Novel microorganisms that break down fatty acid-containing oils and fats

Abstract

To provide a novel microorganism capable of degrading lipids and fats. [Solution] In one embodiment, the microorganism belongs to the Burkholderiae family. In one embodiment, the microorganism belongs to the Burkholderia genus. In one embodiment, the microorganism is Burkholderia arboris or Burkholderia cepacia complex, etc. In one embodiment, the microorganism of the disclosure has the ability to assimilate trans fatty acids. In one embodiment, the microorganism of the disclosure has the ability to decompose trans fatty acids. In one embodiment, the microorganism of the disclosure has the ability to decompose trans fatty acid-containing oils and fats. In one embodiment, the microorganism of the disclosure has the ability to assimilate trans fatty acid-containing oils and fats.

Description

[Technical Field]

[0001] This disclosure relates to microorganisms having the ability to degrade oils and / or fatty acids and their use. More specifically, it relates to novel microorganisms that degrade trans fatty acid-containing oils. [Background technology]

[0002] Wastewater from food processing plants and oil factories contains large amounts of oil. This oil content causes various degradations in biological treatment functions, including reduced treatment capacity by activated sludge, failure of solid-liquid separation due to decreased settling properties, membrane fouling in membrane bioreactors (MBR), and inhibition of methane fermentation in anaerobic digestion. Therefore, as a preliminary step to biological treatment of high-oil-content wastewater, oil is removed using methods such as pressurized flotation separators. Similarly, kitchen wastewater from the food service industry also contains a lot of oil, so grease traps are installed to remove it. Both pressurized flotation separators and grease traps have problems such as being sources of foul odors and pests, the costs of recovering and transporting the separated oil and disposing of it as industrial waste, and the labor and costs involved in management and cleaning. As a means of solving these problems, microbial oil degradation technology has been considered, and several related microbial preparations are commercially available. However, it is extremely difficult to reduce the oil concentration to the desired level through microbial degradation within the settable residence time. Therefore, currently, pressurized flotation separators and conventional grease traps are used in most cases.

[0003] Furthermore, in the fermentation treatment of food waste, if the oil content is high, problems arise such as inhibition of fermentation and high oil content in wastewater from elimination-type treatment machines. In addition, oily sludge separated and recovered by pressurized flotation separators and grease traps becomes industrial waste, and its treatment incurs significant costs. Therefore, the decomposition of these oils by microorganisms is being considered, but as with the wastewater treatment mentioned above, the reality is that there are limits to the decomposition capacity of microorganisms.

[0004] As mentioned above, the rate of decomposition is a problem when removing oils using microorganisms, and the decrease in activity due to low temperatures, especially in winter, often makes the application of microorganisms difficult. In particular, at low winter temperatures, the rate of oil decomposition by microorganisms is extremely slow, and it is considered impossible to treat wastewater or waste using specific microorganisms. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Journal of Bioscience and Biotechnology,Vol.107,No.4 401-408,2009 [Overview of the Initiative] [Means for solving the problem]

[0006] As a result of diligent research, the inventors have discovered a novel microorganism belonging to the Burkholderia family that decomposes trans fatty acid-containing oils and fats. It was also found that this microorganism can efficiently decompose oils and / or fatty acids even at low temperatures of around 15°C. This disclosure also relates to the applications of this microorganism, such as oil treatment.

[0007] This disclosure provides a novel microorganism having the ability to decompose oils and fats, and a method for decomposing oils and / or fatty acids using this microorganism.

[0008] Therefore, this disclosure provides the following: (Item 1) A bacterium of the family Burkholderiae that has the ability to utilize trans fatty acid-containing oils. (Item 2) A bacterium of the Burkholderia family that has the ability to utilize trans fatty acids. (Item 3) A bacterium of the family Burckholderia that has the ability to break down trans fatty acids. (Item 4) A Burkholderia bacterium having the ability to decompose oils and fats containing trans fatty acids. (Item 5) A Burkholderia bacterium having the ability to decompose oils and fats at 15°C. (Item 6) The Burkholderia bacterium according to any one of Items 1 to 4, wherein the ability to assimilate or decompose is retained at 15°C. (Item 7) The bacterium according to any one of Items 1 to 6, which is a bacterium of the genus Burkholderia. (Item 8) The bacterium according to any one of Items 1 to 7, which is a microorganism belonging to Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex. (Item 9) The bacterium according to any one of Items 1 to 8, which is the Burkholderia bacterium KH-1 strain (the strain specified by the accession number NITE BP-02731), KH-1AL1 strain (the strain specified by the accession number NITE ABP-02977), KH-1AL2 strain (the strain specified by the accession number NITE ABP-02978), or KH-1AL3 strain (the strain specified by the accession number NITE ABP-02979), or an induced strain thereof, and the induced strain has the characteristics of the bacterium according to any one or more of Items 1 to 7. (Item 10) An oil decomposing agent containing the bacterium according to any one of Items 1 to 9. (Item 11) The oil decomposing agent according to Item 10, further containing an additional oil treatment component. (Item 12) An oil decomposition kit comprising the bacterium according to any one of Items 1 to 9, or the oil decomposing agent according to Item 10, and an additional oil treatment component. (Item 13) An oil decomposition and removal method including allowing the bacterium according to any one of Items 1 to 9, or the oil decomposing agent according to Item 10 or 11, to act on a treatment target. (Item 14) The method according to item 13, wherein the subject of processing includes trans fatty acids or oils and fats containing trans fatty acids.

[0009] In this disclosure, the one or more of the above features are intended to be provided in combinations other than those explicitly stated. Further embodiments and advantages of this disclosure will be apparent to those skilled in the art, by reading and understanding the detailed description below as necessary. [Effects of the Invention]

[0010] The microorganisms and compositions containing the same, in that they can decompose trans fatty acids and oils containing trans fatty acids, are particularly capable of treating oil-containing materials such as wastewater discharged from food factories, can handle a wide range of oil concentrations, and can be used at lower temperatures than conventional methods. Therefore, they are applicable to a wide range of situations, including the remediation of environmental pollution caused by oils, food waste treatment, composting, wastewater treatment, and other waste treatment and composting processes.

[0011] This disclosure can also solve the problem of difficult-to-decompose oils and fats, i.e., oil types. The microorganisms and compositions containing the same of this disclosure can decompose trans fatty acids and the oils containing them that are produced in the hydrogenation process of oils and fats, and in particular can process margarine, fat spreads, shortening, etc., which contain large amounts of such fatty acids and oils, and which could not be processed by conventional microorganisms. In particular, this disclosure provides the effect of achieving trans fatty acid decomposition, which can be used at a practical level as bacteria that will be the main microorganisms for wastewater treatment and waste treatment. Furthermore, the microorganisms and compositions containing the same of this disclosure can enable the decomposition of oils and fatty acids at low temperatures. [Brief explanation of the drawing]

[0012] [Figure 1] The results of culturing the Burkholderia arboris KH-1 strain on a canola oil-supplemented medium at 15°C for 5 days are shown. [Figure 2]This shows a comparison of the elaidic acid decomposition activity of KH-1 and BioRemove3200 (BR3200) (Novozymes, Denmark). The samples were cultured at 28°C and 130 rpm for 24 hours. (A) This is a photograph of fatty acid analysis in the culture medium by TLC. The left column is KH-1, and the right column is BR3200. (B) This shows the results of measuring the oil content equivalent to n-hexane using an oil content measurement reagent kit. The left column is BR3200, and the right column is KH-1. Error bars indicate the standard deviation. [Figure 3A] This shows the elaidic acid-containing lipid (trielaidin) degradation activity of KH-1. The culture was performed at 28°C and 130 rpm for 5 days. The image shows the results of TLC analysis of glycerides and fatty acids in the culture medium. The left column shows the results for TBS buffer, and the right column shows the results for KH-1. [Figure 3B] This shows the elaidic acid-containing lipid (trielaidin) degradation activity of the culture supernatant of KH-1. Incubation was performed at 28°C and 130 rpm for 24 hours. The images show the results of TLC analysis of glycerides and fatty acids in the solution. The left image shows the results for TBS buffer, and the right image shows the results for KH-1. [Figure 4] This paper compares the oil decomposition capabilities of KH-1 and BioRemove3200 (BR3200) using actual wastewater. Samples were cultured at 28°C and taken after 24 and 48 hours. (A) Photographs of residual oil content in wastewater analyzed by TLC (left: 24 hours after start of culture, right: 48 hours after start of culture). Each column, from left to right, shows the results for no microorganism addition, BR3200 addition, and KH-1 addition. (B) Results of measuring oil content equivalent to n-hexane value using an oil content measurement reagent kit. From left to right, the results are shown for no microorganism addition, BR3200 addition, and KH-1 addition, respectively. [Figure 5A] This shows the decomposition of canola oil by KH-1 under incubation at 15°C (5L volume fermenter, 250 rpm, 200 ml / min air reflux, pH 7.0). The oil content equivalent to n-hexane was measured using an oil content measurement reagent kit. From left to right, the results are shown at 0, 24, 48, and 72 hours after the start of incubation, respectively. The vertical axis shows the percentage with the 0-hour measurement set to 100%, along with the standard deviation. [Figure 5B] This shows the degradation of canola oil by KH-1 under incubation at 15°C (5L volume fermenter, 250 rpm, 200 ml / min air reflux, pH 7.0). The total fatty acids (sum of fatty acids in triglycerides and free fatty acids) at 0, 24, 48, and 72 hours after the start of incubation are quantified by gas chromatography. Error bars indicate the standard deviation. [Figure 5C] This shows the ability of the KH-1 strain to assimilate elaidic acid or trielaidin under 15°C culture. The results are from culturing for 5 days at 15°C in a medium with elaidic acid or trielaidin as the sole carbon source. From left to right, the images show elaidic acid medium with KH-1 added, elaidic acid medium without microorganisms, trielaidin medium with KH-1 added, and trielaidin medium without microorganisms. [Figure 6A] This shows the resolution of KH-1 for oils (canola oil) under 28°C culture. The image is a photograph of the residual oil content in the culture medium analyzed by thin-layer chromatography (TLC). Each column, from left to right, shows the results at the start of culture, 24 hours after the start of culture, and 48 hours after the start of culture. [Figure 6B] This shows the lipid-degrading ability of KH-1 under 28°C incubation. The total fatty acids (sum of fatty acids in triglycerides and free fatty acids) were quantified by gas chromatography at 0, 24, and 48 hours after the start of incubation. Error bars indicate the standard deviation. [Figure 6C] This shows the lipid decomposition capacity of KH-1 under 28°C incubation. The oil content equivalent to n-hexane values ​​was measured using an oil content measurement reagent kit. From left to right, the results are shown for 0 hours, 24 hours, and 48 hours after the start of incubation, respectively. The vertical axis shows the percentage with the standard deviation, with the measurement value at 0 hours set to 100%. [Figure 7]This shows a comparison between KH-1 and detergents. The photos show a range hood filter with grease stains soaked in KH-1 culture supernatant, an oil-removing detergent, and a general detergent. The left image shows the filter before treatment. The middle and right columns show the results as follows: top row: KH-1 treatment (30 minutes in the middle column, 1 hour in the right column), middle row: oil-removing detergent treatment (2 hours in the middle column, 4 hours in the right column), and bottom row: general detergent treatment (2 hours in the middle column, 4 hours in the right column). [Figure 8] This shows a comparison of the palmiteridic acid and vaccenic acid degradation activities of KH-1 and BioRemove3200 (BR3200) (Novozymes, Denmark). The images show the fatty acid analysis of the culture medium by TLC after incubation at 15°C for 48 or 72 hours. From left to right, the panels represent palmiteridic acid cultured for 48 hours, vaccenic acid cultured for 48 hours, palmiteridic acid cultured for 72 hours, and vaccenic acid cultured for 72 hours. In each panel, the left is the negative control (BS), the center is BR3200, and the right is KH-1. [Figure 9] This shows a comparison of the palmiteridic acid and vaccenic acid degradation activities of KH-1 and BioRemove3200 (BR3200) (Novozymes, Denmark). The images show the fatty acid analysis of the culture medium by TLC after incubation at 28°C for 24 hours. The left panel shows the palmiteridic acid culture, and the right panel shows the vaccenic acid culture. In each panel, the left is the negative control (BS), the center is BR3200, and the right is KH-1. [Figure 10] This shows the degradation of canola oil by KH-1AL1 under 15°C incubation. Results are shown at 0, 24, 48, 72, and 96 hours after the start of incubation. The left image is a photograph of residual oil content in the culture medium analyzed by TLC. The upper right image shows the result of quantifying total fatty acids (sum of fatty acids in triglycerides and free fatty acids) by gas chromatography, with the total fatty acid concentration on the vertical axis (error bars indicate standard deviation). The lower right image shows the result of measuring oil content equivalent to n-hexane value using an oil content measurement reagent kit, with the vertical axis showing the percentage of residual oil content when the measurement value at 0 hours is set to 100% (error bars indicate standard deviation). [Figure 11]This shows the degradation of canola oil by KH-1AL2 in a 15°C culture. Results are shown at 0, 24, 48, 72, and 96 hours after the start of culture. The left image is a photograph of residual oil content in the culture medium analyzed by TLC. The upper right image shows the result of quantifying total fatty acids (sum of fatty acids in triglycerides and free fatty acids) by gas chromatography, with the total fatty acid concentration on the vertical axis (error bars indicate standard deviation). The lower right image shows the result of measuring oil content equivalent to n-hexane value using an oil content measurement reagent kit, with the vertical axis showing the percentage of residual oil content when the measurement value at 0 hours is set to 100% (error bars indicate standard deviation). [Figure 12] This shows the degradation of canola oil by KH-1AL3 under 15°C culture. Results are shown at 0, 24, 48, 72, and 96 hours after the start of culture. The left image is a photograph of residual oil content in the culture medium analyzed by TLC. The upper right image shows the result of quantifying total fatty acids (sum of fatty acids in triglycerides and free fatty acids) by gas chromatography, with the total fatty acid concentration on the vertical axis (error bars indicate standard deviation). The lower right image shows the result of measuring oil content equivalent to n-hexane value using an oil content measurement reagent kit, with the vertical axis showing the percentage of residual oil content when the measurement value at 0 hours is set to 100% (error bars indicate standard deviation). [Figure 13] These images show the TLC analysis of triellidine degradation in the culture supernatants of KH-1, KH-1AL1, KH-1AL2, and KH-1AL3 cultured at 28°C. From left to right, the results are shown for the sterile group (control), KH-1, KH-1AL1, KH-1AL3, and KH-1AL2, respectively. [Figure 14] This shows the assimilation and degradation of elaidic acid by KH-1, KH-1AL1, KH-1AL2, and KH-1AL3 under 28°C incubation. The left image is a photograph of residual elaidic acid in the culture medium analyzed by TLC. The right image shows the results of measuring oil content equivalent to n-hexane using an oil content measurement reagent kit, with the residual oil concentration (mg / L) shown on the vertical axis. From left to right, the results are shown for the sterile group (control), KH-1, KH-1AL1, KH-1AL3, and KH-1AL2, respectively (error bars indicate standard deviation). [Figure 15] This shows the assimilation and degradation of elaidic acid by KH-1, KH-1AL1, KH-1AL2, and KH-1AL3 under 15°C incubation. The left image is a photograph of residual elaidic acid in the culture medium analyzed by TLC. The right image shows the results of measuring oil content equivalent to n-hexane using an oil content measurement reagent kit, with the residual oil concentration (mg / L) on the vertical axis. From left to right, the results are shown for the sterile group (control), KH-1, KH-1AL1, KH-1AL2, and KH-1AL3, respectively (error bars indicate standard deviation). [Modes for carrying out the invention]

