Gas emission measurement system and method, methane generation inhibitor and its evaluation method, and bacterial quantification method

Abstract

This invention enables accurate measurement of greenhouse gas emissions from animals. It also allows for easy evaluation of the reduction effect of greenhouse gas emissions from animals. Furthermore, it provides a methane emission inhibitor that suppresses methane production from Methanobrevibacter bacteria. [Solution] A bacterial quantification method for quantifying Methanobrevibacter species contained in the rectal feces of animals includes the steps of: collecting rectal feces from an animal; holding the rectal fecal sample in an anaerobic porter under anaerobic conditions; replacing the gas in the anaerobic porter with nitrogen gas, diluting the sample with a culture medium, and culturing the bacteria in a test tube; recovering the cultured bacteria; extracting DNA from the recovered bacteria; and quantifying the Methanobrevibacter species from the extracted DNA by quantitative PCR or a gene analysis method using next-generation sequencing analysis.

Claims

1. A method for quantifying Methanobrevibacter species in the rectal feces of animals, The process of collecting animal rectal feces, The process involves holding the rectal stool sample in an anaerobic porter under anaerobic conditions, The process involves replacing the gas in the anaerobic porter with nitrogen gas, diluting the sample with a culture medium, and culturing the bacterial flora in a test tube. The process of recovering the cultured bacterial flora, The process involves extracting DNA from the recovered bacterial flora, A step of quantifying the Methanobrevibacter species from the extracted DNA by quantitative PCR or next-generation sequencing analysis, A method for quantifying bacteria, including the method described above.

2. A method for determining bacterial quantity according to claim 1, further comprising the step of collecting rectal feces from the animal, The process involves feeding the first animal with a first feed containing the test substance, A step of feeding a second animal of the same species as the first animal with a second feed that does not contain the test substance, Includes, The step of quantifying the Methanobrevibacter species is, A step of determining the first quantity by quantifying the Methanobrevibacter species in bacteria collected and cultured from the rectal feces of the first animal, The process involves determining the second quantity by quantifying the Methanobrevibacter species in bacteria collected and cultured from the rectal feces of the second animal, A step of evaluating the increase or decrease of the Methanobrevibacter species due to the addition of a methane generation inhibitor based on the variation of the first amount relative to the second amount, including, Bacterial quantification method.

3. A method for determining bacterial quantity according to claim 2, A method for quantifying bacteria, wherein the test substance contains at least one of an organic acid, an organic acid salt, or an oligosaccharide as an active ingredient.

4. A method for determining bacterial quantity according to claim 3, A method for determining the number of bacteria, wherein the organic acid comprises at least one of citric acid, sorbic acid, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, and phosphoric acid.

5. A method for determining bacterial quantity according to claim 3, A method for determining bacterial quantity, wherein the organic acid salt comprises at least one of citric acid, sorbic acid, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, phosphoric acid, and potassium, calcium, zinc, and magnesium salts thereof.

6. A method for determining bacterial quantity according to claim 3, A method for determining bacterial quantity, wherein the organic acid salt comprises at least one of potassium diformate or sodium formate.

7. A method for determining bacterial quantity according to claim 3, A method for quantifying bacteria, wherein the oligosaccharide comprises at least one of galactooligosaccharides or fructooligosaccharides.

8. A method for determining bacterial quantity according to any one of claims 2 to 6, A method for determining bacterial count, wherein the test substance is an additive added to the feed of a mammal or bird selected from pigs, dogs, or cats.

9. A method for measuring greenhouse gas emissions from animals, or from animal excrement or metabolites, A gas emission measurement system having a cage-like section that defines a breeding space for housing animals, comprising the steps of placing an animal into the breeding space, The process of surrounding the cage portion with a light-transmitting cover portion, The process involves connecting an air supply unit through an air intake hole opened in a part of the surface constituting the cover portion, thereby continuously supplying air into the cover portion, and discharging a portion of the gas inside the cover portion to the outside through a plurality of exhaust holes opened in another part of the surface constituting the cover portion. The steps include: connecting a detection unit through a detection hole opened in another part of the surface constituting the cover portion to initiate pre-detection of at least greenhouse gases among the gases contained within the cover portion; The process includes ending the pre-detection when the amount of greenhouse gases pre-detected by the detection unit has stabilized over time, and starting the main detection by the detection unit, A method for measuring gas emissions, including those mentioned above.

10. A method for measuring gas emissions according to claim 9, A method for measuring gas emissions in which the step of starting the detection in the detection unit is performed 30 minutes or more after the animal is placed in the cage, covered with the cover unit, and the supply of air is started in the air supply unit.

11. A method for measuring gas emissions according to claim 9 or 10, A method for measuring gas emissions, wherein the animal is a livestock, companion animal, or exhibit animal, and is a monogastric animal that does not ruminate.

