Method for extracting lignin contained in herbaceous plants, composition containing isolated lignin derived from herbaceous plants, and method for producing same

Abstract

A method for extracting lignin contained in herbaceous plants, the method comprising feeding wood-eating termites with a herbaceous plant.

Description

Method for extracting lignin contained in herbs, composition containing isolated lignin derived from herbs, and method for producing the same

[0001] The present invention relates to a method for extracting lignin contained in herbs, a composition containing isolated lignin derived from herbs, and a method for producing the composition.

[0002] Lignin is a polymeric compound formed by enzymatic oxidative polymerization of lignin monomers called monolignols, and is one of the main components of plant cell walls. Although lignin has attracted attention as a renewable resource, it is difficult to isolate from plants because it forms a strong composite material with polysaccharides (cellulose and hemicellulose) in plant cell walls (Non-Patent Documents 1 and 2).

[0003] Methods for separating (extracting) lignin contained in herbs have been discovered to date, including a method using black liquor discharged during paper pulp production (Patent Document 1), and a method involving enzymatic saccharification of lignocellulosic raw materials followed by heat treatment in a specific solvent to obtain a lignin decomposition product (Patent Document 2).

[0004] JP 2012-214432 A JP 2015-6998 A

[0005] Network Polymer, Vol. 31, No. 5, pp. 213-223, 2010 Humanosphere Research, No. 13, pp. 10-18, 2017

[0006] The present inventors have discovered that herbs can be fed to termites that consume herbs as food, and that lignin contained in herbs can be extracted by a method comprising feeding the herbs to termites that consume herbs as food. The present invention is based on this novel finding, and aims to provide a method for extracting lignin contained in herbs, as well as a composition containing isolated lignin derived from herbs and a method for producing the same.

[0007] One aspect of the present invention relates to a method for extracting lignin contained in herbs, which comprises feeding herbs on termites that eat herbs.

[0008] The inventors' research has revealed that it is possible to feed termites that use herbs as food. It has also been discovered that by feeding termites that use herbs as food, lignin can be concentrated and separated as excrement (feces) from the termites. Thus, lignin contained in herbs can be extracted by a method that includes feeding termites that use herbs as food.

[0009] Another aspect of the present invention relates to a method for producing a composition comprising lignin from an isolated herbaceous plant, comprising feeding termites that are food-eating agents on the herbaceous plant.

[0010] Yet another aspect of the present invention relates to a composition comprising isolated herbaceous lignin.

[0011] According to the present invention, it is possible to provide a method for extracting lignin contained in herbs, a composition containing separated lignin derived from herbs, and a method for producing the same.

[0012] 1 is a graph showing the results of an experiment in which large termites were reared on artificial bait.

[0013] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0014] As used herein, "isolated" lignin means that the lignin is in a state different from that of lignin in plants such as herbs. More specifically, lignin is tightly combined with polysaccharides (cellulose, hemicellulose) in plants, and lignin is referred to as "isolated" lignin when it is at least partially separated from these polysaccharides.

[0015] In one embodiment, a method for extracting lignin contained in herbs includes feeding herbs to termites that eat herbs.

[0016] Food-eating termites are termites that primarily feed on woody plants and are not known to feed on herbaceous plants. However, the inventors' studies have revealed that it is possible to feed food-eating termites on herbaceous plants. Termites can decompose and absorb the polysaccharides in complexes of polysaccharides and lignin, and excrete the resulting separated lignin derived from herbaceous plants as excrement (feces). Therefore, by feeding food-eating termites on herbaceous plants, the lignin contained in the complex with polysaccharides in the herbaceous plants can be extracted into the excrement of the food-eating termites.

[0017] Conventionally, to separate lignin contained in herbs, the herbs are treated with an acid or alkali at high temperature and high pressure, and the decomposed lignin is extracted into an acid or alkali liquid. The lignin recovered by such a method is soluble in an acid or alkali at least under high temperature or high pressure, and is highly decomposed and broken down into smaller molecules.

[0018] On the other hand, in the method according to the present embodiment, lignin is isolated by termites selectively consuming the polysaccharides in the lignin-polysaccharide complex in the herbaceous plant. Therefore, the method according to the present embodiment allows lignin to be extracted without further decomposition and with minimal chemical modification.

[0019] Furthermore, the method according to this embodiment utilizes the digestive function of termites and does not require high-temperature, high-pressure treatment, so lignin can be extracted at low cost and with low energy.

