Method for obtaining bacteria in sterile environments that develop spores resistant to high temperatures

Abstract

The invention relates to a method for increasing the sporulation of bacteria such as Bacillus velezensis and Paenibacillus polymyxa using a liquid medium comprising glucose, ammonium sulfate, magnesium chloride and non-ionic surfactant polymer compounds. This method for increasing sporulation is used in a method for generating a large quantity of spores and selecting spores more resistant to higher temperatures than the spores of wild strains. The spores with higher heat resistance than those of the wild strains can be used in a method for eliminating contamination in fermentations.

Description

[0001] METHOD FOR OBTAINING BACTERIA IN STERILE ENVIRONMENTS THAT DEVELOP SPORES RESISTANT TO HIGH TEMPERATURES

[0002] Field of Invention

[0003] The present invention falls within the field of biotechnology. Specifically, it relates to a method for obtaining bacteria that develop temperature-resistant spores, maintain biofungicidal activity, and allow for better contamination control during their production by fermentation.

[0004] Background of the Invention

[0005] Sporulation is an important characteristic for manufacturing products with microorganism-based formulas that can withstand unfavorable conditions both in the environment and in artificial systems, as well as during their manufacturing stage in bioreactors. Spores are resistant forms of bacteria that allow some organisms to withstand high temperatures or high concentrations of chemical compounds that can be toxic or lethal.

[0006] On the other hand, the best-positioned biofungicide products on the market are those that contain microorganisms in their formulation that have the ability to sporulate, thus allowing them to survive longer in the environment, increasing their shelf life and better tolerating the conditions in which plant crops are grown. In this regard, there are bacteria that have demonstrated biofungicidal capacity against various phytopathogenic fungi when applied in agriculture, and which have the characteristic of generating spores, such as species of the genera Bacillus and Paenibacillus. Therefore, improving the resistance of these strains to high temperatures reduces production costs, as they can withstand higher temperatures during production and limit crop contamination.However, for certain agro-industrial sectors, the use of genetically modified organisms is restricted, so the methods for obtaining improved strains must be carried out without the use of recombinant DNA technologies.

[0007] As an alternative to genetic modification, methods can be explored that use various conditions to allow a higher percentage of the microbial population to sporulate without employing chemical agents that specifically alter the microorganism's genetics and DNA. Similarly, in the production of strains with biofungicidal capacity, cultures can sometimes be contaminated with other microorganisms during their production or formulation. Generally, when a bioreactor becomes contaminated, its contents must be discarded, and the reactor must be washed and sterilized before a new fermentation cycle can begin. This results in economic losses, both in terms of materials and the time lost preparing a new production batch.

[0008] Currently, crops with a 100% organic or reduced-nutrient base are gaining widespread acceptance in the food production market, as the resulting products are free of or contain fewer pesticides and agrochemicals. Some of the most important crops in Mexico and the United States are...

[0009] America includes lettuce, strawberries, potatoes, tomatoes, Brussels sprouts, among others.

[0010] One of the most significant problems these crops face is caused by phytopathogenic fungi; these fungi cause infections in plants that can lead to the loss of thousands of hectares of the harvest, resulting in losses of millions of dollars.

[0011] Lettuce crops can be attacked by various fungal diseases and oomycetes, the main ones being downy mildew, gray mold, and sclerotinia. Other diseases can also be very damaging in certain growing conditions, either regularly or occasionally, such as ringspot, powdery mildew, and Pythium root rot.

[0012] Strawberries are one of California's most important crops by sales volume; their production is concentrated mainly in three coastal regions: Salinas, Watsonville, Santa Maria, and Oxnard. These regions have a temperate climate that facilitates year-round strawberry production.

[0013] Of the numerous pathogens that cause strawberry crop diseases, gray mold is the most widespread and important in California and other strawberry-producing regions worldwide. The disease is called Botrytis fruit rot or gray mold. Botrytis fruit rot is found in strawberries throughout California and can cause significant fruit loss before and after harvest because the disease can develop in the field, during storage, and during shipping. It remains one of the most difficult pathogens to control. In 2006, Fusarium wilt was first detected in Ventura County. In subsequent years, Fusarium wilt was found in Santa Barbara, Monterey, and Santa Cruz counties. Fusarium wilt is economically important and can eventually affect significant portions of strawberry fields.

[0014] Furthermore, in California alone, over 900,000 acres of potatoes were cultivated in 2019, with a value of nearly $4 billion. It is the only state that grows potatoes in the spring, as it has three growing seasons annually. Several diseases have also been detected that affect potato production in California, the most significant being those caused by Colletotrichum coccodes, Macrophomina phaseolina, Alternaria solani, Fusarium sambucinum, F. solani, F. culmorum, F. avenaceum, and Phytophthora infestans.

[0015] In 2020, 234,000 acres of processing tomatoes were planted in California. Total production was 11.3 million tons, valued at $1.2 billion. The three main fungal diseases affecting tomatoes are late blight and Botrytis stem blight, although early blight, Fusarium wilt, Septoria leaf spot, and Verticillium wilt are also common.

[0016] Organic agriculture seeks to use environmentally friendly pest and disease control methods that do not rely on harmful synthetic chemicals. Among the authorized fungicides are those whose active ingredient is a microorganism, especially those that do not contain genetically modified organisms (GMOs).

[0017] On the other hand, some polymeric compounds made from ethylene oxide and / or propylene oxide are used as non-ionic surfactants or antifoaming agents. These molecules have hydrophobic and hydrophilic ends that exhibit specific affinities and can act as solubilizing agents for the bacterial cell membrane or wall, disrupting cellular processes and causing cellular stress.