[0013] The present disclosure is described below in best form. Throughout this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified. Accordingly, singular articles (e.g., "a," "an," "the" in English) should be understood to include the concept of their plural form unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0014] The following provides definitions of terms used specifically in this specification and / or basic technical concepts as appropriate.

[0015] (Definition, etc.) In this specification, "lipase" refers to a type of esterase that reversibly catalyzes the hydrolysis of neutral fats (glycerol esters) into fatty acids and glycerol. For example, triglycerol lipase, classified as EC3.1.1.3 by enzyme number (EC number), is an example of lipase.

[0016] In this specification, "oils and fats" refers to oily substances, and oils and fats include ester group-containing compounds formed by the dehydration condensation of a hydroxyl group-containing compound and a fatty acid. Typically, this hydroxyl group-containing compound is glycerin, but other examples include polyglycerin. In the same sense as commonly used in this art, in this specification, an ester group-containing compound formed by the dehydration condensation of glycerin and a fatty acid is called a "glyceride." If this hydroxyl group-containing compound has multiple hydroxyl groups, it falls under the definition of an ester group-containing compound in this specification if at least one of those hydroxyl groups dehydrates and condenses with a fatty acid to form an ester.

[0017] Oils and fats are found, for example, in kitchen wastewater from the food service industry and wastewater from food processing plants. Grease traps and pressurized flotation separators, which are treatment facilities that remove oils by solid-liquid separation, are sources of foul odors and pests. Considering the labor and costs involved in the recovery, transportation, and cleaning of separated oils, as well as the cost of coagulants, the microorganisms or compositions of this disclosure are used to eliminate oil in grease traps and factory wastewater treatment facilities.

[0018] This disclosure provides microbial formulations for grease traps and industrial wastewater containing the oleolytic bacteria described herein. In particular, when applied to industrial wastewater, it can reduce or even replace the operating rate of pressurized flotation separators. Food processing plant wastewater often contains trans fatty acids, which are frequently not completely removed and can cause problems as residual contamination; however, the microorganisms described herein can address these issues as well. Furthermore, kitchen wastewater in the food service industry typically contains high concentrations of oleolytics, sometimes exceeding 1 g / L, and sometimes exceeding 10 g / L. Moreover, the retention time of wastewater in many grease traps is extremely short, around 10 minutes; however, the microorganisms described herein can be used even in such environments.

[0019] Oils and fats are abundant in food waste, livestock waste, and sludge from wastewater treatment plants. Many of these contain trans fatty acids. Microorganisms are often used to treat such solid waste, but treatment becomes difficult when the oil content is high, or oil may remain. The microorganisms or compositions of this disclosure can also be applied to the decomposition treatment of oil in such waste.

[0020] In this specification, "fatty acid" refers to a compound having 2 to 100 carbon atoms and at least one carboxyl group. Typically, the carbon chain in a fatty acid is a straight chain, but it may be branched or contain a ring. Typically, a fatty acid contains one carboxyl group, but it may contain multiple carboxyl groups. The carbon chain in a fatty acid may contain a C=C double bond. "Trans fatty acid" is used in the sense commonly used in the art and refers to an unsaturated fatty acid having a trans double bond. In this specification, "trans fatty acid-containing oil" refers to a compound formed by the dehydration condensation of a trans fatty acid and a compound containing a hydroxyl group. Trans fatty acids include elaidic acid and vaccenic acid, but there are no particular restrictions on the type of trans fatty acid when referred to in this specification. The proportion of trans fatty acids present in a trans fatty acid-containing oil is not particularly limited. "Trans" and "cis" double bonds are used in the sense commonly used in the art and refer to the following structure in which four substituents (R1, R2, R3, and R4) are bonded to the two carbon atoms forming the double bond. [ka] In this context, when R1 and R2 or R3 and R4 are non-hydrogen groups and the remaining two substituents are hydrogen atoms, it is called the cis type, and when R1 and R4 or R3 and R2 are non-hydrogen groups and the remaining two substituents are hydrogen atoms, it is called the trans type. Trans fatty acids exist in nature in trace amounts as conjugated linoleic acid and vaccenic acid, and are relatively abundant in the fats of ruminant animals, for example. Trans fatty acids can be produced during the hydrogenation process to produce saturated fatty acids from unsaturated fatty acids, and during the refining of vegetable oils that are rich in unsaturated fatty acids. Therefore, margarine, fat spreads, shortening, etc., may contain relatively high amounts of trans fatty acids.

[0021] In this specification, the "n-Hex value" refers to the amount of non-volatile substances extracted by n-hexane, and is an indicator of the amount of oil (fats and oils, their hydrolysis products, etc.) in water. The n-Hex value can be determined, for example, according to JIS K 0102. It can also be determined using a simple measurement reagent kit for polynipamp extractant measurement.

[0022] In this specification, "assimilation" means using a substance as a source of nutrition, and the substance that is assimilated (e.g., fats and oils) will be lost as a result.

[0023] In this specification, when used in reference to fats and / or fatty acids, "decomposition" means that the fat and / or fatty acid in question becomes smaller molecules, for example, by separating into glycerol and (free) fatty acids. Decomposition also refers to the conversion of fatty acids into fatty acids with fewer carbon atoms, or even into carbon dioxide and water.

[0024] In this specification, "ability to assimilate trans fatty acid-containing oils and fats" refers to the activity of assimilating trans fatty acid-containing oils and fats. In this specification, "to assimilate trans fatty acid-containing oils and fats" is used in the sense commonly used in this art, and means that microorganisms take in trans fatty acid-containing oils and fats as a nutrient source such as a carbon source. "Assimilation" includes not only hydrolysis into glycerol and free fatty acids, but also transformation into part of other substances. The ability to assimilate trans fatty acid-containing oils and fats can be measured and identified by the following tests. • A test to determine whether growth is possible in a culture medium containing trans fatty acid-containing oils as the sole carbon source. • A test to determine whether colonies form in a culture medium containing trans fatty acid-containing oils as the sole carbon source. • A test to measure the decrease in n-hexane levels in the culture supernatant as growth occurs. This test involves converting all fatty acids (the sum of fatty acids in the oil and free fatty acids) in the culture supernatant during growth to methyl esters, and then measuring the total amount by gas chromatography. • A test to measure the amount of oils and free fatty acids in the culture supernatant as growth progresses using thin-layer chromatography. Individual, more detailed measurement methods are provided herein, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0025] In this specification, "ability to decompose trans fatty acid-containing oils and fats" refers to the activity of hydrolyzing trans fatty acid-containing oils and fats into glycerol and free fatty acids. The ability to decompose trans fatty acid-containing oils and fats can be measured and identified by the following tests. • A test to confirm whether or not the organism has the ability to utilize trans fatty acid-containing oils and fats. • A test to measure the amount of oils and free fatty acids in the culture supernatant using thin-layer chromatography. • A test in which trans fatty acid-containing oils are used as a carbon source for cultivation, and the decrease in the n-hexane level in the culture supernatant is measured. • A test to measure the concentration of free fatty acids in the culture supernatant using gas chromatography. • A test to measure the concentration of free fatty acids in the culture supernatant using GC-MS. • A test to measure the concentration of free fatty acids in the culture supernatant using HPLC. This test checks whether a clear zone is observed around colonies formed on an agar medium containing trans fatty acid-containing oils. • A test to prepare a water sample containing trans fatty acid-containing oils as the main organic matter (for example, 70% or more by weight of the total organic matter) and measure its biochemical oxygen demand (BOD). Individual, more detailed measurement methods are provided herein, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0026] In this specification, "ability to assimilate trans fatty acids" refers to the ability to assimilate trans fatty acids. The ability to assimilate trans fatty acids can be measured and identified by the following tests. • A test to determine whether growth is possible in a culture medium containing trans fatty acids as the sole carbon source. • A test to determine whether colonies form in a culture medium containing trans fatty acids as the sole carbon source. • A test to measure the decrease in n-hexane levels in the culture supernatant as growth occurs. • A test to measure the concentration of trans fatty acids in the culture supernatant using gas chromatography as growth progresses. • A test to measure the concentration of trans fatty acids in the culture supernatant using HPLC as growth progresses. • A test to measure the amount of trans fatty acids in the culture supernatant as growth progresses using thin-layer chromatography. Individual, more detailed measurement methods are provided herein, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0027] In this specification, "ability to break down trans fatty acids" refers to the ability to break down trans fatty acids. The ability to break down trans fatty acids can be measured and identified by the following tests. • A test to determine whether or not a person has the ability to utilize trans fatty acids. • A test to measure the amount of trans fatty acids in the culture supernatant using thin-layer chromatography. • A test in which trans fatty acids are used as a carbon source for cultivation, and the decrease in n-hexane levels in the supernatant is measured. • A test to measure the concentration of trans fatty acids in the culture supernatant using gas chromatography. • A test to measure the concentration of trans fatty acids in the culture supernatant using GC-MS. • A test to measure the concentration of trans fatty acids in the culture supernatant using HPLC. • A test to check whether a clear zone is observed around colonies formed on an agar medium containing trans fatty acids. • A test to prepare a water sample containing trans fatty acids as the main organic substance (for example, 70% or more by weight of the total organic substance) and measure its biochemical oxygen demand (BOD). Individual, more detailed measurement methods are provided herein, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0028] In this specification, "ability to decompose fats and oils at 15°C" refers to the activity of hydrolyzing fats and oils into glycerol and free fatty acids at low temperatures. The ability to decompose fats and oils at 15°C can be measured and identified by the following tests. It is sufficient for decomposition ability to be demonstrated in any of the following tests; decomposition must not necessarily be observed in all tests. A test to confirm whether the substance has the ability to utilize fats and oils at 15°C. This test checks whether a clear zone is observed around colonies formed on an agar medium containing oil at 15°C. • A test in which oils and fats are used as a carbon source, cultured at 15°C, and the decrease in the n-hexane level in the culture supernatant is measured. This test involves culturing at 15°C using oil as a carbon source and measuring the time-dependent changes in the amount of oil and free fatty acids in the culture supernatant using thin-layer chromatography. A decrease in the amount of oil over time indicates decomposition ability. Alternatively, an increase in the amount of free fatty acids indicates decomposition ability. This test involves culturing at 15°C using oil as a carbon source, and measuring the concentration of free fatty acids in the culture supernatant using instrumental analysis such as gas chromatography, GC-MS, and HPLC. An increase in the concentration of free fatty acids indicates the ability to decompose them. • A test to prepare a water sample containing oils and fats as the main organic matter (for example, 70% or more by weight of the total organic matter) and measure its biochemical oxygen demand (BOD). Individual, more detailed measurement methods are provided herein, and those skilled in the art can perform these measurements using any other equipment and conditions.