[0020] Feeding food-eating termites with herbs may include feeding food-eating termites with bait containing herbs (hereinafter also simply referred to as "bait").

[0021] The herbaceous plant may be a monocotyledonous plant. Examples of monocotyledonous plants include herbs belonging to the Poales order. Examples of herbaceous plants belonging to the Poales order include herbs of the Poaceae family, Juncaceae family, Cyperaceae family, and Typhaceae family. Examples of herbaceous plants in the Poaceae family include herbs of the Oryzae genus, Bromus genus, Brassica genus, Avena genus, Rehmannia genus, Imperata genus, Miscanthus genus, Saccharum genus, Sasa genus, Hordeum genus, Zea mays, Echinochloa genus, Paspalum genus, Goosegrass genus, Digitaria genus, Setaria genus, Albizia genus, Sphagnum genus, Sorghum genus, and Panicum genus. Examples of herbaceous plants in the Oryza genus include rice. An example of herbaceous plants in the Bromus genus is Brassica napus. An example of herbaceous plants in the Brassica genus is Brassica napus. An example of herbaceous plants in the Avena genus is Avena sativa. An example of herbaceous plants in the Retina genus is Retina. An example of herbaceous plants in the Orchard genus is Orchard grass. An example of herbaceous plants in the Triticum genus is Wheat. An example of herbaceous plants in the Phragmites genus is Phragmites reticulata. An example of herbaceous plants in the Job's tears genus is Job's tears. An example of herbaceous plants in the Imperata genus is Imperata imperata. An example of herbaceous plants in the Miscanthus genus is Miscanthus sinensis. An example of herbaceous plants in the Saccharum genus is Sugarcane. An example of herbaceous plants in the Psathyrium genus is Psathyrium stenophylla. An example of herbaceous plants in the Hordeum genus is Barley. An example of herbaceous plants in the Maize genus is Maize. The sugarcane may be bagasse (residue left over from sugarcane extraction).

[0022] The feed may be composed of herbs or may contain components other than herbs. The content of herbs in the feed may be 30% by volume or more, 40% by volume or more, or 45% by volume or more, or may be 100% by volume or less, based on the total volume of the feed.

[0023] The food may include woody plants, examples of which include conifers such as cedar, cypress, and pine, and broad-leaved trees such as konara oak, sawtooth oak, and mizunara oak.

[0024] The content of woody plants in the bait may be 1% by volume or more, or 5% by volume or more, and may be 80% by volume or less, 60% by volume or less, or 55% by volume or less, based on the total volume of the bait.

[0025] The feed may contain other ingredients in addition to herbs and woody plants, such as soybean pulp, crab shells, shrimp shells, ant soil, waste mushroom beds, and bamboo powder.

[0026] The content of other components other than herbs and woody plants in the feed may be 0.1% by volume or more, 10% by volume or less, or 5% by volume or less, based on the total volume of the feed.

[0027] The moisture content of the bait may be 10% by mass or more, or 30% by mass or more, and may be 90% by mass or less, or 80% by mass or less, based on the total mass of the bait.

[0028] The bait can be prepared, for example, by compressing a raw material containing herbs and, if necessary, non-herb ingredients, for example, by placing the raw material in a mold and applying pressure using a bench press.

[0029] Food-eating termites are termites that primarily feed on woody plants, and may be lower termites or higher termites. Examples of lower termites include termites of the families Isotermesidae, Paratermiteidae, Cephalotermiteidae, Chafertermiteidae, Scissortermiteidae, and Rhinotermitidae. Examples of termites of the Isotermesidae family include the Formosan termite and the Nevada termite. Examples of termites of the Chafertermite family include the Formosan termite. Examples of termites of the Chafertermite family include the Formosan termite and the Formosan termite. Examples of termites of the Scissortermite family include the Formosan termite. Examples of termites of the Rhinotermitidae family include the Reticulitermes speratus and the Formosan termite.

[0030] Giant termites prefer rotten wood, and in nature they only live in deep forests with heavy rainfall, and they rarely damage wooden buildings. Furthermore, they have large body sizes and consume a large amount of food per individual. Therefore, giant termites are particularly suitable as food-use termites in one embodiment. The use of giant termites has the advantage that the production of winged termites can be completely suppressed under artificial management, preventing the escape of winged termites from the breeding environment.