[0018] Generally, for a bacterium to form spores, it must be subjected to some change in its environment that is detrimental to its growth. Under these conditions, the bacterium may choose to form spores. Not all bacteria form spores; only some, such as Gram-positive bacteria of the genera Bacillus and Clostridium, have the natural ability to do so.

[0019] Information from other inventions indicates that the use of organisms resistant to low and high temperatures is beneficial for certain processes. This is the case, for example, in patent CN103773791 A, which uses E. coli (which normally grows at 37°C) that is genetically modified using molecular biology techniques to insert genes responsible for withstanding high temperatures, based on mechanisms present in thermophilic bacteria. It is reported that they achieved cell resistance temperatures of 46°C, resulting in greater efficiency in enzymatic reactions and thus reducing the energy costs associated with cooling the bioreactor during fermentation. The use of high temperatures is limited by the tolerance of some vegetative cells (e.g., E. coli, Bacillus, etc.).) which have a lower resistance threshold than spores, since spores can withstand conditions above 60 °C, given this characteristic, working with spores presents an advantage in fermentation processes.

[0020] Brief description of the invention

[0021] The present invention aims to develop a method that allows increasing the efficiency of sporulation in bacterial cultures and that these are produced faster than under normal culture conditions using polymeric compounds made from ethylene oxide and / or propylene oxide.

[0022] Also, the present invention aims to develop a method that generates varieties of bacteria whose spores can withstand temperatures higher than those they naturally withstand.

[0023] On the other hand, the present invention aims to generate variants of bacteria that withstand high temperatures but maintain biofungicidal activity.

[0024] Furthermore, the present invention aims to generate a method for obtaining bacterial variants that withstand high temperatures and can also withstand heating cycles during fermentations, where this heating is intended to eliminate biological contamination. Additionally, the present invention aims to develop a method for bacterial variants of the genera Bacillus and Paenibacillus to develop spores more rapidly and to withstand higher temperatures than they naturally tolerate.

[0025] In turn, the present invention aims to develop varieties of the bacteria Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682, which are capable of sporulating more rapidly and withstanding higher temperatures than they naturally withstand.

[0026] In turn, the present invention aims to develop a method that generates variants of the bacteria Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682, which are capable of sporulating faster and withstanding higher temperatures than they naturally withstand, and which also maintain their biofungicidal activity.

[0027] On the other hand, the present invention aims to develop a method where the spores of the descendant strains of the bacteria Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682 can withstand high temperature cycles during fermentation processes.

[0028] In one embodiment of the present invention, the bacteria used are selected from the wild strains of Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682.

[0029] Brief Description of the Figures Figure 1. Shows a diagram of spore generation using polymeric compounds, in which 1 represents the start, 2 represents the growth of the bacteria, 3 represents the separation of the biomass, 4 represents sporulation and 5 represents the germination of spores.

[0030] Figure 2. Shows a diagram of the generation of high-temperature resistant spores, in which 6 represents the start, 7 represents the growth of the bacteria, 8 represents the separation of the biomass, 9 represents sporulation, 10 represents the selection of temperature-resistant spores, 11 represents the germination of spores, 12 represents the selection of temperature-resistant spores, and 13 represents the cell death temperature of the new strains.

[0031] Figure 3 shows a confrontation test on solid medium of Fusarium oxysporum with Bacillus velezensis LCT-632. The bacterium B. velezensis LCT-632 has completely covered the fungus, inhibiting its growth, which demonstrates the biofungicidal capacity that this bacterium has against one of the most aggressive fungi for different plants.

[0032] Figure 4. Shows a diagram of the removal of contamination in cultures, in which 14 represents the start, 15 represents the inoculation of the reactor, 16 represents the rise in temperature, 17 represents the decrease in temperature, and 18 represents the determination of the axenity of the culture.

[0033] DETAILED DESCRIPTION OF THE INVENTION In order to better understand the object of the present invention, the following definitions and abbreviations are established.

[0034] The term “agrocultivation” refers to the cultivation of edible plant species such as potatoes, strawberries, raspberries, lemons, apples, etc.

[0035] The term “organic agriculture” refers to a crop that does not use chemical compounds such as pesticides, fertilizers, or any other chemical compound necessary to keep it productive; instead, the use of living organisms is permitted that give it the ability to fix nitrogen, fix phosphorus, be more resistant to pests, etc.

[0036] The terms “phytopathogenic fungus” or “phytopathogenic fungi” refer to a fungus or fungi with the ability to infect a plant, causing disease, reduced growth, reduced yield, or even death.

[0037] The terms “wild strain” or “wild strains” refer to a fungus or bacteria isolated from a geographical location, where these fungi or bacteria have not undergone genetic change by any method or technique performed by humans.

[0038] The terms “descendant strain” or “descendant strains” refer to bacteria that have been subjected to an environmental condition that allows them to generate resistance or an evolutionary advantage over the wild strain without the use of recombinant DNA techniques or genetic engineering.

[0039] The term “spores” refers to biological resistance structures developed by bacteria and fungi that contain genetic material, which have virtually no metabolism and can withstand long periods without water or nutrients, in conditions of extreme heat or cold.

[0040] The term “sporulate” refers to the ability of an organism to generate spores.

[0041] The term “biofungicidal capacity” refers to the ability of a bacterium to reduce, decrease, inhibit, and / or stop the growth of a fungus. This capacity can originate from the production of chemical substances that are naturally produced by bacteria.

[0042] The term “cell death temperature” refers to a temperature at which bacteria are unable to reproduce or divide, and when this temperature is lowered to regain the ability to divide or reproduce, the bacteria are unable to develop, grow, or divide again.

[0043] The terms “colony forming units” and / or “CFU” refer to a method known in the art for counting the amount of bacteria present in a given sample.

[0044] The terms “colony” and “colonies” refer to the way in which a microorganism grows in a semi-solid medium, generally it will be circular and larger than 0.5 mm in size.