[0029] In this specification, “oil treatment component” means a component that assists in the assimilation and decomposition of oils and / or fatty acids. Specifically, this includes components that promote the dispersion of oils and / or fatty acids, such as biosurfactants, components that decompose oils into fatty acids and glycerol, as well as components that decompose fatty acids, components that decompose glycerol, and components that adsorb oil and remove it from the object to be treated. In one aspect, the oil treatment component may include biosurfactants produced by the microorganisms of this disclosure.

[0030] In this specification, “oil-degrading agent” means a formulation capable of degrading oils and / or fatty acids, comprising the microorganisms of this disclosure as an active ingredient. In this disclosure, the oil-degrading agent may be used in combination with an oil treatment component. In this case, the timing of the combined use of the oil-degrading agent and the oil treatment component may be simultaneous or one may be used first. Furthermore, the oil-degrading agent may further contain components that enhance the activity of the bacterial strain or lipase derived from the bacterial strain (e.g., carbon source, nitrogen source), surfactants, drying protectants, components for maintaining bacteria for a long period of time, preservatives, excipients, fortifiers, antioxidants, etc.

[0031] As used herein, “inducible strain,” “similar strain,” or “mutant strain” preferably contains a gene (e.g., 16S rDNA) that is substantially homologous to the DNA of the microorganism in question, and such strain has a whole genome sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical when aligned with the whole genome sequence of the original strain using a computer homology program known in the art. This means a microorganism modified by gene mutation, substitution, deletion, and / or addition, in which the induced strain still exhibits the biological functions of the original microorganism, though not necessarily to the same degree. For example, gene mutations can be introduced using any known mutagen, UV, plasma, etc. In one embodiment, the “inducible strain,” “similar strain,” or “mutant strain” is a strain of the same genus and / or species as the original strain. For example, the biological functions of such a microorganism can be investigated by appropriate and available in vitro assays described herein or known in the art.

[0032] In this specification, "homology" of genes refers to the degree of identity between two or more gene sequences, and generally, having "homology" means having a high degree of identity or similarity. Therefore, the higher the homology of two genes, the higher the identity or similarity of their sequences. Whether two types of genes have homology can be investigated by direct comparison of sequences, or, in the case of nucleic acids, by hybridization under stringent conditions. When two gene sequences are directly compared, genes have homology if their DNA sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In this specification, "similarity" of genes or base sequences refers to the degree of similarity between two or more gene sequences, and means having a high degree of similarity between sequences other than identity. "Similarity" is a numerical value that takes into account not only identity but also similar bases, where similar bases refer to cases where some of the mixed bases (for example, R=A+G, M=A+C, W=A+T, S=C+G, Y=C+T, K=G+T, H=A+T+C, B=G+T+C, D=G+A+T, V=A+C+G, N=A+C+G+T) are identical.

[0033] Amino acids may be referred to herein by either their generally known three-letter code or the one-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their generally recognized one-letter codes. In this specification, comparisons of similarity, identity, and homology of amino acid sequences and base sequences are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.7.1 (published October 19, 2017). The value of "identity" in this specification usually refers to the value obtained when aligning using BLAST under default conditions. However, if a higher value is obtained by changing the parameters, the highest value will be used as the identity value. If identity is evaluated in multiple regions, the highest value among them will be used as the identity value. "Similarity" is a numerical value that takes into account similar amino acids in addition to identity.

[0034] In one embodiment of this disclosure, the numerical value of identity, etc., "70% or more" may be, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or more, and may be within the range of any two of the starting numerical values. The above "identity" is calculated by determining the proportion of homologous amino acids or bases in two or more amino acid or base sequences according to the known method described above. Specifically, before calculating the proportion, the amino acids or base sequences of the amino acid or base sequence group to be compared are aligned, and gaps are introduced in parts of the amino acids or base sequences if necessary to maximize the proportion of identical amino acids or bases. Methods for alignment, methods for calculating proportions, methods for comparison, and related computer programs are conventionally well known in the art (e.g., BLAST as described above). In this specification, "identity" and "similarity" may be expressed as values ​​measured by NCBI's BLAST unless otherwise specified. When comparing amino acid or base sequences with BLAST, the Blastp algorithm can be used by default. The measurement results are quantified as Positives or Identities. In this case, when "similarity" is used instead of "identity," the numerical value also takes into account those that meet the definition of "similar" "amines" or "bases" as described herein.

[0035] In this specification, “biological function” refers to a specific function that a microorganism may possess, including, but is not limited to, the breakdown of fats and oils (e.g., breakdown of trans fatty acid-containing fats and oils). In this disclosure, examples include, but is not limited to, the breakdown of trans fatty acid-containing fats and oils, the breakdown of cis fatty acid-containing fats and oils, the breakdown of cis fatty acids, the breakdown of saturated fatty acid-containing fats and oils, and the breakdown of saturated fatty acids. In this specification, biological function can be exerted by the corresponding “biological activity.” In this specification, “biological activity” refers to the activity that a microorganism may possess in a given environment, and includes activities that exert various functions (e.g., activity to break down trans fatty acid-containing fats and oils). Such biological activity can be measured by techniques well known in the art. Therefore, “activity” refers to various measurable indicators that influence the response (i.e., have a measurable effect in response to some exposure or stimulus), and may include, for example, the amount of upstream or downstream proteins or other similar functions of the microorganisms in this disclosure after some stimulus or event.

[0036] As used herein, the “amount” of an analyte in a sample generally refers to an absolute value that reflects the mass of the analyte detectable in the volume of the sample. However, the amount may also refer to a relative amount compared to another analyte. For example, the amount of an analyte in a sample may be greater than the control level or normal level of the analyte normally present in the sample.

[0037] The term "approximately" refers to a range of plus or minus 10% from the given value.

[0038] In this specification, “kit” means a unit in which the parts to be provided (e.g., a composition containing the microorganisms of this disclosure, additional components, buffers, instructions, etc.) are provided, usually divided into two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided mixed for stability or other reasons, but is preferably mixed immediately before use. Such a kit is preferably advantageous to include instructions or a manual describing how to use or handle the provided parts (e.g., a composition containing the microorganisms, additional components, etc.). When a kit is used in this specification, the kit usually includes instructions describing how to use the microorganisms or compositions of this disclosure.

[0039] In this specification, “Instructions” are instructions for the user on how to use the Disclosure. These Instructions contain language that directs the use of the Disclosure. If necessary, these Instructions will be prepared in accordance with the format prescribed by the supervisory authority in the country where the Disclosure is implemented (e.g., the Ministry of Health, Labour and Welfare or the Ministry of Agriculture, Forestry and Fisheries in Japan, or the Food and Drug Administration (FDA) or the Department of Agriculture (USDA) in the United States) and will be clearly stated to have been approved by that supervisory authority. Instructions may, but are not limited to, be provided in paper form, and may also be provided in electronic form, for example, on a website provided on the Internet, or by email.

[0040] (Preferred embodiment) Preferred embodiments of the Disclosure are described below. The embodiments provided below are provided for a better understanding of the Disclosure, and it will be understood that the scope of the Disclosure should not be limited to the descriptions below. Accordingly, it will be obvious that those skilled in the art can make appropriate modifications within the scope of the Disclosure, taking into consideration the descriptions herein. It will also be understood that the embodiments of the Disclosure below can be used individually or in combination.

[0041] (Oil-degrading microorganisms) In one aspect, the present disclosure provides novel microorganisms capable of degrading lipids. In particular, the microorganisms of the present disclosure have one or more characteristics, including the ability to assimilate trans fatty acid-containing lipids, the ability to assimilate trans fatty acids, the ability to degrade trans fatty acids, the ability to degrade trans fatty acid-containing lipids, and / or the ability to assimilate and / or degrade lipids and / or fatty acids at 15°C.

[0042] In one embodiment, the microorganism of the disclosure is a bacterium of the Burkholderiaceae family. In one embodiment, the microorganism of the disclosure is a bacterium of the Burkholderia genus. The genus Burkholderia is a Gram-negative, non-spore-forming, aerobic, polar-flagellated rod-shaped bacterium and is the type genus of the Burkholderiaceae family. In one embodiment, the microorganism of the disclosure is Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia, preferably Burkholderia arboris or Burkholderia ambifaria. In one embodiment, the microorganism of the disclosure is a microorganism belonging to the Burkholderia cepacia complex. The Burkholderia cepacia complex is a classification of microorganisms belonging to the genus Burkholderia that are genetically very close, and includes amphifaria, anthina, arboris, cenocepacia, cepacia, contaminans, diffusa, dolosa, lata, latens, metallica, multivorans, pseudomultivorans, puraquae, pyrrocinia, semialis, stabilis, stagnalis, territorii, ubonensis, and vietnamiensis (Martina P et al., Int J Syst Evol Microbiol. 2018 Jan;68(1):14-20.). In another embodiment, the Burgholderia bacteria of the present disclosure may be metallica, seminalis, anthina, amphifaria, diffusa, ubonensis, multivorans, latens, cenocepacia, vietnamiensis, pyrrocinia, stabilis, glumae, gladioli, plantarii, oklahomensis, thailandensis, mallei, pseudomallei, or phytofirmans.The inventors identified a new microorganism (KH-1) as Burgholderia arboris by sequencing its 16S ribosomal DNA and performing phylogenetic analysis. This microorganism was deposited with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE) as an international deposit under the Budapest Convention, and was received on June 4, 2018, with a deposit certificate issued on June 12, 2018. The deposit number is NITE ABP-02731. Furthermore, the inventors identified other bacteria of the genus Burgholderia (KH-1AL1, KH-1AL2, and KH-1AL3) and deposited them with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE) as an international deposit under the Budapest Convention, and were received on June 26, 2019. The deposit numbers are NITE ABP-02977, NITE ABP-02978, and NITE ABP-02979, respectively. Furthermore, certificates of acceptance for KH-1AL1, KH-1AL2, and KH-1AL3 were issued on July 8, 2019, and were assigned the respective acceptance numbers NITE BP-02977 (acceptance number NITE ABP-02977), NITE BP-02978 (acceptance number NITE ABP-02978), and NITE BP-02979 (acceptance number NITE ABP-02979). In one embodiment, the microorganism of the present disclosure is Burgholderia strain KH-1 (the strain identified by accession number: NITE BP-02731 / receipt number NITE ABP-02731), strain KH-1AL1 (the strain identified by receipt number NITE ABP-02977), strain KH-1AL2 (the strain identified by receipt number NITE ABP-02978), or strain KH-1AL3 (the strain identified by receipt number NITE ABP-02979), or an derived strain thereof.

