 rpm, and culturing time of 4 to 60 hours to obtain probiotic seed solution;
[0028] Step S2: inoculating probiotics, bacteriophages and phage host bacteria at the same time, mixing them and co-fermenting them; wherein the probiotics and / or their metabolites can kill the phage host bacteria, and the phages and probiotics are fermented at different times;
[0029] Step S3: After the fermentation culture is completed, centrifugal separation is performed, the supernatant is filtered and the phage product is collected, and the precipitate is spray-dried or freeze-dried to obtain the probiotic product;
[0030] Step S4: Aseptic packaging: Aseptically packaging the fermentation sterilization clear liquid in step 3 to obtain an aqueous solution of a mixture of bacteriophages and probiotic metabolites;
[0031] Step S5: adding a protective agent and / or a carrier in an amount of 1 to 15 times the amount of the bacteria in step 3, emulsifying and mixing, drying, crushing and sieving, and aseptically packaging to obtain a powder of a mixture of bacteriophages and probiotics.
[0032] Preferably, step S1 specifically includes that the probiotic is Bacillus subtilis BLCC1-0155, whose deposit number is CCTCCNO: M2015278;
[0033] The phages selected were Salmonella typhimurium phage φSa-1 and Aeromonas hydrophila phage BLCC16-001;
[0034] Among them, the deposit number of Salmonella typhimurium phage φSa-1 is CCTCCNO: M2017217;
[0035] The deposit number of Aeromonas hydrophila phage BLCC16-001 is CCTCCNO: M2020456.
[0036] Preferably, step S2 specifically includes inoculating the phage and host bacteria seed liquid into the fermentation medium, fermenting for 4 to 60 hours to obtain the phage fermentation liquid, sterilizing the fermentation liquid with a 0.1 μm to 0.22 μm membrane, inoculating the probiotic seed liquid into the sterile phage fermentation liquid, fermenting for 6 to 120 hours, and obtaining the phage and probiotic fermentation liquid.
[0037] Preferably, step S3 specifically includes: the fermentation liquid in step 3 is separated by one or more separation processes including flocculation, centrifugation or membrane filtration to obtain fermentation sterilization clear liquid and bacterial cells.
[0038] Preferably, step S2 specifically comprises: inoculating the phage and host bacteria seed solution into the fermentation medium, and adding trace elements FeCl3, vitamin B12 and biotin, with concentrations of 0.01 g / L, 0.002 g / L and 0.001 g / L respectively, fermenting for 4-30 hours, and obtaining the phage fermentation solution.
[0039] Embodiment 2
[0040] A combined fermentation process of bacteriophage and probiotics comprises the following steps:
[0041] Step S1: inoculating the activated host bacteria into TSB culture medium at an inoculation ratio of 1% to 20%, culturing temperature of 25 to 45° C., rotating speed of 20 to 240 rpm, and culturing time of 4 to 60 hours to obtain host bacteria seed solution; inoculating the activated probiotic bacteria into seed culture medium at an inoculation ratio of 1% to 20%, culturing temperature of 25 to 45° C., rotating speed of 20 to 240 rpm, and culturing time of 4 to 60 hours to obtain probiotic seed solution;
[0042] Step S2: inoculating probiotics, bacteriophages and phage host bacteria at the same time, mixing them and co-fermenting them; wherein the probiotics and / or their metabolites can kill the phage host bacteria, and the phages and probiotics are fermented at different times;
[0043] Step S3: After the fermentation culture is completed, centrifugal separation is performed, the supernatant is filtered and the phage product is collected, and the precipitate is spray-dried or freeze-dried to obtain the probiotic product;
[0044] Step S4: Aseptic packaging: Aseptically packaging the fermentation sterilization clear liquid in step 3 to obtain an aqueous solution of a mixture of bacteriophages and probiotic metabolites;
[0045] Step S5: adding a protective agent and / or a carrier in an amount of 1 to 15 times the amount of the bacteria in step 3, emulsifying and mixing, drying, crushing and sieving, and aseptically packaging to obtain a powder of a mixture of bacteriophages and probiotics.
[0046] Preferably, step S1 specifically includes that the probiotic is Bacillus subtilis BLCC1-0155, whose deposit number is CCTCCNO: M2015278;
[0047] The phages selected were Salmonella typhimurium phage φSa-1 and Aeromonas hydrophila phage BLCC16-001;
[0048] Among them, the deposit number of Salmonella typhimurium phage φSa-1 is CCTCCNO: M2017217;
[0049] The deposit number of Aeromonas hydrophila phage BLCC16-001 is CCTCCNO: M2020456.
[0050] Preferably, step S2 specifically includes inoculating the phage and host bacteria seed liquid into the fermentation medium, fermenting for 4 to 60 hours to obtain the phage fermentation liquid, sterilizing the fermentation liquid with a 0.1 μm to 0.22 μm membrane, inoculating the probiotic seed liquid into the sterile phage fermentation liquid, fermenting for 6 to 120 hours, and obtaining the phage and probiotic fermentation liquid.