[0045] The term “biomass” refers to the total amount of organic matter that makes up the culture and corresponds to a single type of microorganism, in this case the producing strain and its exponential growth resulting from the fermentation process. Biomass is determined spectrophotometrically by optical density at 600 nm and by dry weight using a thermobalance, expressed in g / L.

[0046] The term “inoculum” refers to the initial portion of biomass corresponding to the strain of interest to start the fermentation process.

[0047] The term “germination” refers to the process by which spores form viable cells capable of growing and reproducing.

[0048] The term “germination medium” refers to a culture medium where germination can be carried out properly.

[0049] The term “uA” refers to a measured value of the absorbance of radiation that a substance absorbs when electromagnetic waves strike it, generally in the visible region of a certain wavelength, in this case it is 600 nanometers.

[0050] The term “medium for measuring biofungicidal activity” refers to a culture medium where both a bacterium and a phytopathogenic fungus are placed, in which the inhibition zone generated by the bacterium on the fungus is determined.

[0051] The terms “liquid sporulation medium” and “LSM” refer to a liquid culture medium where bacteria are placed to promote sporulation.

[0052] The term "inhibition zone" refers to the area within a semi-solid medium that a fungus cannot occupy because a bacterium has produced substances that inhibit fungal growth. The term "pouring plate on semi-solid medium" is a technique used in microbiology to inoculate macroorganisms. It involves placing a volume of 10 to 50 microliters into a Petri dish containing pre-solidified medium and spreading the culture with a sterile rod or beads.

[0053] The terms “ethylene oxide-propylene oxide copolymer ether with glycerol” and “Pluracol 2019 / 1 Polyol” refer to a copolymer made from glycerin, ethylene oxide, and propylene oxide. The CAS number for this copolymer is 9082-00-2.

[0054] The terms “oxirane, 2-methyl-, polymer with oxirane, monobutyl ether” and “butoxypolypropylene glycol” refer to a polymer made from propylene oxide butyl ether. The CAS number for this polymer is 9003-13-8.

[0055] The terms “2-methyloxirane, oxirane polymer”, “Kolliphor® P407”, and “Pluronic F-127” refer to a polymer made from ethylene oxide and propylene oxide. The CAS number for this polymer is CAS 9003-11-6.

[0056] The terms “oxirane, 2-methyl-, oxirane-polymer, triblock” and “Poloxamer 188” refer to a polymer made from ethylene oxide and propylene oxide. The CAS number for this polymer is 691397-13-4.

[0057] The terms “poly(oi-1,2-ethanedule), a-dodecyl-oj-hydroxy”, “decaethylene glycol monododecyl ether”, and “polyethylene glycol monododecyl ether” refer to a polymer made from ethylene oxide and lauryl alcohol. The CAS number for this polymer is 9002-

[0058] 92-0. The terms “ethanol, 2-[2-(dodecyl)ethoxy]”, “diethylene glycol monododecyl ether”, and “diethylene glycol monolauryl ether” refer to a polymer made from ethylene oxide and lauryl alcohol. The CAS number for this polymer is 3055-93-4.

[0059] The term “liquid culture medium” refers to the solution containing the nutrients necessary to allow the growth of organisms, whether fungi or bacteria. Known media in the state of the art include M9, LB, 2YT, PDA, or any other media reported in the state of the art that may be useful for the growth of the strain of interest.

[0060] The term “sporulation medium” refers to the solution that contains the chemicals necessary to enable organisms to generate spores.

[0061] The term “cell culture” refers to the controlled growth of a certain microorganism or microorganisms, under conventional or natural growth techniques known in the state of the art.

[0062] The term “biological contamination” refers to the presence of organisms other than the target wild-type strains and / or descendant strains in cell cultures, culture media, or sporulation media.

[0063] In one embodiment, the present invention relates to a method that creates conditions to increase the number of spores generated by a bacterium, where polymeric compounds made from ethylene oxide and / or propylene oxide are used, which can alter the membrane or cell wall of the bacteria, generating a better sporulation response by the bacterium that is greater than that generated when these compounds are absent.

[0064] In another embodiment of the present invention, it relates to a method that allows increasing the sporulation efficiency of bacteria, comprising the following steps: a) Starting from an inoculum of a bacterial strain with a value of 2 uA, previously preserved in a cryovial at -70 °C with glycerol, inoculate a flask with a liquid culture medium and leave it incubated at 37 °C and agitation of 150 rpm until reaching a value of 1 uA, a density that guarantees having viable cells; b) Separate the biomass from the liquid culture medium through a physical method, selected from centrifugation or filtration;c) Induce sporulation of the bacteria by placing it in a medium with polymeric compounds at a temperature of 37 °C for 12 hours, where during that period, no other compound is added, nor is the pH controlled with buffer solutions, and d) By means of the microbiological technique of pouring plates, place 50 microliters of the sporulated medium in a semi-solid germination medium, incubate the plates at 37 °C for a period of 12 to 24 hours.

[0065] Furthermore, the present invention relates to a method for generating spores of bacteria of the genera Bacillus and Paenibacillus with greater temperature resistance compared to spores of wild strains, wherein the polymeric compounds used in this method are based on ethylene oxide and / or propylene oxide. Also, the present invention relates to a method that allows generating spores of bacteria of the genera Bacillus and Paenibacillus with greater temperature resistance compared to spores of wild strains, wherein the polymeric compounds used are ethylene oxide-propylene oxide copolymer ether with glycerol (CAS 9082-00-2), Butoxypolypropylene glycol (CAS 9003-13-8), Pluronic F-127 (CAS 9003-11-6), Poloxamer 188 (CAS 691397-13-4), decaethylene glycol monododecyl ether (CAS 9002-92-0) and diethylene glycol monolauryl ether (CAS 3055-93-4).