[0031] Examples of higher termites include termites of the Termitidae family, including the Formosan termite and the Takasago termite.

[0032] The method for extracting lignin contained in herbs according to one embodiment may further include collecting excrement of the termites that are used as food materials. Collecting the excrement of the termites that are used as food materials can be carried out, for example, by collecting ant soil formed by the termites that are used as food materials.

[0033] As described above, the excrement (feces) of termites that consume herbs contains lignin derived from isolated herbs. In other words, the above method can be used to produce a composition containing lignin derived from isolated herbs. Therefore, the above method can also be considered as a method for producing a composition containing lignin derived from isolated herbs. As specific aspects of the method, the above-mentioned aspects can be applied without any particular limitations.

[0034] Another embodiment of the present invention is a composition containing isolated lignin derived from herbs. Such a composition can be produced, for example, by the production method described above. The composition may contain excrement from termites that feed on herbs. Specific examples of termites that feed on herbs are as described above. The composition may contain, for example, excrement from lower termites that feed on herbs. Lower termites have bacteria and protozoa as intestinal symbionts. On the other hand, higher termites do not have protozoa as intestinal symbionts. Therefore, the excrement of lower termites and the excrement of higher termites differ, particularly in terms of components (such as DNA) derived from intestinal symbionts.

[0035] The isolated herbaceous lignin is less modified than lignin obtained by high-temperature, high-pressure treatment using acid or alkali, as decomposition and degradation of the lignin from the herbaceous lignin are suppressed.

[0036] The weight average molecular weight (Mw) of the separated lignin contained in the composition may be 18,000 or more, 19,000 or more, 20,000 or more, 21,000 or more, 22,000 or more, 23,000 or more, 24,000 or more, 26,000 or more, 28,000 or more, 30,000 or more, 32,000 or more, 34,000 or more, 36,000 or more, 38,000 or more, 40,000 or more, more than 40,000, 41,000 or more, 42,000 or more, 43,000 or more, 44,000 or more, 45,000 or more, 46,000 or more, 47,000 or more, 48,000 or more, 49,000 or more, or 50,000 or more, and may be 100,000 or less, 90,000 or less, or 80,000 or less. As used herein, the Mw of the isolated lignin in the composition refers to a value determined by gel permeation chromatography using polyethylene glycol standards and polyethylene oxide standards as standards. The weight-average molecular weight (Mw) of the isolated herbaceous lignin in the composition may be within the ranges described above for the weight-average molecular weight (Mw) of the isolated lignin in the composition.

[0037] Lignin is a polymer containing monolignols (p-hydroxycinnamic alcohol analogues) as monomer units. There are three types of monolignols: coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol. The aromatic nucleus units of lignin derived from each monolignols are called guaiacyl nucleus (G nucleus), syringyl nucleus (S nucleus), and p-hydroxyphenyl nucleus (H nucleus), respectively (Non-Patent Document 1). Lignin may also contain at least one of p-coumaric acid esters (e.g., p-coumaric acid esters of coniferyl alcohol, p-coumaric acid esters of sinapyl alcohol, etc.) and tricine as monomer units (Non-Patent Document 2).

[0038] The structure of lignin contained in plants varies depending on the plant (Non-Patent Document 2). For example, lignin contained in coniferous trees contains coniferyl alcohol as a monomer unit, while lignin contained in broad-leaved trees contains coniferyl alcohol and sinapyl alcohol as monomer units. Furthermore, lignin contained in monocotyledonous plants such as grasses contains coniferyl alcohol, sinapyl alcohol, p-coumaric acid esters, and tricine as monomer units.

[0039] In the composition, the mass ratio of the acid-insoluble lignin content to the acid-soluble lignin content (acid-insoluble lignin content / acid-soluble lignin content) may be 1 / 1 or more, 5 / 1 or more, 10 / 1 or more, 12 / 1 or more, or 15 / 1 or more, and may be 50 / 1 or less, 40 / 1 or less, 30 / 1 or less, or 20 / 1 or less. In this specification, the acid-soluble lignin content and the acid-insoluble lignin content refer to values ​​determined as dry mass by the Klason method, and more specifically, refer to values ​​measured by the method described in the Examples.

[0040] In the composition, the content (dry mass) of acid-insoluble lignin may be 20% by mass or more, 30% by mass or more, 40% by mass or more, or 43% by mass or more, based on the total mass (dry mass) of the composition, and may be 100% by mass or less, 90% by mass or less, or 80% by mass or less.