[0051] Preferably, step S3 specifically includes: the fermentation liquid in step 3 is separated by one or more separation processes including flocculation, centrifugation or membrane filtration to obtain fermentation sterilization clear liquid and bacterial cells.
[0052] Preferably, step S2 specifically comprises: inoculating the phage and host bacteria seed solution into the fermentation medium, and adding trace elements FeCl3, vitamin B12 and biotin, with concentrations of 0.03 g / L, 0.002 g / L and 0.001 g / L respectively, fermenting for 4-30 hours, and obtaining the phage fermentation solution.
[0053] Embodiment 3
[0054] A combined fermentation process of bacteriophage and probiotics comprises the following steps:
[0055] Step S1: inoculating the activated host bacteria into TSB culture medium at an inoculation ratio of 1% to 20%, culturing temperature of 25 to 45° C., rotating speed of 20 to 240 rpm, and culturing time of 4 to 60 hours to obtain host bacteria seed solution; inoculating the activated probiotic bacteria into seed culture medium at an inoculation ratio of 1% to 20%, culturing temperature of 25 to 45° C., rotating speed of 20 to 240 rpm, and culturing time of 4 to 60 hours to obtain probiotic seed solution;
[0056] Step S2: inoculating probiotics, bacteriophages and phage host bacteria at the same time, mixing them and co-fermenting them; wherein the probiotics and / or their metabolites can kill the phage host bacteria, and the phages and probiotics are fermented at different times;
[0057] Step S3: After the fermentation culture is completed, centrifugal separation is performed, the supernatant is filtered and the phage product is collected, and the precipitate is spray-dried or freeze-dried to obtain the probiotic product;
[0058] Step S4: Aseptic packaging: Aseptically packaging the fermentation sterilization clear liquid in step 3 to obtain an aqueous solution of a mixture of bacteriophages and probiotic metabolites;
[0059] Step S5: adding a protective agent and / or a carrier in an amount of 1 to 15 times the amount of the bacteria in step 3, emulsifying and mixing, drying, crushing and sieving, and aseptically packaging to obtain a powder of a mixture of bacteriophages and probiotics.
[0060] Preferably, step S1 specifically includes that the probiotic is Bacillus subtilis BLCC1-0155, whose deposit number is CCTCCNO: M2015278;
[0061] The phages selected were Salmonella typhimurium phage φSa-1 and Aeromonas hydrophila phage BLCC16-001;
[0062] Among them, the deposit number of Salmonella typhimurium phage φSa-1 is CCTCCNO: M2017217;
[0063] The deposit number of Aeromonas hydrophila phage BLCC16-001 is CCTCCNO: M2020456.
[0064] Preferably, step S2 specifically includes inoculating the phage and host bacteria seed liquid into the fermentation medium, fermenting for 4 to 60 hours to obtain the phage fermentation liquid, sterilizing the fermentation liquid with a 0.1 μm to 0.22 μm membrane, inoculating the probiotic seed liquid into the sterile phage fermentation liquid, fermenting for 6 to 120 hours, and obtaining the phage and probiotic fermentation liquid.
[0065] Preferably, step S3 specifically includes: the fermentation liquid in step 3 is separated by one or more separation processes including flocculation, centrifugation or membrane filtration to obtain fermentation sterilization clear liquid and bacterial cells.
[0066] Preferably, step S2 specifically comprises: inoculating the phage and host bacteria seed solution into the fermentation medium, and adding trace elements FeCl3, vitamin B12 and biotin, with concentrations of 0.01 g / L, 0.001 g / L and 0.001 g / L respectively, fermenting for 4-30 hours, and obtaining the phage fermentation solution.
[0067] Embodiment 4
[0068] A combined fermentation process of bacteriophage and probiotics comprises the following steps:
[0069] Step S1: inoculating the activated host bacteria into TSB culture medium at an inoculation ratio of 1% to 20%, culturing temperature of 25 to 45° C., rotating speed of 20 to 240 rpm, and culturing time of 4 to 60 hours to obtain host bacteria seed solution; inoculating the activated probiotic bacteria into seed culture medium at an inoculation ratio of 1% to 20%, culturing temperature of 25 to 45° C., rotating speed of 20 to 240 rpm, and culturing time of 4 to 60 hours to obtain probiotic seed solution;
[0070] Step S2: inoculating probiotics, bacteriophages and phage host bacteria at the same time, mixing them and co-fermenting them; wherein the probiotics and / or their metabolites can kill the phage host bacteria, and the phages and probiotics are fermented at different times;
[0071] Step S3: After the fermentation culture is completed, centrifugal separation is performed, the supernatant is filtered and the phage product is collected, and the precipitate is spray-dried or freeze-dried to obtain the probiotic product;
[0072] Step S4: Aseptic packaging: Aseptically packaging the fermentation sterilization clear liquid in step 3 to obtain an aqueous solution of a mixture of bacteriophages and probiotic metabolites;
[0073] Step S5: adding a protective agent and / or a carrier in an amount of 1 to 15 times the amount of the bacteria in step 3, emulsifying and mixing, drying, crushing and sieving, and aseptically packaging to obtain a powder of a mixture of bacteriophages and probiotics.