[0066] In another embodiment of the present invention, a method is used to increase the sporulation efficiency of the bacterium Bacillus velezensis LCT-632, comprising the following steps: a) Starting with an inoculum of the bacterium Bacillus velezensis LCT-632 with a value of 2 uA, previously preserved in a cryovial at -70 °C with glycerol, inoculate a flask containing LB liquid culture medium and incubate at 37 °C with shaking at 150 rpm until reaching a value of 1 uA, a density that guarantees viable cells; b) Separate the biomass from the liquid culture medium by means of a physical method, selected from centrifugation or filtration; c) Induce sporulation of the bacterium Bacillus velezensis LCT-632 by placing it in a medium with polymeric compounds at a temperature of 30 °C for 12 hours, where during that period no other compound is added nor is the pH controlled with buffer solutions,(d) Using the microbiological pour plate technique, place 50 microliters of the sporulated medium in a semi-solid germination medium and incubate the plates at 37°C for a period of 12 to 24 hours. Another embodiment of the present invention relates to a method that allows increasing the sporulation efficiency of the bacterium Paenibacillus polymyxa LCT-682, comprising the following steps: (a) Starting with an inoculum of the bacterium Paenibacillus polymyxa LCT-682 with a value of 2 µA, previously preserved in a cryovial at -70°C with glycerol, inoculate a flask containing LB liquid culture medium and incubate at 37°C with shaking at 150 rpm until reaching a value of 1 µA, a density that guarantees viable cells; (b) Separate the biomass from the liquid culture medium using a physical method,selected from centrifugation or filtration; c) Induce sporulation of the bacterium Paenibacillus polymyxa LCT-682 by placing it in a medium with polymeric compounds at a temperature of 37 °C for 12 hours, where during that period no other compound is added nor is the pH controlled with buffer solutions, and d) Using the microbiological pour plate technique, place 50 microliters of the sporulated medium in a semi-solid germination medium, incubate the plates at 37 °C for a period of 24 to 36 hours.

[0067] Furthermore, the present invention relates to a method that allows the generation of spores of bacteria of the genera Bacillus and Paenibacillus with greater temperature resistance compared to spores of wild strains, without the use of recombinant DNA technologies or genetic engineering.

[0068] In another embodiment of the present invention, a method is used to generate bacterial spores with greater temperature resistance compared to spores of wild-type bacterial strains, comprising the following steps: a) Starting with an inoculum of a wild-type bacterial strain with a value of 2 µA, previously preserved in a cryovial at -70 °C with glycerol, inoculate a flask containing LB liquid culture medium and incubate at 37 °C with shaking at 150 rpm until reaching a value of 1 µA, a density that guarantees viable cells; b) Separate the biomass from the liquid culture medium using a physical method, selected from centrifugation or filtration; c) Place the recovered biomass in a liquid medium formulated with polymeric compounds, incubate in a baffled flask at 37 °C with shaking at 150 rpm to allow spore formation;d) Select the most temperature-resistant spores, leaving them for at least 5 minutes at a temperature higher than 37 °C, the maximum temperature the bacteria can withstand; e) Using the pour plate technique, place 50 microliters of the medium with the spores on a plate with semi-solid medium, incubate at 37 °C until colonies are visible, and f) Repeat steps “a)”, “b)”, “c)”, “d)” and “e)”, increasing the temperature of step d) by 2 °C while cell growth is still detected in step “e)”.;

[0069] In another embodiment of the present invention, bacteria grown from spores with greater temperature resistance withstand at least 10 °C higher than bacteria from wild-type strains, and wherein the bacteria are selected from among

[0070] Bacillus velezensis LCT-1932 and Paenibacillus polymyxa LCT-1982. In another embodiment of the present invention, the bacteria developed from the spores with greater temperature resistance maintain their biofungicidal activity against phytopathogenic fungi of the genera Fusarium, Colletotrichum, Phytophthora and Botrytis.

[0071] In another embodiment of the present invention, bacteria grown from spores with greater temperature resistance maintain their biofungicidal activity against the phytopathogenic fungi Alternaria brassicicola, Alternaria solani, Cladosporium sphaerospermum, Fusarium fujikuroi, Fusarium oxysporum, Colletotrichum gloeosporioides, Sclerotina sclerotiorum, Phytophthora infestans and Botrytis cinerea.

[0072] In another embodiment of the present invention, a method is used to increase the temperature resistance of spores of wild-type Bacillus velezensis LCT-632 strains, comprising the following steps: a) Starting with an inoculum of a wild-type Bacillus velezensis LCT-632 strain with a value of 2 uA, previously preserved in a cryovial at -70 °C with glycerol, inoculate a flask with LB liquid culture medium and incubate at 37 °C with shaking at 150 rpm until reaching a value of 1 uA, a density that guarantees viable cells; b) Separate the biomass from the liquid culture medium by a physical method, selected from centrifugation or filtration; c) Place the recovered biomass in a liquid medium formulated with polymeric compounds, incubate in a baffled flask at a temperature of 37 °C and agitation of 150 rpm to allow the formation of spores;d) Select the most temperature-resistant spores, leaving them for at least 5 minutes at a temperature higher than 37 °C, the maximum temperature the bacteria can withstand; e) Place the spores on a plate using the pour plate technique, 50 microliters of the medium, incubate at 37 °C until colonies are visible; and f) Repeat steps “a)”, “b)”, “c)”, “d)” and “e)”, increasing the temperature of step d) by 2°C while cell growth is still detected in step “e)”.;