[0043] In one embodiment, the microorganism of the present disclosure is an derived strain of Burkholderia strain KH-1 (the strain identified by accession number NITE BP-02731 / acceptance number NITE ABP-02731), KH-1AL1 (the strain identified by acceptance number NITE ABP-02977), KH-1AL2 (the strain identified by acceptance number NITE ABP-02978), or KH-1AL3 (the strain identified by acceptance number NITE ABP-02979). Here, the derived strain does not need to be a strain obtained from Burkholderia strain KH-1, KH-1AL1, KH-1AL2, or KH-1AL3, but refers to a microorganism that exhibits the biological functions of these Burkholderia strains, although not necessarily to the same degree. In one embodiment, the microorganism that is an derived strain of the present disclosure exhibits a biological function selected from the group consisting of the ability to assimilate (decompose) oils and fats (e.g., trans fatty acid-containing oils and fats) at low temperatures (e.g., below 25°C, below 20°C, below 15°C, below 10°C, below 5°C, etc.) and the ability to assimilate (decompose) trans fatty acids, similar to the Burgholderia strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3, but the degree of its biological function may differ from that of the KH-1, KH-1AL1, KH-1AL2, or KH-1AL3 strains. In one embodiment, the microorganism that is an inducement of the present disclosure is a bacterium of the family Burkholderiaceae, more specifically a bacterium of the genus Burkholderia, and even more specifically a microorganism belonging to Burkholderia arboris, Burkholderia ambifaria, or Burkholderia cepacia complex.

[0044] The microorganisms of this disclosure (including derivative strains of KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) may be isolated on inorganic salt agar medium containing oil as the sole carbon source and adjusted to a pH of 6-8. In one embodiment, the microorganisms of this disclosure (including derivative strains of KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) may be distinguishable by observing the formation of a clear zone (halo) around the colonies that have formed on the agar medium.

[0045] In one embodiment, the microorganisms of the Disclosure (including derivatives of strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) have the ability to assimilate trans fatty acids or trans fatty acid-containing oils and fats. In one embodiment, the microorganisms of the Disclosure (including derivatives of strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) have the ability to degrade trans fatty acids. In one embodiment, the microorganisms of the Disclosure (including derivatives of strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) have the ability to degrade trans fatty acid-containing oils and fats.

[0046] In one embodiment, the microorganisms of the Disclosure (including derivatives of strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) have the ability to decompose oils and / or fatty acids at 15°C. In one embodiment, the microorganisms of the Disclosure (including derivatives of strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) retain the ability to assimilate or decompose trans fatty acids or oils containing trans fatty acids at 15°C.

[0047] In one embodiment, the microorganisms of this disclosure (including derivatives of strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) may secrete biosurfactants when cultured in a medium containing oils or fatty acids.

[0048] In one embodiment, the microorganisms of this disclosure (including derivative strains of KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) are placed in an inorganic salt medium containing 10 g / L of canola oil, with a final concentration of OD 660 When microorganisms are inoculated at a density of 0.03 or 0.02 and cultured under 200 ml / min of air reflux at pH 7.0 and 15°C, the lipid-degrading ability is such that the oil content equivalent to n-hexane in the supernatant after 24 or 48 hours is less than 9 g / L, less than 8 g / L, less than 7 g / L, less than 6 g / L, less than 5 g / L, less than 4 g / L, less than 3 g / L, less than 2 g / L, less than 1 g / L, less than 0.7 g / L, less than 0.5 g / L, less than 0.2 g / L, less than 0.1 g / L, less than 0.07 g / L, less than 0.05 g / L, less than 0.02 g / L, or less than 0.01 g / L. In particular, the microorganisms of this disclosure (including derivative strains of the KH-1 strain) have lipid-degrading ability when judged under these conditions. 660 It is preferable that the OD has the ability to degrade oils, reducing the oil content to less than approximately 6 g / L, equivalent to the n-hexane value, when inoculated at a density of 0.02 and cultured for 48 hours. 660 It is particularly preferable that the microorganisms possess a lipid-degrading ability that reduces the oil content equivalent to the n-hexane value to less than 6 g / L when inoculated at a density of 0.03 and cultured for 24 hours. Microorganisms with such low-temperature lipid-degrading properties can be usefully used in various applications of this disclosure.

[0049] In one embodiment, the microorganisms of the present disclosure (including derivatives of strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) may have a lipid-degrading ability to assimilate and / or degrade trans fatty acid-containing oils in a medium at pH 7.0 and 15°C or 28°C containing trans fatty acid-containing oils (e.g., elaidic acid, palmiteraisic acid, vaccenic acid).

[0050] In one embodiment, the microorganisms of this disclosure (including derivatives of strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) are fed into an inorganic salt medium containing Triton X-100 and elaidic acid at concentrations of 0.25% by weight and 0.2% by weight, respectively, with a final concentration of OD. 660When microorganisms are inoculated at a density such that =0.08 and cultured at pH 7.0 and 15°C, the elaidic acid concentration in the supernatant after 72 hours is less than 0.15%, less than 0.12%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.015%, less than 0.01%, less than 0.007%, less than 0.005%, less than 0.002%, less than 0.001%, less than 0.0007%, less than 0.0005%, less than 0.0002%, or less than 0.0001% by weight, indicating that the microorganism has the ability to decompose trans fatty acids. The microorganisms of this disclosure (including derivatives of strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) preferably have the ability to reduce the elaidic acid concentration in the supernatant to less than 0.15%, less than 0.12%, less than 0.1%, or less than 0.05%, particularly to less than 0.12%, when judged under these conditions. Microorganisms with such trans fatty acid degrading properties can be usefully used in various applications of this disclosure.

[0051] In one embodiment, the microorganisms of this disclosure (including derivatives of strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) are fed into an inorganic salt medium containing Triton X-100 and elaidic acid at concentrations of 0.25% by weight and 0.2% by weight, respectively, with a final concentration of OD. 660When microorganisms are inoculated at a density such that =0.04 and cultured at pH 7.0 and 28°C, the elaidic acid concentration in the supernatant after 24 hours is less than 0.15%, less than 0.12%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.015%, less than 0.01%, less than 0.007%, less than 0.005%, less than 0.002%, less than 0.001%, less than 0.0007%, less than 0.0005%, less than 0.0002%, or less than 0.0001% by weight, indicating that the organism has the ability to decompose trans fatty acids. The microorganisms of this disclosure (including derivatives of strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) preferably have the ability to reduce the elaidic acid concentration in the supernatant to less than 0.15%, less than 0.12%, less than 0.1%, less than 0.05%, less than 0.02%, or less than 0.01%, particularly less than 0.1%, when judged under these conditions. Microorganisms with such trans fatty acid degrading properties can be usefully used in various applications of this disclosure.

[0052] In one embodiment, the microorganisms of this disclosure (including derivative strains of KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) are placed in an inorganic salt medium containing 10 g / L of canola oil, with a final concentration of OD 660When microorganisms are inoculated at a density of 0.03 and cultured under 200 ml / min of air reflux at pH 7.0 at 15°C and 28°C, respectively, the oil content equivalent to n-hexane in the supernatant after 24 hours of culture at 15°C is 1000% or less, 800% or less, 600% or less, 400% or less, 200% or less, 150% or less, 100% or less, 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, or 5% or less compared to the oil content equivalent to n-hexane in the supernatant after 24 hours of culture at 28°C. The microorganisms of this disclosure (including derivative strains of the KH-1 strain) preferably have a lipid degradation ability such that, when judged under these conditions, the lipid retention rate in 15°C culture is 800% or less, 700% or less, 600% or less, 500% or less, 400% or less, and especially 700% or less compared to 28°C culture. Microorganisms with such low-temperature lipid degradation ability can be usefully used in various applications of this disclosure.

[0053] In one embodiment, the microorganisms of this disclosure (including derivative strains of KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) are placed in an inorganic salt medium containing 10 g / L of canola oil, with a final concentration of OD 660 When microorganisms are inoculated at a density of 0.03 and culture is started under 200 ml / min of air reflux at pH 7.0 at 15°C and 28°C, respectively, the total fatty acid degradation rate in the 15°C culture is 1000% or more, 800% or more, 600% or more, 400% or more, 200% or more, 150% or more, 100% or more, 80% or more, 60% or more, 50% or more, 40% or more, 30% or more, 20% or more, 10% or more, or 5% or more compared to the 28°C culture, the microorganisms of this disclosure (including derivative strains of the KH-1 strain) preferably have a lipid degradation ability such that, when judged under these conditions, the total fatty acid degradation rate in the 15°C culture is 50% or more, 40% or more, 30% or more, 20% or more, or 10% or more, particularly 30% or more, compared to the 28°C culture, and microorganisms having such low-temperature lipid degradation ability can be usefully used in various applications of this disclosure.

[0054] In one embodiment, the ability of microorganisms to decompose and assimilate oils and fatty acids can be evaluated by quantifying the fatty acids contained in the oils remaining in the culture medium and the free fatty acids produced by decomposition using gas chromatography. The specific quantification procedure is as follows: First, 1 mL of the culture supernatant is acidified with hydrochloric acid, and 2 mL of chloroform is added. After stirring for 2 minutes, the mixture is centrifuged, and 1 mL of the chloroform layer is transferred to a separate container to evaporate the solvent and concentrate it. 2 mL of methanolysis solution (methanol:sulfuric acid = 17:3) is added, and the mixture is heated at 100°C for 2 hours to methylate the oils and free fatty acids. Subsequently, 2 mL of chloroform and 1 mL of pure water are added, and after stirring, the chloroform layer is analyzed by gas chromatography to quantify the methyl esters of all fatty acids.

[0055] In one embodiment, the ability of microorganisms to decompose and assimilate lipids and fatty acids can be evaluated by analyzing the lipids and fatty acids remaining in the culture medium by thin-layer chromatography (TLC). Specifically, the procedure involves first extracting lipids by adding an equal volume of chloroform to the culture supernatant. 5 μl of this extract is then developed onto a silica gel-coated plate using a developing solvent containing chloroform, acetone, and methanol in a volume ratio of 96:4:1. The plate is then treated with molybdate n-hydrate to color the lipids and / or fatty acids.

[0056] In one embodiment, the ability of microorganisms to decompose and assimilate oils and fatty acids can be evaluated by examining their growth ability in culture media that use each as the sole carbon source.

[0057] In one embodiment, the microorganisms of the present disclosure (including derivatives of strain KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) may or may not be capable of secreting lipase.

[0058] In one embodiment, the microorganisms of this disclosure (including derivatives of strains KH-1, KH-1AL1, KH-1AL2, or KH-1AL3) may be able to grow and decompose lipids under weakly acidic conditions (e.g., pH approximately 5.5 to 6.0).