[0074] Preferably, step S1 specifically includes: the probiotic is Bacillus subtilis BLCC1-0155, whose deposit number is CCTCCNO: M2015278;
[0075] The phages selected were Salmonella typhimurium phage φSa-1 and Aeromonas hydrophila phage BLCC16-001;
[0076] Among them, the deposit number of Salmonella typhimurium phage φSa-1 is CCTCCNO: M2017217;
[0077] The deposit number of Aeromonas hydrophila phage BLCC16-001 is CCTCCNO: M2020456.
[0078] Preferably, step S2 specifically includes inoculating the phage and host bacteria seed liquid into the fermentation medium, fermenting for 4 to 60 hours to obtain the phage fermentation liquid, sterilizing the fermentation liquid with a 0.1 μm to 0.22 μm membrane, inoculating the probiotic seed liquid into the sterile phage fermentation liquid, fermenting for 6 to 120 hours, and obtaining the phage and probiotic fermentation liquid.
[0079] Preferably, step S3 specifically includes: the fermentation liquid in step 3 is separated by one or more separation processes including flocculation, centrifugation or membrane filtration to obtain fermentation sterilization clear liquid and bacterial cells.
[0080] Preferably, step S2 specifically comprises: inoculating the phage and host bacteria seed solution into the fermentation medium, and adding trace elements FeCl3, vitamin B12 and biotin, with concentrations of 0.01 g / L, 0.001 g / L and 0.001 g / L respectively, fermenting for 4-30 hours, and obtaining the phage fermentation solution.
[0081] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.
[0082] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A combined fermentation process of bacteriophage and probiotics, characterized in that: The following steps are involved: Step S1: inoculating the activated host bacteria into TSB culture medium at an inoculation ratio of 1% to 20%, culturing temperature of 25 to 45° C., rotating speed of 20 to 240 rpm, and culturing time of 4 to 60 hours to obtain host bacteria seed solution; inoculating the activated probiotic bacteria into seed culture medium at an inoculation ratio of 1% to 20%, culturing temperature of 25 to 45° C., rotating speed of 20 to 240 rpm, and culturing time of 4 to 60 hours to obtain probiotic seed solution; Step S2: inoculating probiotics, bacteriophages and phage host bacteria at the same time, mixing them and co-fermenting them; wherein the probiotics and / or their metabolites can kill the phage host bacteria, and the phages and probiotics are fermented at different times; Step S3: After the fermentation culture is completed, centrifugal separation is performed, the supernatant is filtered and the phage product is collected, and the precipitate is spray-dried or freeze-dried to obtain the probiotic product; Step S4: Aseptic packaging: Aseptically packaging the fermentation sterilization clear liquid in step 3 to obtain an aqueous solution of a mixture of bacteriophages and probiotic metabolites; Step S5: adding a protective agent and / or a carrier in an amount of 1 to 15 times the amount of the bacteria in step 3, emulsifying and mixing, drying, crushing and sieving, and aseptically packaging to obtain a powder of a mixture of bacteriophages and probiotics.
2. The combined fermentation process of bacteriophage and probiotics according to claim 1, characterized in that: Step S1 specifically includes: the probiotic is Bacillus subtilis BLCC1-0155, whose deposit number is CCTCCNO: M2015278; The phages selected were Salmonella typhimurium phage φSa-1 and Aeromonas hydrophila phage BLCC16-001; Among them, the deposit number of Salmonella typhimurium phage φSa-1 is CCTCCNO: M2017217; The deposit number of Aeromonas hydrophila phage BLCC16-001 is CCTCCNO: M2020456.
3. The combined fermentation process of bacteriophage and probiotics according to claim 1, characterized in that: Step S2 specifically includes inoculating the phage and host bacteria seed liquid into the fermentation medium, fermenting for 4 to 60 hours to obtain the phage fermentation liquid, sterilizing the fermentation liquid with a 0.1 μm to 0.22 μm membrane, inoculating the probiotic seed liquid into the sterile phage fermentation liquid, fermenting for 6 to 120 hours, and obtaining the phage and probiotic fermentation liquid.
4. The combined fermentation process of bacteriophage and probiotics according to claim 1, characterized in that: Step S3 specifically includes: the fermentation liquid in step 3 is separated by one or more separation processes including flocculation, centrifugation or membrane filtration to obtain fermentation sterilization clear liquid and bacterial cells.
5. The combined fermentation process of bacteriophage and probiotics according to claim 1, characterized in that: Step S2 specifically includes: inoculating the phage and host bacteria seed solution into the fermentation medium, and adding trace elements FeCl3, vitamin B12 and biotin, with concentrations of 0.01g / L, 0.002g / L and 0.001g / L respectively, fermenting for 4-30h to obtain phage fermentation solution.