[0073] In another embodiment of the present invention, a method is used to increase the temperature resistance of spores of wild-type strains of Paenibacillus polymyxa LCT-682, comprising the following steps: a) Starting with an inoculum of a wild-type strain of Paenibacillus polymyxa LCT-682 with a value of 2 uA, previously preserved in a cryovial at -70 °C with glycerol, inoculate a flask containing LB liquid culture medium and incubate at 37 °C with shaking at 150 rpm until reaching a value of 1 uA, a density that guarantees viable cells; b) Separate the biomass from the liquid culture medium by means of a method selected from centrifugation or filtration; c) Place the recovered biomass in a liquid medium formulated with polymeric compounds, incubate in a baffled flask at a temperature of 37 °C and agitation of 150 rpm to allow the formation of spores;d) Select the most temperature-resistant spores, leaving them for at least 5 minutes at a temperature higher than 37 °C, the maximum temperature the bacteria can withstand; e) Place the spores using the pour plate technique, 50 microliters of the medium on a plate with semi-solid medium, incubate at 37 °C until colonies are visible; and f) Repeat steps “a)”, “b)”, “c)”, “d)” and “e)”, increasing the temperature of step d) by 2 °C while cell growth is still detected in step “e)”.;

[0074] In another embodiment of the present invention, the liquid medium formulated with the polymeric compounds comprises: a liquid sporulation medium (LSM) having a composition selected from glucose 1 g / L, ammonium sulfate 0.2 g / L, magnesium chloride 0.05 g / L, wherein the composition is adjusted to pH 7 with sodium hydroxide 1 M, and one or more polymeric compounds (having a CAS number) selected from 9082-00-2, 9003-13-8, 9003-11-6, 691397-13-4, 9002-92-0 and 3055-93-4, in a concentration selected from 0.1 g / L, 1 g / L and 10 g / L.

[0075] In turn, the present invention relates to bacteria that withstand high temperatures and also maintain the original biofungicidal activity.

[0076] In another embodiment of the present invention, the bacteria Bacillus velezensis LCT-1932 and Paenibacillus polymyxa LCT-1982 are heat-resistant and retain their original biofungicidal activity. In another embodiment of the present invention, the bacteria Bacillus velezensis LCT-1932 and Paenibacillus polymyxa LCT-1982 are heat-resistant and retain their biofungicidal activity against the fungi Fusarium oxysporum, Colletotrichum gloeosporioides, Phytophthora infestans, and Botrytis cinerea.

[0077] In another embodiment of the present invention, a method is used to eliminate contamination from fermentations using high-temperature resistant bacteria developed in the present invention, comprising the following steps: a) Inoculating a heating reactor with the high-temperature resistant bacteria from cryovials with a value of 2 µA and preserved at -70 °C with glycerol; b) Raising the temperature 2 °C below the cell death temperature and maintaining it for 10 minutes upon detecting contamination by another organism; c) Lowering the temperature to the fermentation temperature; and d) Determining the culture's agility using microbiological or genetic techniques.

[0078] In another embodiment of the present invention, it relates to a method that allows the removal of contamination from fermentations using the bacterium Bacillus velezensis.

[0079] LCT-1932 resistant to high temperatures comprising the following steps: a) Inoculating a reactor with Bacillus velezensis LCT-1932 resistant to high temperatures from cryovials with a value of 2 uA and preserved at -70 °C with glycerol; b) Raising the temperature 2 °C below the cell death temperature and maintaining it for 10 minutes upon detecting contamination by another organism; c) Decreasing the temperature to the fermentation temperature, and d) Determining the culture's axenity using microbiological or genetic techniques.

[0080] In another embodiment of the present invention, a method is used to eliminate contamination from fermentations using the high-temperature resistant bacterium Paenibacillus polymyxa LCT-1982, comprising the following steps: a) Inoculating a reactor with high-temperature resistant Paenibacillus polymyxa LCT-1982 from cryovials with a value of 2 µA and preserved at -70 °C with glycerol; b) Raising the temperature 2 °C below the cell death temperature and maintaining it for 10 minutes upon detecting contamination by another organism; c) Lowering the temperature to the fermentation temperature; and d) Determining the culture's axenity using microbiological or genetic techniques.

[0081] EXAMPLES The following examples are intended to clarify the novelty and inventive step of the present invention. It should be understood that the following examples do not constitute a limitation on the scope of the present invention. From the description of the invention, as well as from the following examples, a person skilled in the field of the invention may make some modifications, which in any case remain within the scope protected by the present invention.

[0082] Example 1. Spore generation using polymeric compounds.

[0083] For the present invention, two previously isolated bacteria (Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682) were used, both of which are deposited in the Microorganism Collection of the CNRG (National Center for Genetic Resources). This example describes the results obtained with Bacillus velezensis LCT-632, although very similar results were obtained for either of the bacteria tested. The steps of this method were as follows (Figure 1): a) Bacterial growth

[0084] 100 microliters of an inoculum previously preserved in glycerol at -70 °C of the Bacillus velezensis LCT-632 strain were placed in different liquid culture media: potato dextrose broth, LB and M9, which are widely known in the state of the art (Madigan et al., 2020). The tubes were incubated at 37 °C and shaken at 150 rpm for a period of 10 hours until the biomass reached an optical density greater than 1; b) Biomass separation. The biomass was separated from the liquid culture medium by centrifugation at 14,000 rpm for 3 minutes using a Hermle Z 327 K refrigerated centrifuge. Subsequently, the culture medium was decanted; c) Sporulation

[0085] The biomass was transferred to a liquid sporulation medium (LSM). Different LSMs were tested, all with the same composition (glucose 1 g / L, ammonium sulfate 0.2 g / L, magnesium chloride 0.05 g / L, pH 7 adjusted with 1 M sodium hydroxide), except for the polymer compound and its concentration used in each treatment. Three concentrations were used for each polymer compound (0.1 g / L, 1 g / L, and 10 g / L), as described in Table 1. A control LSM, which did not contain any polymer compound, was also used.