[0041] In the composition, the content (dry mass) of acid-soluble lignin may be 1 mass % or more, 1.5 mass % or more, 2 mass % or more, or 2.3 mass % or more, based on the total mass (dry mass) of the composition, and may be 50 mass % or less, 30 mass % or less, or 10 mass % or less.

[0042] In the composition, the content (dry mass) of alcohol / benzene soluble matter may be 1 mass% or more, 1.5 mass% or more, 2 mass% or more, or 2.3 mass% or more, and may be 10 mass% or less, 5 mass% or less, or 3 mass% or less, based on the total mass (dry mass) of the composition. In this specification, the alcohol / benzene soluble matter content means a value determined in accordance with JAPAN TAPPI Paper Pulp Testing Method No. 63:2000.

[0043] The lignin content (dry mass) in the composition, as measured by the detergent method, may be 36% by mass or more, 40% by mass or more, 44% by mass or more, or 48% by mass or more, based on the total mass (dry mass) of the composition, and may be 100% by mass or less, or 95% by mass or less.

[0044] The cellulose content (dry mass) in the composition, measured by the detergent method, may be 1% by mass or more, 5% by mass or more, 10% by mass or more, or 20% by mass or more, and may be 40% by mass or less, 30% by mass or less, or 25% by mass or less, based on the total mass (dry mass) of the composition.

[0045] The hemicellulose content (dry mass) in the composition, as measured by the detergent method, may be 1% by mass or more, 3% by mass or more, 5% by mass or more, or 7% by mass or more, based on the total mass (dry mass) of the composition, and may be 20% by mass or less, 15% by mass or less, or 11% by mass or less.

[0046] The mass ratio of the lignin content (dry mass) to the cellulose content (dry mass) in the composition (lignin content / cellulose content), as measured by the detergent method, may be 0.5 or more, 1 or more, 1.5 or more, or 2 or more, and may be 50 or less, 30 or less, 10 or less, 5 or less, or 3 or less.

[0047] The mass ratio of the lignin content (dry mass) to the hemicellulose content (dry mass) in the composition (lignin content / hemicellulose content), as measured by the detergent method, may be 1 or more, 2 or more, 3 or more, 4 or more, 4.5 or more, or 5 or more, and may be 50 or less, 30 or less, 20 or less, 10 or less, or 5.5 or less.

[0048] Generally, compositions containing lignin are obtained by subjecting lignin-containing plants to chemical treatment with alkali or physical treatment such as explosion treatment and hydrothermal treatment. In these cases, moisture is added during the chemical or physical treatment, resulting in a composition containing a large amount of moisture. On the other hand, a composition according to one embodiment can be obtained by a method that does not require the addition of moisture, as described above. Therefore, a composition according to one embodiment may have a relatively low moisture content. A composition according to one embodiment may be a solid.

[0049] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0050] <Preparation of artificial bait> The following materials were prepared for the artificial bait: Bagasse Cedar powder Okara

[0051] (Bait A) Using a bench press and a mold, the bagasse was compression molded to prepare a block of bait A. The moisture content of bait A was 30 to 70% by mass based on the total mass of bait A.

[0052] (Bait B) A mixture of bagasse and cedar powder was compression molded using a bench press and a mold to produce block-shaped bait B. The volume ratio of the bagasse to cedar powder mixture was bagasse:cedar powder = 1:1. The moisture content of bait B was 30 to 70 mass% based on the total mass of bait B.

[0053] (Bait C) A mixture of bagasse, cedar powder, and soybean pulp was compression-molded using a bench press and a mold to produce block-shaped bait C. The volume ratio of the bagasse, cedar powder, and soybean pulp to the soybean pulp was bagasse:cedar powder:soybean pulp (volume ratio) = 1:1:0.1. The moisture content of bait C was 30 to 70% by mass based on the total mass of bait C.

[0054] <Experiment for rearing giant termites using artificial bait> A colony of giant termites was transplanted onto each of the baits A to C and reared at room temperature (25° C.) for 4 weeks. After that, the giant termites and the termite soil (solid matter) were collected.

[0055] <Component Analysis of Terrier Soil> The components of the terrier soil collected in the system using bait A were analyzed as follows. The terrier soil contained feces of the formosan termites.