[0059] In one embodiment, the growth capacity of a microbial organism is expressed as the optical density of the bacterial cells, measured by the absorbance (turbidity) at 660 nm (OD). 660 This can be investigated by methods such as measuring the number of cells (CFU) or measuring the number of colony-forming units (CFU). In the latter method, a fixed amount of the stock solution and diluted solution of the culture medium are spread on an agar plate, and the colonies formed by static culture are counted.

[0060] In one embodiment, the microorganisms of this disclosure may secrete lipase. The lipase secretion ability of the microorganisms can be evaluated by measuring the lipase activity of the culture supernatant obtained by centrifugation of the microbial culture medium. Lipase activity can be determined by performing an enzymatic reaction using 4-nitrophenyl palmitate (4-NPP), an ester of palmitic acid and 4-nitrophenol, as a substrate, and measuring the amount of 4-nitrophenol produced by hydrolysis of the ester by measuring the absorbance at 410 nm. First, 4-NPP (18.9 mg) is added to 3% (v / v) Triton X-100 (12 ml) and dissolved at 70°C to obtain a substrate solution. Place 1 mL of substrate solution, 0.9 mL of deionized water, and 1 mL of 150 mM GTA buffer (prepared to pH 7 by adding NaOH or HCl to 150 mM 3,3-dimethylglutaric acid, 150 mM Tris, and 150 mM 2-amino-2-methyl-1,3-propanediol) into a cell and incubate at 28°C for 5 minutes. Add 0.1 mL of the culture supernatant and measure the value at 410 nm while stirring.

[0061] A person skilled in the art can use the above measurement method appropriately to test derivative strains of KH-1 strain, KH-1AL1 strain, KH-1AL2 strain, or KH-1AL3 strain to obtain a derivative strain having the above-mentioned biological function (and its degree).

[0062] (Composition containing microorganisms) In one aspect, the Disclosure provides a composition comprising the microorganisms of the Disclosure. In one aspect, the Disclosure provides a composition comprising the culture supernatant of the microorganisms of the Disclosure. The microorganisms of the Disclosure can be produced by culturing them by any suitable method. In one embodiment, the composition is an oil degrading agent. In one embodiment, the composition is a fatty acid degrading agent. Treatment with the fatty acid degrading agent of the Disclosure can produce compounds containing fewer carbon atoms than those in fatty acids. In one embodiment, the composition is a trans fatty acid degrading agent.

[0063] (Applicable to) In one embodiment, examples of oils and fats to which the oil-dissolving agent of this disclosure is applied include, but are not limited to, vegetable oils and fats (cottonseed oil, rapeseed oil, soybean oil, corn oil, olive oil, safflower oil, rice oil, sesame oil, palm oil, coconut oil, peanut oil, etc.), animal oils and fats (lard, beef tallow, milk fat, etc.), fish oil, processed products of these oils and fats (margarine, shortening, butter, etc.), insulating oils, lubricating oils, etc. The oils and fats may exist in the form of an emulsion or in a free state.

[0064] Specific embodiments include oils and fats to which the oil-degrading agent of this disclosure is applied, such as oils and fats containing trans fatty acids. Examples of such oils and fats include, but are not limited to, processed products such as oils and fats produced by hydrogenation (margarine, shortening, butter, etc.). Adding hydrogen reduces the number of double bonds in unsaturated fatty acids and increases the proportion of saturated fatty acids, which can result in the formation of trans fatty acids. Trans fatty acids are said to be present in margarine, fat spreads, shortening produced by hydrogenation, as well as in Western-style confectionery such as bread, cakes, donuts, and fried foods that use these as raw materials. In the process of refining oils extracted from plants and fish, they are treated at high temperatures to remove undesirable odors. During this process, trans fatty acids are formed from cis-type unsaturated fatty acids contained in the oil, so refined vegetable oils such as salad oil are also said to contain trace amounts of trans fatty acids.

[0065] The applications of the oil-degrading agents or fatty acid-degrading agents of this disclosure are not particularly limited and include, but are not limited to, industrial wastewater, household wastewater, industrial waste, household waste (such as food scraps), livestock waste, aquaculture farms (and their wastewater), livestock barns (and their wastewater), slaughterhouses (and their wastewater), soil contaminated with oils and fats, water contaminated with oils and fats (such as the sea, ponds, rivers, and drinking water for animals), the body surface of animals, aquariums (for aquaculture, ornamental purposes, etc.), any oil-contaminated products (such as tableware and machine parts), grease traps installed in kitchens, fatbergs, drain pipes, and insulating oil or deteriorated insulating oil leaked from transformers, etc. Any of these applications may include oils and fats containing trans fatty acids and / or trans fatty acids, and the oil-degrading agents or fatty acid-degrading agents of this disclosure can be suitably applied to them. A "grease trap" is a device for separating and collecting oil in wastewater, and typically consists of three tanks. The first tank is equipped with a basket to capture food scraps and leftovers. In the second tank, oil and water are separated. The wastewater, separated from the oil, is sent to a third tank where sedimentary debris is removed. Grease traps are mandatory in commercial kitchens such as restaurants, hospitals, and hotels. When applying to grease traps, a separate decomposition tank may be installed, but it is also possible to directly introduce oil decomposing agents or microorganisms into the grease trap and decompose the grease within the trap itself.

[0066] Because the microorganisms of this disclosure are highly efficient at low temperatures, there may be embodiments in which low-temperature treatment is desirable. For example, industrial wastewater, household wastewater, industrial waste, household waste (such as food scraps), soil contaminated with oils and fats, and water contaminated with oils and fats (such as seawater, ponds, rivers, and drinking water for animals) are examples of materials that are expected to be treated at temperatures below 20°C (e.g., 15°C), and these are preferred examples of materials that can be treated by this disclosure.

[0067] Specifically, this involves introducing or adding formulations or other substances, or installing carriers immobilized with the microorganisms described herein in drainage routes, drainage storage tanks, grease traps, etc. Alternatively, a separate, dedicated decomposition tank may be provided outside the grease trap.

[0068] In one embodiment, the wastewater may include, but is not limited to, wastewater from restaurants, hospitals, hotels, etc., domestic wastewater, industrial wastewater discharged from food processing factories, oil processing factories, and the like.

[0069] (Usage form) The form of the microorganism or composition of the present disclosure may include, for example, a liquid state, a solid state, etc. Examples of the microorganism or composition in a liquid state include a culture solution of the microorganism, and after collecting the microorganism from the culture solution by centrifugation or the like, it is redispersed in water, a buffer solution, a culture solution, or the like. Examples of the microorganism or composition in a solid state include those dehydrated by centrifugation, press compression, etc., those in a paste-like state or mayonnaise-like state similar to the intermediate between solid and liquid, and dried bodies dried (for example, vacuum drying, freeze drying). Examples of the shape of the solid include powder, granule, tablet, etc. Further, the composition may be provided in a state where the microorganism or the culture supernatant is fixed to a carrier.

[0070] In one embodiment, the microorganism or composition of the present disclosure is about 1×10 8 cells / mL, about 1×10 7 cells / mL, about 1×10 6 cells / mL, about 1×10 5 cells / mL, about 1×10 4 cells / mL, about 1×10 3 cells / mL, about 1×10 2 cells / mL or may be added to a liquid to a density of about 10 cells / mL.

[0071] (Applicable environment) The microorganism or composition of the present disclosure can be used under any suitable environment. In one embodiment, the microorganism or composition of the present disclosure can be used in an environment of 0 to 100°C, 5 to 70°C, less than 10 to 50°C, 15 to 40°C, 20 to 35°C, less than 70°C, less than 60°C, less than 50°C, less than 40°C, less than 30°C, less than 25°C, less than 20°C, less than 15°C, less than 10°C, less than 5°C, less than 0°C, about 70°C, about 60°C, about 50°C, about 40°C, about 30°C, about 25°C, about 15°C, about 10°C, about 5°C, or about 0°C.

[0072] In one embodiment, the microorganisms or compositions of the present disclosure may be used in environments with pH 3 to 13, pH 4 to 12, pH 5 to 11, pH 6 to 10, pH 7 to 9, pH 5.5 to 8.5, approximately pH 3, approximately pH 4, approximately pH 5, approximately pH 6, approximately pH 7, approximately pH 8, approximately pH 9, approximately pH 10, approximately pH 11, approximately pH 12, or approximately pH 13.

[0073] In one embodiment, the microorganisms or compositions of the present disclosure may be used in environments with dissolved oxygen (DO) concentrations of 0.05 mg / L or higher, 0.1 mg / L or higher, 0.5 mg / L or higher, or 1 mg / L or higher.

[0074] In one embodiment, the microorganisms or compositions of the present disclosure may be used in wastewater with n-hexane values ​​of 100-40,000 mg / L, 200-30,000 mg / L, and 300-30,000 mg / L. In solid waste (which may contain water), such as sludge slurry or food waste, higher concentrations of oils and fats may be present, and in one embodiment, the microorganisms or compositions of the present disclosure may also be usefully applied to such solid waste.

[0075] In one embodiment, the microorganisms or compositions of the present disclosure may be added to a subject containing 50% or more by weight, 20% or more by weight, 10% or more by weight, 7% or more by weight, 5% or more by weight, 2% or more by weight, 1% or more by weight, 0.7% or more by weight, 0.5% or more by weight, 0.2% or more by weight, 0.1% or more by weight, 0.07% or more by weight, 0.05% or more by weight, 0.02% or more by weight, 0.01% or more by weight, 0.007% or more by weight, 0.005% or more by weight, 0.002% or more by weight, or 0.001% or more by weight of trans fatty acids.

[0076] In one embodiment, the microorganism or composition of the present disclosure may be added to a subject in which trans fatty acids (the sum of free fatty acids and fatty acids in ester group-containing compounds) account for 50% or more by weight, 20% or more by weight, 10% or more by weight, 7% or more by weight, 5% or more by weight, 2% or more by weight, 1% or more by weight, 0.7% or more by weight, 0.5% or more by weight, 0.2% or more by weight, 0.1% or more by weight, 0.07% or more by weight, 0.05% or more by weight, 0.02% or more by weight, 0.01% or more by weight, 0.007% or more by weight, 0.005% or more by weight, 0.002% or more by weight, or 0.001% or more by weight.

[0077] In one embodiment, the microorganisms or compositions to which the present disclosure are added may contain nitrogen in a form available to the microorganisms, preferably in the form of ammonium salts, nitrates, sulfates, or organic nitrogen compounds, more preferably in the form of ammonium sulfate, urea, amino acids, or peptides such as peptones, tryptones, or casamino acids. The amount of nitrogen present may be in the range of C / N = 2 to 50 (where C refers to carbon derived solely from n-Hex), preferably in the range of C / N = 2 to 30, and more preferably in the range of C / N = 2 to 20, where C / N is the weight ratio of n-Hex-derived carbon atoms to nitrogen atoms contained in the wastewater. In one embodiment, nitrogen may be further added to bring the total amount within these ranges.

[0078] In one embodiment, the microorganism or composition to which the present disclosure is added may contain phosphorus (P) in a form available to the microorganism, preferably in the form of a phosphate or nucleic acid, more preferably in the form of a phosphate. The amount of phosphorus present may be such that N / P = 1 to 20 relative to nitrogen, where N / P is the weight ratio of nitrogen atoms to phosphorus atoms contained in the wastewater. In one embodiment, phosphorus may be further added to achieve this range.

[0079] In one embodiment, the microorganisms or compositions of the present disclosure may be used under conditions in which salt, surfactant, light, electric current, stirring, aeration, or any combination thereof is present.

[0080] In one embodiment, the microorganism or composition of the Disclosure may be applied after removing substances that kill or inhibit the growth of the microorganism (such as chlorine or antibiotics).