[0086] Table 1. Liquid sporulation media

[0087] Using the methods described in Table 1, the time at which 20% of spores were quantified by malachite green staining was determined, as well as the percentage of sporulation after 24 hours; d) Spore germination

[0088] The spores were transferred to a germination medium or semi-solid medium described in Tables 2.

[0089] Table 2. Germination medium

[0090] The cultures were incubated at 37 °C to allow cell growth in a Thermo brand Heratherm model incubator.

[0091] Tables 3 and 4 show the results obtained using this method. It can be seen that all the tested polymeric compounds resulted in greater spore formation than the control medium (Table 3) and a shorter spore development time (Table 4). However, the best compound was Pluronic F-127 (CAS 9003-11-6), which allowed for faster spore development using a lower concentration in the sporulation medium.

[0092] It was also observed that in some polymeric compounds a high concentration generated conditions of toxicity and cell death.

[0093] Table 3. Number of spores formed after 24 h.

[0094] *The amount of spores obtained in the MEL control was used as 100%.

[0095] Table 4. Time to formation of 20% of spores.

[0096] ND: No growth detected. This example demonstrates that the method proposed in the present invention allowed spore generation to occur at least 40% faster compared to a control culture where the polymeric compound was not added. Additionally, there was at least a 15% increase in spore conversion in the cells compared to the same control.

[0097] Example 2. Generation of spores resistant to high temperatures.

[0098] For the generation of spores resistant to high temperatures, the bacteria Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682 were used, using the different liquid sporulation media described in Example 1, although for the purposes of this example only the results obtained with the MEL 3A described in Table 1 of Example 1 will be shown. To perform the selection of temperature resistant spores, the temperature at which the spores did not show germination (growth) in liquid LB medium was first determined (Table 5).

[0099] Table 5. Cell death temperature.

[0100] Once the cell death temperature was determined, the following steps were carried out for the selection of temperature-resistant spores (Figure 2); a) Growth of the bacteria 100 microliters of an inoculum previously preserved in glycerol a -

[0101] 70 °C of Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682 in LB liquid culture medium, until the biomass reached an optical density greater than 1; b) Separation of the biomass

[0102] The biomass of the bacteria Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682 was separated from the liquid culture medium by centrifugation at 14,000 rpm for 3 minutes using a Hermle Z 327 K refrigerated centrifuge, after which the culture medium was decanted; c) Sporulation

[0103] The biomass of the bacteria Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682 was transferred to MEL 3A containing Pluronic F-127 (CAS 9003-11-6) and spore formation was allowed for 16 hours; d) Selection of temperature-resistant spores

[0104] Spores of the bacteria Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682 were placed for at least 5 minutes at a temperature 2 °C lower than the cell death temperature described in Table 5. This allowed the selection of spores that could survive at a temperature slightly lower than the cell death temperature; e) Spore germination The spores were placed in the germination medium described in Table

[0105] 2 of Example 1 at a temperature 2 °C lower than the cell death temperature described in Table 5. This made it possible to obtain strains with an ability to tolerate temperatures very close to that of cell death; f) Selection of strains resistant to high temperatures

[0106] Steps “a)” to “e)” were repeated, but in each repetition the temperature was increased by 2 °C. With this methodology, strains resistant to high temperatures were generated, which were named Bacillus velezensis LCT-1932 and Paenibacillus polymyxa LCT-1982. Table 6 shows the cell death temperature of the new strains obtained.

[0107] Table 6. Cell death temperature of the new strains.

[0108] This example demonstrates that the method described herein is efficient in generating bacteria that can withstand at least 10 °C higher than the original cell death temperature of wild-type strains.

[0109] Example 3. Determination of biofungicidal activity.

[0110] In order to determine the effectiveness of the bacteria Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682, as well as the varieties generated in the present invention Bacillus velezensis LCT-1932 and Paenibacillus polymyxa LCT-1982 against thiopathogenic fungi and to be able to use them as a biofungicide, confrontation tests were carried out on solid medium against strains of Fusarium oxysporum, Colletotrichum gloeosporioides, Phytophthora infestans and Botrytis cinerea.

[0111] PDA medium was prepared in petri dishes with a bacterial concentration of 1x10 9 CFU / ml. Subsequently, a square of agar with fungal mycelium was placed in the center of the plate and incubated for 7 days at 28°C.

[0112] Biofungicidal activity was determined by measuring the mycelium growth radius on each plate (Figure 3) and comparing it to a control where no solution of the bacteria Bacillus velezensis LCT-632, Paenibacillus polymyxa LCT-682, Bacillus velezensis LCT-1932, and Paenibacillus polymyxa LCT-1982 was added. The relationship between the mycelium growth of each plate and the control is represented as a percentage of effectiveness and the results are summarized in Table 7.

[0113] Table 7. Effectiveness of wild-type strains and high-temperature resistant strains

[0114] This example demonstrates that the method for withstanding high temperatures using polymeric compounds with which the Bacillus velezensis LCT-1932 and Paenibacillus polymyxa LCT-1982 strains were obtained did not affect the biofungicidal activity of the bacteria.