12. A method for evaluating the methane emission suppression ability of a test substance added to feed given to an animal, which evaluates the effect of the test substance on the greenhouse gas emissions emitted by the animal, A step of collecting rectal feces from animals that have been fed a basic diet that does not contain the aforementioned test substance, The process involves holding the rectal stool sample in an anaerobic porter under anaerobic conditions, The process involves replacing the gas in the anaerobic porter with nitrogen gas, diluting the rectal stool sample with a culture medium, and culturing the bacterial flora in test tubes with and without the test substance added. The process of recovering the cultured bacterial flora, The process involves extracting DNA from the recovered bacterial flora, A step of evaluating the increase or decrease of Methanobrevibacter species from the extracted DNA by quantitative PCR or next-generation sequencing analysis, A method for evaluating methane emission suppression ability, including the ability to suppress methane generation.

13. A gas emission measurement system capable of measuring greenhouse gas emissions from animals, or from animal excrement or metabolites, A cage-like section that defines a breeding space for housing animals inside, A translucent cover portion for surrounding the cage portion, An air supply unit connected to the cover portion surrounding the cage portion is provided for continuously supplying air into the cover portion. A flow meter capable of measuring the amount of air supplied to the cover portion by the air supply unit, A detection unit capable of detecting at least greenhouse gases among the gases contained within the cover portion, A gas emission measurement system comprising: The cover portion has a part of the surface that constitutes the cover portion, An air intake port for connecting to the aforementioned air supply unit, A detection hole for connecting to the aforementioned detection unit, Multiple exhaust holes for discharging a portion of the gas inside the cover to the outside, We have established a system, A gas emission measurement system in which the inside of the cover is maintained at a higher pressure than the outside.

14. A gas emission measurement system according to claim 13, A gas emission measurement system comprising the plurality of exhaust holes, each opening on an opposing side of the cover portion.

15. A gas emission measurement system according to claim 13, A gas emission measurement system wherein the plurality of exhaust holes, the intake holes, and the detection holes are arranged on the upper part of the opposing sides of the cover portion, at positions where they are spaced apart on each side.

16. A gas emission measurement system according to claim 13, A gas emission measurement system in which the opening area of ​​the plurality of exhaust holes or detection holes is formed to be smaller than that of the intake holes.

17. A gas emission measurement system according to claim 13, A gas emission measurement system in which the diameters of the plurality of exhaust holes or detection holes are 2 to 22 mm.

18. A gas emission measurement system according to claim 13, A gas emission measurement system comprising the detection unit configured to start detection when at least greenhouse gases among the gases contained in the cover are in a stable state.

19. A gas emission measurement system according to any one of claims 13 to 16, The aforementioned detection unit is composed of a photoacoustic system, which is used to measure gas emissions.

20. A gas emission measurement system according to any one of claims 13 to 16, A gas emission measurement system in which the animal housed in the cage is a micro miniature pig or a pig raised for food.

21. A gas emission measurement system according to any one of claims 13 to 16, The aforementioned cage section is A slatted floor section, A receiving portion provided on the lower surface of the floor portion, A gas emission measurement system equipped with [a specific feature].

22. A gas emission measurement system according to any one of claims 13 to 16, The gas emission measurement system comprises a cover portion formed in the shape of a box with an open bottom, and a packing provided at the open end of the bottom.

23. A gas emission measurement system according to claim 20, The aforementioned cover portion is a gas emission measurement system having a handle provided on a part of its opposing side.

24. A methane production inhibitor for addition to animal feed, A methane emission inhibitor containing at least one of the following as an active ingredient: an organic acid, an organic acid salt, or an oligosaccharide.

25. A methane generation inhibitor according to claim 24, A methane-producing inhibitor that has an inhibitory rate of 80% or more on Metanobrevibacter bacteria.

26. A methane generation inhibitor according to claim 24, A methane generation inhibitor that causes a change in pH of less than 1 before and after addition to feed.

27. A methane generation inhibitor according to claim 24, A methane generation inhibitor comprising at least one of the following organic acids: citric acid, sorbic acid, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, and phosphoric acid.

28. A methane generation inhibitor according to claim 24, A methane generation inhibitor comprising at least one of the following organic acid salts: citric acid, sorbic acid, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, phosphoric acid, and potassium, calcium, zinc, and magnesium salts thereof.

29. A methane generation inhibitor according to claim 24, A methane generation inhibitor comprising at least one of potassium diformate and sodium formate as the organic acid salt.

30. A methane generation inhibitor according to claim 24, A methane generation inhibitor comprising at least one of galactooligosaccharides or fructooligosaccharides as the oligosaccharide.

31. A methane generation inhibitor according to any one of claims 24 to 30, A methane-producing agent that is an additive added to the feed of monogastric animals, which are livestock, companion animals, or exhibition animals.

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