[0056] (Acid-insoluble lignin) The content of acid-insoluble lignin in the ant soil was determined by acid decomposition-gravimetric method (Klason method). As a result, the content (dry mass) of acid-insoluble lignin was 44.9% by mass based on the total amount (dry mass) of the ant soil. The lower limit of quantification by this analytical method was 0.1% by mass in dry mass.

[0057] (Acid-soluble lignin) The content of acid-soluble lignin in the ant soil was determined by acid decomposition-absorption spectrophotometry (Klason method). As a result, the content of acid-soluble lignin (dry mass) was 2.6% by mass based on the total amount (dry mass) of the ant soil. The lower limit of quantification by this analytical method was 0.2% by mass in dry mass.

[0058] (Alcohol-benzene soluble content) The alcohol-benzene soluble content was determined in accordance with JAPAN TAPPI Paper Pulp Test Method No. 63:2000. As a result, the alcohol-benzene soluble content (dry mass) was 2.4% by mass based on the total mass (dry mass) of the ant soil. The lower limit of quantification by this analytical method was 0.1% by mass on a dry mass basis.

[0059] In addition, for each of the systems using diets A to C, we investigated (a) worker survival rate, (b) weight loss rate per worker, (c) feed consumption rate, and (d) apparent digestibility. The calculation formulas are as follows: (a) Worker survival rate (%) = 100 × (number of workers surviving after rearing) / (number of workers before rearing) (b) Weight loss per worker (mg) = {(sum of all worker weights before rearing (mg) / (number of workers before rearing)} - {(sum of all worker weights after rearing (mg) / (number of workers after rearing)} (c) Feed consumption rate (%) = 100 × (feed intake (dry mass g)) / (feed amount before rearing (dry mass g)) (d) Apparent digestibility (ADC) (%) = 100 × {(feed intake (dry mass g)) - (feces weight (dry mass g))} / (feed intake (dry mass g))

[0060] The above-mentioned rearing experiment was conducted in five test groups for each strain, and (a) to (d) were calculated for each. Statistical analysis was performed using a generalized linear mixed model (GLMM) for (a) and a linear mixed model (LMM) for (b) to (c), followed by a post-hoc test using the Tukey HSD Test (P<0.05). The results are shown in Figures 1(a) to 1(d). In each graph in Figures 1(a) to 1(d), the data represent, from left to right, the results for the strain using diet A, the strain using diet B, and the strain using diet C. Each data point is assigned a letter, and data points assigned different letters were deemed to be significantly different. Error bars indicate standard error.

[0061] <Preparation of bait D> Bait D was prepared by mixing bagasse and cedar powder and burying cedar wood in the bagasse and cedar powder mixture. The volume ratio of the bagasse to cedar powder mixture was bagasse:cedar powder (volume ratio) = 1:1. The cedar wood accounted for 20 to 40% by volume of the total volume of bait D. The moisture content of bait D was 30 to 70% by mass of the total mass of bait D.

[0062] <Experiment for Rearing Giant Termites Using Bait D> A colony of giant termites was transplanted onto the above-mentioned bait D and reared at room temperature (25° C.) for 4 months. After that, the giant termites and the termite soil (solid matter) were collected.

[0063] <Component Analysis of Ant Soil Recovered in a System Using Bait D> The detergent method was used to determine the cellulose, hemicellulose, and lignin contents of the ant soil recovered in a system using bait D. The ant soil contained the feces of formosan termites. Based on the total dry mass of the ant soil, the cellulose content (dry mass) was 22% by mass, the hemicellulose content (dry mass) was 9.6% by mass, and the lignin content (dry mass) was 49% by mass. The lower limit of quantification using this analytical method was 0.5% by dry mass. Generally, the cellulose content in plants is considered to be 40-50% by mass, and the hemicellulose and lignin contents are approximately 15-35% by mass each (see Non-Patent Document 2, RSC Adv., 12, 3946-3956, 2013). The lignin content in the above composition was high, confirming that lignin had been extracted.

Claims

1. A method for extracting lignin contained in herbs, comprising feeding herbs on termites that eat said herbs.

2. The method according to claim 1, wherein the herbaceous plant is a grass plant.

3. A method for producing a composition comprising isolated herbaceous lignin, comprising feeding on the herbaceous plant to food-eating termites.

4. The method according to claim 3, wherein the herbaceous plant is a grass family herbaceous plant.

5. A composition comprising isolated herbaceous lignin.