[0115] Example 4. Removal of contamination using the bacteria Bacillus velezensis LCT-1932 and Paenibacillus polvmvxa LCT-1982

[0116] This example aims to demonstrate that by applying the Bacillus velezensis LCT-1932 and Paenibacillus polymyxa LCT-1982 strains, contamination of fermentations caused by other bacteria can be eliminated. To this end, fermentations were carried out in a bioreactor with each of the developed bacteria, and contamination with another bacterium was simulated. This example describes the results obtained with the Bacillus velezensis LCT-1932 strain and the bacteria Escherichia coli, Pseudomonas aeruginosa, and Paenibacillus polymyxa LCT-682. It should be noted that the results obtained were satisfactory for all four bacteria developed in this invention. The steps of this method are as follows (Figure 4): a) Reactor inoculation

[0117] A reactor was inoculated with the Bacillus velezensis LCT-1932 strain in M9 medium with 20 g / L of sucrose. Fermentation conditions were 35°C and pH 6.7, with 20% dissolved oxygen. When the culture reached a cell concentration of 1 g / L, a culture containing a mixture of the bacteria E. coll., P. aeruginosa, and Paenibacillus polymyxa LCT-682 was added, each bacterium at a concentration of 0.3 g / L. The culture was left for 1 hour to allow competition among the three bacteria. b) Temperature Increase: The temperature was raised 2°C below the cell death temperature of the strains generated in the previous examples (55°C, Table 6) and maintained for 10 minutes. c) Temperature Decrease

[0118] The temperature was lowered to 35°C to continue fermentation. Cell growth was detected 7 hours after the temperature change, and the sucrose was depleted; d) Determination of culture axenity

[0119] Using biochemical tests and molecular biology techniques such as 16SrDNA PCR, RFLPs and sequencing, it was determined that the only bacteria that survived the temperature change was Bacillus velezensis LCT-1932.

[0120] The results demonstrate that the bacteria obtained in the present invention are capable of withstanding temperatures that contaminating bacteria cannot. Therefore, these bacterial varieties can be subjected to high temperatures in an industrial process to eliminate contamination and thus prevent the loss of production batches. This generates a competitive advantage over traditional biotechnological processes where contaminated batches would have to be discarded. REFERENCES

[0121] - Madigan MT, Bender KS, Buckley DH, Sattley WM, Stahl DA. 2020. Brock Biology of Microorganisms 16th edition. Pearson.

[0122] - Daniel E. Otzen, Biosurfactants and surfactants interacting with membranes and proteins: Same but different, Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1859, Issue 4, 2017, Pages 639-649, ISSN 0005-2736, https: / / doi.Org / 10.1016 / j.bbamem.2O16.09.024.

[0123] - Carroll KC, Morse SA, Mietzner T, Miller S. Microbiología Médica. Jawetz, Melnick y Adelberg. 27° ed. México DF. Mcgraw-Hill / lnteramericana Editores, 2016.

[0124] - Zhang D, Jiang J, Shi H, Lu L, Zhang M, Lin J, Lü T, Huang J, Zhong Z, Zhao H. Nonionic surfactant Tween 80-facilitated bacterial transport in porous media: A nonmonotonic concentration-dependent performance, mechanism, and machine learning prediction. Environ Res. 2024 Mar 16;251 (Pt 2):118670. doi: 10.1016 / j.envres.2024.118670. Epub ahead of print. PMID: 38493849.

[0125] - Yu-An Chen, Yan Zhou, Yanlin Qin, Dehua Liu, Xuebing Zhao, Evaluation of the action of Tween 20 non-ionic surfactant during enzymatic hydrolysis of lignocellulose: Pretreatment, hydrolysis conditions and lignin structure, Bioresource Technology, Volume 269, 2018, Pages 329-338ISSN 0960-8524, https: / / doi.Org / 10.1016 / j.biortech .2018.08.11

[0126] - Seo, Youngwoo & Bishop, Paul. (2007). Influence of Nonionic Surfactant on Attached Biofilm Formation and Phenanthrene Bioavailability during Simulated Surfactant Enhanced Bioremediation. Environmental science & technology. 41.

[0127] 7107-13. 10.1021 / es0701154. - Barbosa, T., Claudia, R.S., LaRagione, R, Martí, J.W. &Adriano, H.0.2005. "Screening for Bacillus isolates in the broiler gastrointestinal tract". Applied and Environmental Microbiology.71 (2):968-978.

[0128] - Bawer, A., Kirvy, M., Sherris, J. &Turck, M.1996. "Antibiotic susceptibility testing by standardized single disk method. "J. American of Clinical Pathological. 45:493-496.

[0129] - Joseph F. Frank, Rose A. Koffi, Surface-adherent Growth of Listeria monocytogenes is Associated with Increased Resistance to Surfactant Sanitizers and Heat, Journal of Food Protection, Volume 53, Issue 7, 1990, Pages 550-554, ISSN 0362-028X, https: / / doi.Org / 10.4315 / 0362-028X-53.7.550.

[0130] - Dutta, Dhiraj & Gaur, Nisha & Ghosh, Debottam & Dubey, Rama & Kumar, Sanjai. (2022). A Review on the Degradation of Ionic and Non-lonic Surfactants in Water. Defence Life Science Journal. 7. 103-117. 10.14429 / dlsj.7.17309.

[0131] - Dyer, A. 1979. The culture of fern gametophytes for experimental investigation. In: The experimental biology of ferns (Ed. A. Dyer), pp. 254-305. Academic Press, Londres.

[0132] - Edwards, M.E. & J.H. Miller. 1972. Growth regulation by ethylene in fern gametophytes III. Inhibition of spore germination. American Journal of Botany 59: 458-465.

[0133] - Hennipman, E., P. Veldhoen & K. U. Kramer. 1990. Polypodiaceae. In: The families and genera of vascular plants, vol. 1 (Ed K. Kubitzki), pp. 203-230. Springer- Verlag, Berlin.

[0134] - Klekowsky, E.J. & R.M. Lloyd. 1968. Reproductive biology of the Pteridophyta I. General considerations and a study of Onoclea sensibilis L. Journal of the Linnean Society, Botany 60: 315-324.

[0135] - 2014. Molecular regulation method for improving heat resistance of escherichia coli; CN103773791 A.