[0081] In one embodiment, the microorganism or composition of this disclosure may be used together with a carrier capable of immobilizing the microorganism. Using such a carrier can effectively avoid washout. The material of the carrier is not particularly limited as long as it can immobilize the microorganism, and examples include carbon fiber (PAN-based, pitch-based, phenolic resin-based, etc.), polyethylene resin, polypropylene resin, polyurethane resin, polystyrene resin, polyvinyl chloride resin, polyvinyl acetate resin, polyvinyl alcohol resin, polyethylene glycol resin, acrylic resin, gelatin, sodium alginate, carrageenan, dextrin, ceramics, silicon, metal, charcoal, activated carbon, minerals (zeolite, diatomaceous earth, etc.), and composites thereof. It is preferable to use a porous or fibrous carrier in order to increase the immobilization rate and the efficiency of the microorganism's action. Microorganisms may also be contained within a gel-like carrier. Examples of carrier shapes include cubic, rectangular parallelepiped, cylindrical, spherical, disc-shaped, sheet-shaped, and membrane-shaped. For information on microbial immobilization technology, please refer to works such as "Wastewater Treatment by Microbial Immobilization Method (edited by Ryuichi Sudo, Industrial Water Research Association)" and "Water Treatment by Microbial Immobilization Method - Carrier Immobilization Method, Comprehensive Immobilization Method, Biological Activated Carbon Method (New Water Treatment Series (1)) (authored by Kazuhiro Mochizuki, Katsutoshi Hori, and Hideki Tachimoto, NTS Corporation)."

[0082] (Additional ingredients) In one embodiment, the microorganism or composition of the Disclosure may be used in combination with additional components. In one embodiment, the additional components may be added to the composition or used separately from the microorganism or composition, and if used separately, they may be provided as a kit.

[0083] In one embodiment, additional components may include, but are not limited to, components that enhance the activity of the microorganisms used (e.g., carbon sources, nitrogen sources), surfactants, desiccant protectants, components for maintaining the microorganisms for a long period of time, preservatives, excipients, fortifiers, antioxidants, and other microorganisms. Any suitable components may be used.

[0084] In one embodiment, other microorganisms include those having lipid-degrading ability, those producing lipase, those that degrade (assimilate) fatty acids and / or glycerol (which are lipid-degrading products of lipase), and those that degrade (assimilate) proteins, amino acids, nucleic acids, or polysaccharides (e.g., cellulose). It is preferable that the other microorganisms are symbiotic with the microorganisms of this disclosure.

[0085] Examples of lipase-producing microorganisms include bacteria, yeasts, and filamentous fungi, with bacteria and yeasts being preferred, and Gram-positive bacteria and proteobacteria being more preferred. Examples of bacteria include those of the genera Bacillus, Corynebacterium, Rhodococcus, Burkholderia, Acinetobacter, Pseudomonas, Alcaligenes, Rhodobacter, Ralstonia, Acidovorax, Serratia, and Flavobacterium. Examples of proteobacteria include alphabacteria, betabacteria, and gammabacteria.

[0086] As microorganisms that decompose (assimilate) glycerol, for example, eubacteria, yeasts, and filamentous fungi can be used. Preferably, Candida yeast is used. A specific example of Candida yeast is Candida cylindracea SL1B2 strain (deposited with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation under accession number NITE P-714). This strain has excellent glycerol assimilation ability and is also capable of symbiosis with Burkholderia arboris. Therefore, using Candida cylindracea SL1B2 strain in combination is particularly preferable in embodiments where Burkholderia arboris is used. By using microorganisms that decompose (assimilate) glycerol in combination, the decrease in the rate of fat decomposition due to glycerol accumulation can be prevented, and more efficient fat decomposition can be achieved.

[0087] Examples of microorganisms that decompose (assimilate) fatty acids include bacteria, yeasts, and filamentous fungi, with bacteria and yeasts being preferred, and yeasts being more preferred. Examples of yeasts include Yarrowia yeasts, Cryptococcus yeasts, Trichosporon yeasts, and Hansenula yeasts.

[0088] (Methods using microorganisms) In one aspect, the Disclosure provides a method for the decomposition and removal of fats and / or fatty acids, which involves acting the microorganisms or compositions of the Disclosure on a target for treatment. The target for treatment may include trans fatty acids or fats and oils containing trans fatty acids. The target for treatment may be any specified target to which the microorganisms or compositions of the Disclosure can be applied. The method for the decomposition and removal of fats and / or fatty acids of the Disclosure can be carried out in any specified environment to which the microorganisms or compositions of the Disclosure can be applied. The method for the decomposition and removal of fats and / or fatty acids of the Disclosure may use any additional components specified in the Specification that can be used in combination with the microorganisms or compositions of the Disclosure.

[0089] In one embodiment, the method for decomposing and removing oils and / or fatty acids of the present disclosure includes the step of introducing the microorganisms or composition of the present disclosure into an oil decomposition tank, which may be introduced continuously or sequentially. The HRT (hydraulic residence time) of the oil decomposition tank is usually 12 hours or more, preferably 18 hours or more, more preferably 20 hours or more, and even more preferably 24 hours or more. For wastewater with an n-Hex value exceeding 10,000 mg / L, if an 80% or greater reduction in the n-Hex value is expected, the HRT may be usually 18 hours or more, preferably 20 hours or more, and more preferably 24 hours or more. For wastewater with an n-Hex value of 3,000 mg / L or less, if an 80% or greater reduction in the n-Hex value is expected, the HRT may be usually 8 hours or more, preferably 12 hours or more, and more preferably 18 hours or more.

[0090] The microbial concentration in the grease decomposition tank may depend on the grease and / or fatty acid concentration in the wastewater; the higher the grease and / or fatty acid concentration, the higher the microbial concentration can be maintained. If the grease decomposition tank foams, countermeasures such as shortening the HRT, showering, or adding an antifoaming agent can be taken. However, since antifoaming agents can inhibit microbial growth, it is desirable to set the amount added considering this factor.

[0091] The n-Hex value of the effluent from the oil and fat decomposition tank is preferably 60 mg / L or less, more preferably 30 mg / L or less, for low-concentration wastewater with an n-Hex value of approximately 300 mg / L or less of influent water. For medium-concentration wastewater with an n-Hex value of approximately 3000 mg / L of influent water, it is preferably 600 mg / L or less, more preferably 300 mg / L or less, even more preferably 150 mg / L or less, and most preferably 30 mg / L or less. For high-concentration wastewater with an n-Hex value of approximately 10000 mg / L of influent water, it is preferably 1000 mg / L or less, more preferably 500 mg / L or less, even more preferably 100 mg / L or less, and most preferably 30 mg / L or less. For high-concentration wastewater with an n-Hex value of approximately 30000 mg / L or more of influent water, it is preferably 3000 mg / L or less, more preferably 1000 mg / L or less, and even more preferably 300 mg / L or less.

[0092] In one embodiment, the method of this disclosure can reduce the n-Hex value of oil and / or fatty acid-containing wastewater by preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more. As a result, in many wastewaters, it is possible to reduce the n-Hex value of the effluent from the oil and fat decomposition tank to less than 30 mg / L, which is the discharge standard value into sewage systems in many municipalities. If this standard value is achieved, then, focusing solely on the n-Hex value, even subsequent treatments such as activated sludge treatment may become unnecessary.

[0093] The amount of microorganisms introduced into the effluent from the oil and fat decomposition tank does not need to increase. The amount of microorganisms introduced into the effluent is preferably 0.01 times or more, more preferably 0.1 times or more, even more preferably 0.5 times or more, and most preferably 1 time or more, compared to the amount introduced.

[0094] The method for decomposing and removing oils and / or fatty acids according to this disclosure may include additional steps other than those described above. Such steps include, for example, returning all or part of the effluent from the oil decomposition tank back to the oil decomposition tank. However, since the method according to this disclosure can obtain a sufficient oil and / or fatty acid decomposition effect without such a return process, it is not essential to return all or part of the effluent from the oil decomposition tank back to the oil decomposition tank.

[0095] (General technology) The molecular biological, biochemical, and microbiological methods used herein are well-known and commonly used in their respective fields. For example, see Savli, H., Karadenizli, A., Kolayli, F., Gundes, S., Ozbek, U., Vahaboglu, H. 2003. Expression stability of six housekeeping genes: A proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J.Med.Microbiol. 52:403-408. and Marie-Ange Teste, Manon Duquenne, Jean M Francois and Jean-Luc Parrou 2009. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae. BMC Molecular Biology 10:99, Seiji Ishii, Hiroshi Okumura, Chiyo Matsubara, Fumi Ninomiya, and Hiroshi Yoshioka, 2004, "A Simple Method for Measuring Oil Content in Water Using a Heat-Sensitive Polymer," Vol. 46, No. 12, "Water and Wastewater," etc., and relevant parts (possibly all) of these are referenced in this specification.

[0096] (Note) In this specification, "or" is used when "at least one" of the items listed in the text can be adopted. The same applies to "or else". In this specification, when it is specified that "within the range" of "two values", that range includes the two values ​​themselves.

[0097] References such as scientific literature, patents, and patent applications cited herein are incorporated herein by reference to the same extent as they are specifically described herein.

[0098] The present disclosure has been described above with reference to preferred embodiments for ease of understanding. The present disclosure will now be described based on examples, but the above description and the following examples are provided for illustrative purposes only and not to limit the present disclosure. Accordingly, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. [Examples]

[0099] Examples are described below. Where necessary, the handling of organisms used in the following examples complied with the standards stipulated by Nagoya University, the supervisory authority, and the Cartagena Protocol. Specifically, the reagents used were those described in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Fujifilm, Wako Pure Chemical Industries, Nakai, R&D Systems, USCN Life Science INC, Kanto Chemical, Funakoshi, Tokyo Chemical Industries, Merck, etc.) can be substituted.

[0100] (Example 1: Identification of microorganisms capable of assimilating and decomposing trans fatty acid-containing oils and fats) Samples were collected from a river near a food processing plant that discharges oil-containing wastewater, and microorganisms were isolated from them. To investigate whether the isolated microorganisms could be cultured in a low-temperature environment (15°C), each microorganism was streaked onto an agar plate containing canola oil as the sole carbon source and cultured at 15°C for 5 days. As a result, microorganisms that can be cultured at low temperatures using oil as a nutrient source were found.

[0101] Furthermore, the lipid-degrading ability of each microorganism at low temperatures (15°C) was investigated. Colonies of each microorganism were inoculated using a toothpick into 20 mL of inorganic salt medium containing 10 g / L canola oil as the sole carbon source, and then cultured in a 100 mL Erlenmeyer flask. As a result, microorganisms that decompose and assimilate lipids and grow at low temperatures were identified.

[0102] Next, the ability of each microorganism to decompose and assimilate trans fatty acids was investigated. In 2 mL of inorganic salt medium to which elaidic acid was added at a final concentration of 0.2% as the sole carbon source, the final OD concentration was used. 660Each microorganism was inoculated to achieve a ratio of =0.04. The cells were incubated in 15 mL Harmony centrifuge tubes (LMS, Tokyo) at 28°C and 130 rpm for 24 hours. As a result, microorganisms capable of degrading and utilizing elaidic acid, a trans fatty acid, and subsequently growing were identified.

[0103] These tests revealed that one isolated microbial strain could be cultured in a low-temperature environment using oils and fats as nutrients, could assimilate (decompose) oils and fats at low temperatures, and could decompose and assimilate trans fatty acids. This strain was named KH-1.

[0104] To further characterize KH-1, 16S rDNA gene sequence analysis was performed. As a result, KH-1 was identified as Burkholderia arboris. Furthermore, whole-genome sequence analysis using RAST (Rapid Annotation using Subsystem Technology, http: / / rast.nmpdr.org) compared KH-1 with a previously isolated strain of the same species revealed differences in genome size and mutations with deletions and insertions at dozens of locations. In addition, SNPs / INDEL analysis using CLC Genomics server 9.0 detected variants at hundreds of locations. Based on these results, it was determined that KH-1 is a novel strain of Burkholderia arboris.

[0105] Here, we focused our analysis on KH-1, but by examining the lipid and / or fatty acid degradation capabilities of other strains through similar tests, their lipid and / or fatty acid degradation capabilities can also be identified.