Claims

CLAIMS Having described the invention, the following claims are claimed as property:

1. A method for increasing bacterial sporulation characterized in that it comprises the following steps: a) Starting from an inoculum of a bacterial strain with a value of 2 uA, previously preserved in a cryovial at -70°C with glycerol, inoculate a flask with a liquid culture medium and leave it incubated at 37°C and agitation at 150 rpm until reaching a value of 1 uA, a density that guarantees having viable cells; b) Separate the biomass from the liquid culture medium through a physical method selected from centrifugation or filtration;c) Induce sporulation of the bacteria by placing it in a liquid culture medium with polymeric compounds at a temperature of 37 °C for 12 hours, where during that period no other compound is added nor is the pH controlled with buffer solutions, and d) By means of the microbiological technique of pouring plates, place 50 microliters of the sporulated medium in a semi-solid germination medium, incubate the plates at 37 °C for a period of 12 to 24 hours.

2. Method according to claim 1, characterized in that in step “a)” the bacteria are selected from the genus Bacillus and Paenibacillus.

3. Method according to claim 2, characterized in that in step “a)” the bacteria are selected from the wild strains of Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682.

4. Method according to claim 1, characterized in that in step “c)” the sporulation medium is a liquid sporulation medium (LSM), with a composition selected from glucose 1 g / L, ammonium sulfate 0.2 g / L, magnesium chloride 0.05 g / L, wherein the composition is adjusted to a pH 7 with sodium hydroxide 1 M.

5. Method according to claim 1, characterized in that in step “c)” the polymeric compounds (with a CAS number) are selected from among 9082-00-2, 9003-13-8, 9003-11-6, 691397-13-4, 9002-92-0 and 3055-93-4, in a concentration selected from among 0.1 g / L, 1 g / L and 10 g / L.

6. Method according to claim 1, characterized in that in step “d)” the semi-solid germination medium comprises compounds in a concentration of the following Table 2: Table 2. Germination medium 7. A method for generating bacterial spores with greater temperature resistance compared to spores of wild-type bacterial strains, characterized in that it comprises the following steps: a) Starting with an inoculum of a wild-type bacterial strain with a value of 2 µA, previously preserved in a cryovial at -70 °C with glycerol, inoculate a flask with liquid culture medium and incubate at 37 °C with shaking at 150 rpm until reaching a value of 1 µA, a density that guarantees viable cells; b) Separate the biomass from the liquid culture medium using a physical method selected from centrifugation or filtration; c) Place the recovered biomass in a liquid medium formulated with polymeric compounds, incubate in a baffled flask at 37 °C with shaking at 150 rpm to allow spore formation;d) Select the spores most resistant to temperature, leaving them for at least 5 minutes at a temperature higher than 37 °C, the maximum temperature that the bacteria can withstand; e) Using the pour plate technique, place 50 microliters of the medium with the spores on a plate with semi-solid medium, incubate at 37 °C until colonies are visible, and f) Repeat steps “a)”, “b)”, “c)”, “d)” and “e)”, increasing the temperature of step d) by 2°C while cell growth is still detected in step “e”.

8. Method according to claim 7, characterized in that in step “a)” the bacteria are selected from the genus Bacillus and Paenibacillus.

9. Method according to claim 7, characterized in that in step “a)” the bacteria are selected from the wild strains of Bacillus velezensis LCT-632 and Paenibacillus polymyxa LCT-682.

10. Method according to claim 7, characterized in that in step “c)” the sporulation medium is a liquid sporulation medium (LSM), having a composition selected from glucose 1 g / L, ammonium sulfate 0.2 g / L, magnesium chloride 0.05 g / L, wherein the composition is adjusted to a pH of 7 with sodium hydroxide 1 M.

11. Method according to claim 7, characterized in that in step “c)” the polymeric compounds (with a CAS number) are selected from among 9082-00-2, 9003-13-8, 9003-11-6, 691397-13-4, 9002-92-0 and 3055-93-4, in a concentration selected from among 0.1 g / L, 1 g / L and 10 g / L.

12. Method according to claim 7, characterized in that in step “e)” the semi-solid germination medium comprises compounds in a concentration of the following Table 2: Table 2. Germination medium 13. Method according to claim 7, characterized in that the bacteria grown from the spores with greater temperature resistance are selected from the genera Bacillus and Paenibacillus, and wherein they were not genetically modified by recombinant DNA technologies.

14. Method according to claim 7, characterized in that the bacteria grown from the spores with greater temperature resistance withstand at least 10 °C more than the bacteria from wild-type strains, and wherein The bacteria are selected from among Bacillus velezensis LCT-1932 and Paenibacillus polymyxa LCT-1982.

15. Method according to claim 7, characterized in that the bacteria maintain their biofungicidal activity against phytopathogenic fungi of the genera Fusarium, Colletotrichum, Phytophthora and Botrytis.

16. Method according to claim 7, characterized in that the bacteria maintain their biofungicidal activity against the phytopathogenic fungi Alternaria brassicicola, Alternaria solani, Cladosporium sphaerospermun, Fusarium fujikuroi, Fusarium oxysporum, Colletotrichum gloeosporioides, Sclerotina sclerotiorum, Phytophthora infestans and Botrytis cinerea.

17. A method for removing contamination from fermentations using the high-temperature resistant bacteria of claim 7, characterized in that it comprises the following steps: a) Inoculating a reactor with the high-temperature resistant bacteria from cryovials with a value of 2 uA and stored at -70°C; b) Raising the temperature 2°C below the cell death temperature and maintaining it for 10 minutes upon detecting contamination by another organism; c) Lowering the temperature to the fermentation temperature; and d) Determining the culture's axenity using microbiological or genetic techniques.

18. Method according to claim 17, characterized in that in step “a)” the bacteria are selected from Bacillus velezensis LCT-1932 and Paenibacillus polymyxa LCT-1982.

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