[0106] Burkholderia arboris KH-1 strain was streaked onto agar plates containing canola oil (Nisshin Canola Oil, Nisshin Oillio, Tokyo) as the sole carbon source and cultured at 15°C for 5 days. The results are shown in Figure 1. Since the KH-1 strain proliferated well, it is considered that the KH-1 strain is well-suited to survival in environments containing lipids.

[0107] (Example 2: Comparison of trans fatty acid-containing oil degradation by other microorganisms) Trans fatty acid degradation activity The activity of KH-1 in breaking down elaidic acid (the trans isomer of oleic acid), a trans fatty acid, was compared with that of BioRemove3200 (BR3200) (Novozymes, Denmark) (Figure 2). A stock solution was prepared by dissolving 2% elaidic acid in a 2.5% Triton X-100 (Sigma-Aldrich) solution. Since elaidic acid is solid at room temperature, 4 ml of the stock solution was heated to 65°C and added to 40 ml of inorganic salt medium (Na2HPO4 3.5 g / L, KH2PO4 2.0 g / L, (NH4)2SO4 4.0 g / L, MgCl2·6H2O 0.34 g / L, FeSO4·7H2O 2.8 mg / L, MnSO4·5H2O 2.4 mg / L, CoCl2·6H2O 2.4 mg / L, CaCl2·2H2O 1.7 mg / L, CuCl2·2H2O 0.2 mg / L, ZnSO4·7H2O 0.3 mg / L, and NaMoO4 0.25 mg / L), after which it was autoclaved. The final concentrations of Triton X-100 and elaidic acid were 0.25% and 0.2%, respectively. KH-1 cultured overnight in LB medium was washed twice with PBS medium, and then concentrated using an MX-100 micro-high-speed centrifuge (Tommy Seikou, Tokyo) to obtain the final OD concentration. 660 The above culture medium was inoculated so that the concentration was 0.04 (HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo)). BR3200 was weighed out at 1g along with the base, suspended in 100ml of distilled water, stirred for 30 minutes, and then inoculated so that the initial inoculation concentration was the same as that of KH-1 (the final concentration of each bacterium was 4x10). 5 (The cell count was standardized to cells / ml). The cells were cultured at 28°C and 130 rpm for 24 hours. The samples were analyzed using TLC (Figure 2A) and an oil content measurement reagent kit (Kyoritsu Chemical Laboratory, Tokyo) (measurement reagent kit using polynipamp extractive analysis method) (Figure 2B). As a result, it was found that KH-1 almost completely decomposed and assimilated 0.2% elaidic acid in 24 hours (reducing the concentration to at least 200 mg / L (0.02%) or less). On the other hand, elaidic acid remained in BR3200.

[0108] (Example 3: Comparison of the decomposition rate of trans fatty acid-containing oils and fats) Microbial decomposition of trans fatty acid-containing oils To a TBS buffer (20 mM Tris, 150 mM NaCl, pH 7.0) to which triellidine was added to achieve a final concentration of 0.1%, the final concentration was measured by the optical density of the bacterial cells using a HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo). 660 The KH-1 strain was inoculated to a ratio of 0.04. This was cultured in a Falcon tube (130 rpm, 28°C), and samples were collected after 5 days. The samples were visualized using TLC (applied to a silica gel plate, developed with chloroform:acetone:methanol (96:4:2) solution, and then molybdenum phosphate n-hydrate (2.4 g in 60 ml of ethanol)) (Figure 3A). For control, data for the case where only TBS buffer was added are also shown. These results confirm that KH-1 has the ability to decompose and assimilate trans fatty acid-containing oils, and furthermore, to decompose and assimilate trans fatty acids themselves.

[0109] Decomposition of trans fatty acid-containing lipids in microbial culture supernatant Similarly, 1 mL of the above-mentioned KH-1 strain culture supernatant (estimated lipase concentration; 50 u / ml) or TBS buffer (pH 7.0) and 0.05 mL of triellidine stock solution (2% triellidine, 5% Triton X-100 aqueous solution) were added to a 1.5 mL microtube (Watson, Tokyo) to prepare a reaction solution with a final triellidine concentration of 0.1%. This was incubated at 130 rpm and 28°C for 24 hours to obtain a sample. The sample was extracted with an equal volume of chloroform, and 5 μl was subjected to TLC. The sample was applied to a silica gel plate, developed with chloroform:acetone:methanol (96:4:2) solution, and then visualized with molybd phosphoric acid n hydrate (2.4 g in 60 ml of ethanol) (Figure 3B). These results confirm that it is possible to decompose trans fatty acid-containing oils (triellidines) into trans fatty acids (elaidic acid) even when using the supernatant of KH-1.

[0110] The oil and grease resolution capabilities of KH-1 and BR3200 were compared using actual wastewater (Figure 4). Wastewater samples containing high levels of trans fatty acids from food processing plants using hydrogenated oils were cultured in an inorganic salt medium with nitrogen (ammonium sulfate) and phosphorus added. KH-1 was cultured in LB medium, washed twice with PBS medium, and then cultured to 1 × 10⁻⁶ 6 The cells were inoculated at a concentration of 1 / ml, and BR3200 was used at 10 times the manufacturer's recommended concentration, which is 1 × 10⁶. 8 Cells were inoculated at a concentration of cells / ml. The cells were cultured at 28°C, and samples were collected after 24 and 48 hours. Samples were analyzed using TLC (applied to silica gel plates, developed with chloroform:acetone:methanol (96:4:1) solution, and visualized with molybdenum hydrate) (Figure 4A) and an oil content analysis reagent kit (equivalent to n-hexane extraction, as described above) (Figure 4B). BR3200 showed slow degradation even in excess amounts, while KH-1 demonstrated superior degradation ability.

[0111] (Example 4: Lipid decomposition ability of KH-1 strain at 15℃) Burkholderia arboris KH-1 was placed in 3 L of inorganic salt medium (composition above) containing 10 g / L of canola oil (Nisshin Canola Oil, Nisshin Oillio), and the final concentration was determined by the optical density of the bacterial cells using a HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo). 660 The cells were inoculated to a pH of 0.03. These were cultured in a 5L volume fermenter (250 rpm, 200 ml / min air reflux, pH 7.0, 15°C), and samples were taken over time. The bacterial cells were removed from the sampled culture medium by centrifugation. The oil content equivalent to the n-hexane value in the supernatant was measured using an oil content measurement reagent kit (n-hexane extraction, as described above) (Figure 5A). Total fatty acids (the sum of fatty acids in triglycerides and free fatty acids) were quantified by gas chromatography (Figure 5B). Specifically, 3 ml of the culture supernatant was acidified with hydrochloric acid, and twice the volume of ethyl acetate was added. After stirring for 5 minutes, the mixture was centrifuged, and 1 ml of the ethyl acetate layer was transferred to an organic solvent-resistant tube and completely evaporated. 4 ml of methanol:sulfuric acid = 17:3 methanol esterification solution was added, and the mixture was heated at 100°C for 2 hours to methylate all fatty acids. A chloroform:pure water = 1:1 mixture was added and thoroughly stirred. The chloroform layer and 0.5% methyl octanoate (internal standard) were mixed in a 1:1 ratio, and the mixture was analyzed by gas chromatography (GC-17A (Shimadzu Corporation, Kyoto)) equipped with an FID detector. These results confirm that KH-1 possesses high lipid decomposition and assimilation capabilities even at low temperatures.

[0112] (Example 4A: Absorption capacity of trans fatty acids and their contained oils in KH-1 strain at 15°C) The assimilation ability of the KH-1 strain was evaluated by culturing it in a medium using elaidic acid or trielaidin as the sole carbon source. A stock solution of elaidic acid or trielaidin (2.5% Triton X-100 aqueous solution) was added to 2 mL of an inorganic salt medium (composition as above, pH 7) prepared to a final concentration of elaidic acid or trielaidin of 0.1%. The final concentration was determined by the optical density of the bacterial cells using a HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo). 660 The KH-1 strain was inoculated so that the ratio was 0.08. This was cultured at 15°C for 5 days, and the growth of microorganisms was observed. The results for elaidic acid and trielaidin-supplemented media are shown in Figure 5C. The results for the control group without microorganisms are also shown. In samples inoculated with the KH-1 strain, the culture medium was observed to be cloudy. This indicates that the KH-1 strain can grow even at 15°C in an environment where elaidic acid or trielaidin is the sole carbon source, and that it has the ability to utilize these compounds.

[0113] (Example 5: Lipid decomposition ability of KH-1 strain at 28°C) Similarly, the lipid-degrading ability of KH-1 under 28°C culture was also evaluated (Figure 6). The residual oil content in the culture medium was analyzed by thin-layer chromatography (TLC) (Figure 6A). Specifically, an equal volume of ethyl acetate was added to 3 ml of the sample supernatant and stirred for 5 minutes. After separating the ethyl acetate layer and completely evaporating the solvent, it was dissolved in 300 μL of chloroform. 3 μL of this mixture was applied to a silica gel plate and developed with a chloroform:acetone (96:4) solution. After development, a 4% (w / v) 12-moleb(IV) phosphate ethanol solution was sprayed and heated at 110°C for 12 minutes to visualize the oils and free fatty acids, and the amount of oils and their degradation products (fatty acids) remaining in the medium was compared. As a result of the degradation of triglycerides by lipase secreted by microorganisms, free fatty acids, which are hydrolysis products, were detected (24 h). These fatty acids also degraded over time. KH-1 completely eliminated the free fatty acids after 48 h.

[0114] Similar to Example 4, total fatty acids (the sum of fatty acids in triglycerides and free fatty acids) were quantified by gas chromatography at 28°C (Figure 6B). Similar to Example 4, residual oils and fats were measured at 28°C using an oil content measurement reagent kit (Figure 6C). KH-1 maintained good lipid decomposition capabilities even at 28°C.

[0115] (Example 6: Decomposition of various trans fatty acids) The activity of KH-1 in breaking down the trans fatty acids palmiteradicic acid (16:1) and vaccenic acid (18:1) was compared with that of BioRemove3200 (BR3200) (Novozymes, Denmark). KH-1 and BR3200 were prepared in 5 mL of inorganic salt medium (composition above, pH 7) with a final concentration of palmiteraidic acid or vaccenic acid of 0.2% and a final concentration of Triton X100 of 0.25%, respectively. The final concentration was determined by the optical density of the bacterial cells using a HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo). 660KH-1 was inoculated so that the value was 0.05, and OD 660 = Approximately 5 × 10, which corresponds to 0.05 6 BR3200 was inoculated to achieve a CFU / g concentration. These were then cultured at 15°C for 48 or 72 hours, or at 28°C for 24 hours, with shaking at 130 rpm, and the culture supernatant was obtained for each strain. Control samples without the use of microorganisms were also prepared. Subsequently, the residual oil content in the culture medium was analyzed by thin-layer chromatography (TLC). Specifically, fatty acids were extracted with chloroform at half the volume of the sample, 6 μl of the extract was applied to a silica gel plate, and developed with a chloroform:acetone:methanol (96:4:2) solution. After development, fatty acids were visualized with a 12-molybdenum(IV) phosphate ethanol solution, as in Example 5, and the amount of fatty acids remaining in the culture medium was compared (Figures 8 and 9). KH-1 was able to completely decompose palmiteraisic acid and vaccenic acid within 24 hours at 28°C and within 72 hours at 15°C. On the other hand, BR3200 did not have the ability to decompose these fatty acids. Thus, the microorganisms of this disclosure may be able to decompose various trans fatty acids.

[0116] (Example 7: Comparison of KH-1 strain with detergent) The oil-stained ventilation fan filter was cultured in KH-1 culture supernatant (culture conditions: inorganic salt medium with 1% canola oil added (composition as above), initial concentration OD). 660 The samples were inoculated to a concentration of 0.01 and incubated at 28°C for 24 hours. After incubation, the supernatant was used after removing the bacterial cells by centrifugation. The filters were then soaked at room temperature (25°C) in oil-based detergent (NicoEco Kitchen Natural Enzyme Detergent (NicoEco, Nagano), diluted 143 times with water according to the instructions) and general detergent (Family® (Kao, Tokyo), diluted 666 times according to the instructions) (Figure 7). With general detergent and oil-based detergent, oil stains could not be completely removed even after 4 hours of soaking, but with KH-1, the filter became as white as new after 1 hour of soaking.

[0117] (Example 8: Acquisition of a different strain) Samples were collected from a river near a food processing plant that discharges oil-containing wastewater, and microorganisms were isolated from them. The isolated microorganisms were then investigated to see if they could decompose trans fatty acids or trans fatty acid-containing oils under low-temperature conditions (15°C). As a result, microorganisms capable of decomposing trans fatty acids and trans fatty acid-containing oils at low temperatures were found. These microbial strains were named KH-1AL1, KH-1AL2, and KH-1AL3, respectively.

[0118] To further characterize KH-1AL1, KH-1AL2, and KH-1AL3, we performed 16S rDNA gene sequence analysis. KH-1AL1 was identified as Burkholderia ambifaria because its partial nucleotide sequence of 16rDNA was 100% identical to that of Burkholderia ambifaria. KH-1AL2 exhibited 99.9% homology in a partial nucleotide sequence of its 16rDNA to Burkholderia contaminans, and on the molecular phylogenetic tree, it was classified into the same group as B. seminalis, B. territorii, and B. cepacia (with homology of 99.7%, 99.7%, and 99.8%, respectively, in the partial nucleotide sequence of its 16rDNA). As a result, KH-1AL2 was identified as a bacterium of the Burkholderia cepacia complex. KH-1AL3 exhibited 99.9% homology in a partial nucleotide sequence of its 16rDNA to Burkholderia contaminans, and on the molecular phylogenetic tree, it was classified into the same group as B. seminalis, B. territorii, and B. cepacia (with homology in the partial nucleotide sequence of 16rDNA being 99.7%, 99.7%, and 99.8%, respectively). As a result, KH-1AL3 was identified as a bacterium of the Burkholderia cepacia complex. The Burkholderia cepacia complex is a classification of microorganisms belonging to the genus Burkholderia that are genetically very close, and includes amphifaria, anthina, arboris, cenocepacia, cepacia, contaminans, diffusa, dolosa, lata, latens, metallica, multivorans, pseudomultivorans, puraquae, pyrrocinia, semialis, stabilis, stagnalis, territorii, ubonensis, and vietnamiensis (Martina P et al., Int J Syst Evol Microbiol. 2018 Jan;68(1):14-20.). Analysis revealed that various bacteria belonging to the Burkholderia cepacia complex exhibited high lipid and / or fatty acid degradation capabilities, suggesting that bacteria belonging to the Burkholderia cepacia complex are particularly useful.

[0119] (Example 9: Further oil-degrading ability of three strains at 15°C) KH-1AL1, KH-1AL2, and KH-1AL3 were pre-cultured in LB medium, then subjected to two centrifugations in PBS at 25°C, 3000g for 10 minutes each, followed by washing to prepare samples of each strain. These three bacterial strain samples were placed in an inorganic salt medium (composition above) with a pH of 7 containing 1% canola oil (Nisshin Canola Oil, Nisshin Oillio), and the final concentration was determined by the optical density of the bacterial cells using a HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo). 660The cultures were inoculated to a ratio of 0.02. These were cultured at 15°C for 96 hours, and samples were taken at various time points. The bacterial cells were removed from the sampled cultures by centrifugation. For each supernatant of these three strains, the residual oil content in the culture was analyzed by thin-layer chromatography (TLC), the total fatty acids (total amount of fatty acids in triglycerides and free fatty acids) were quantified by gas chromatography, and the oil content equivalent to the n-hexane value was measured using an oil content measurement reagent kit, following the same procedure as in the above example. The results are shown in Figures 10-12. These results confirm that KH-1AL1, KH-1AL2, and KH-1AL3 can efficiently decompose and assimilate oils and fats even at low temperatures.

[0120] (Example 10A: Inter-strain comparison of trans fatty acid-containing oils and fats with culture supernatant at 28°C resolution) In inorganic salt media (composition as above, pH 7) prepared with KH-1, KH-1AL1, KH-1AL2, and KH-1AL3, respectively, so that the final concentration of triellidine was 0.2%, the final concentration was measured using a HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo) based on the optical density of the bacterial cells. 660 The cells were inoculated to achieve a ratio of 0.08. After incubation at 28°C for 6 days, the culture supernatant of each strain was obtained. A control sample without the use of microorganisms was also prepared. The culture supernatants obtained from each strain were filtered through a cellulose acetate membrane filter with a pore size of 0.45 μm (ADVANTEC, Tokyo). Triellidine and Triton X100 were then added to final concentrations of 0.4% and 0.5%, respectively, and incubated at 37°C for 24 hours with shaking at 130 rpm. Subsequently, the residual oil content in the culture medium was analyzed by thin-layer chromatography (TLC). Specifically, oils and free fatty acids were extracted using chloroform at half the volume of the sample, 5 μl of the extract was applied to a silica gel plate, and developed with a chloroform:acetone:methanol (96:4:2) solution. After development, the oils and free fatty acids were visualized using a 12-molybdenum(IV) phosphate ethanol solution, as in Example 5, and the amount of oils and their degradation products (fatty acids) remaining in the culture medium was compared (Figure 13). As a result, it was confirmed that the culture supernatants of KH-1, KH-1AL1, KH-1AL2, and KH-1AL3 could efficiently decompose trans fatty acid-containing oils. This result is thought to be due to the secretion of lipase by these strains, suggesting that both the microorganisms themselves and their culture supernatants can be usefully used for the decomposition of trans fatty acid-containing oils.

[0121] (Example 10B: Inter-strain comparison of trans fatty acid assimilation and decomposition at 28°C) In inorganic salt media (composition as above, pH 7) prepared by KH-1, KH-1AL1, KH-1AL2, and KH-1AL3 so that the final concentration of elaidic acid was 0.2% and the final concentration of Triton X100 was 0.25%, respectively, and the final concentration by cell optical density was measured using a HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo) and the final concentration was OD 660 The cells were inoculated to a ratio of 0.04. These were then cultured at 28°C for 20 hours with shaking at 130 rpm, and the culture supernatant of each strain was obtained. A control sample without the use of microorganisms was also prepared. Subsequently, the residual oil content in the culture medium was analyzed by thin-layer chromatography (TLC). Specifically, fatty acids were extracted with chloroform at half the volume of the sample, 5 μl of the extract was applied to a silica gel plate, and developed with a chloroform:acetone:methanol (96:4:2) solution. After development, free fatty acids were visualized with a 12-molybdo(IV) phosphate ethanol solution, as in Example 5, and the amount of fatty acids remaining in the culture medium was compared (Figure 14 left). In addition, the residual oil content equivalent to the n-hexane value was measured using an oil content measurement reagent kit, following the same procedure as in Example 4 (Figure 14 right). As a result, it was confirmed that KH-1, KH-1AL1, KH-1AL2, and KH-1AL3 can efficiently decompose trans fatty acids, suggesting that all of these strains are useful for removing oils containing trans fatty acids. Considering this together with the results of Example 10A, it is predicted that trans fatty acid-containing oils (e.g., triellidines) can be completely decomposed by these microorganisms.

[0122] (Example 10C: Inter-strain comparison of trans fatty acid assimilation and decomposition at 15°C) In inorganic salt media (composition as above, pH 7) prepared by KH-1, KH-1AL1, KH-1AL2, and KH-1AL3 so that the final concentration of elaidic acid was 0.2% and the final concentration of Triton X100 was 0.25%, respectively, and the final concentration by cell optical density was measured using a HITACHI U-2810 spectrophotometer (Hitachi, Ltd., Tokyo) and the final concentration was OD 660 The cells were inoculated to a ratio of 0.08. These were then cultured at 15°C for 72 hours with shaking at 130 rpm, and the culture supernatant of each strain was obtained. A control sample without the use of microorganisms was also prepared. Subsequently, the residual oil content in the culture medium was analyzed by thin-layer chromatography (TLC). Specifically, free fatty acids were extracted with chloroform at half the volume of the sample, 5 μl of the extract was applied to a silica gel plate, and developed with a chloroform:acetone:methanol (96:4:2) solution. After development, the free fatty acids were visualized with a 12-molybdo(IV) phosphate ethanol solution, as in Example 5, and the amount of fatty acids remaining in the culture medium was compared (Figure 15 left). In addition, the residual oil content equivalent to the n-hexane value was measured using an oil content measurement reagent kit, following the same procedure as in Example 4 (Figure 15 right). As a result, it was confirmed that KH-1, KH-1AL1, KH-1AL2, and KH-1AL3 can efficiently decompose trans fatty acids, suggesting that all of these strains are useful for removing oils containing trans fatty acids. Considering this together with the results of Example 10A, it is predicted that trans fatty acid-containing oils (e.g., triellidines) can be completely decomposed by these microorganisms.

[0123] (Example 11: Application to food waste treatment) KH-1 1.2 x 10 7 The bacteria were inoculated to a cell / mL concentration and treated at 30°C for 24 hours. The n-hexane levels in the wastewater discharged from the food waste processor were measured. A significant reduction in n-hexane levels (2.22 g / L) was observed compared to a control case without KH-1 (3.26 g / L). The food waste in the disintegrating food waste processor consisted of food waste from a restaurant kitchen, with bones and shells removed. The food waste processing capacity of the disintegrating food waste processor was 20 kg / day, and the water inlet / outlet volume was 38.4 L / day.

[0124] (Example 12: Use within the device) KH-1 was reduced to 2 × 10¹⁶ units in an inorganic salt medium containing 10 mL / L canola oil. 10 Culture until the cell count reaches 1 / mL and use as the stock solution. Dilute this 10-fold to make a microbial preparation (2 × 10⁻¹⁰⁻¹ 9The concentration is set to (cells / mL). This is stored under refrigeration in the microbial storage tank of the automatic amplification and dispensing device to serve as the inoculum. This inoculum is automatically inoculated into the inorganic salt medium in the culture amplification tank of the same device at a rate of 1 / 100th daily, and cultured until the number of microorganisms increases 100-fold, i.e., to the same cell concentration as the microbial preparation. By adding this to the wastewater tank at a rate of 1 / 1000th of the oil decomposition treatment volume, the microbial concentration of decomposing bacteria in the oil treatment water is increased to 2 × 10⁻⁶. 6 The treatment involves decomposing wastewater from a food processing plant that discharges a large amount of trans fatty acid-containing oils, using a cell / mL ratio over a 24-hour period. The season at this time is winter, and the water temperature during treatment fluctuates between 12 and 17°C. As a result, a significant reduction in n-hexane levels was observed compared to a control case where no microorganisms were introduced.

[0125] (Example 13: Other Embodiments) A carrier such as charcoal, various plastics, or ceramic pieces is placed in the grease trap, and an appropriate amount (for example, 1 × 10) is added. 6 KH-1 (cells / mL) is automatically added daily after the cafeteria closes. Water is sampled immediately before the start of operations each day, and the n-hexane value is analyzed. After one week, a significant decrease in n-hexane value is observed compared to a control case where KH-1 is not added, and the appearance of the grease trap itself also shows effects such as reduced oil adhesion and floating.

[0126] (Note) As described above, while the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of this disclosure should be interpreted solely by the claims. Patents, patent applications and other documents cited herein should be incorporated herein by reference as if their contents were specifically described herein. [Industrial applicability]

[0127] This disclosure provides microorganisms that have lipid-degrading properties and compositions containing the same, and by using such microorganisms or compositions, it is possible to reduce the environmental burden caused by food processing plant wastewater containing large amounts of lipids.

Entrusted number

[0128] KH-1 strain (NITE BP-02731) KH-1AL1 strain (NITE ABP-02977) KH-1AL2 strain (NITE ABP-02978) KH-1AL3 strain (NITE ABP-02979)

Claims

[Claim 1] The invention described in part of this specification.

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