JP2017075133A - Method for solubilizing secondary cell walls - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide novel food materials derived from plant secondary cell walls.SOLUTION: A plant cell in which a primary cell wall is removed or crushed with cellulase is heated in an environment of pH 2.5 to 3.5. Then, an insoluble matter is removed by centrifugation or the like to obtain a solution which is a solubilized product according to the present invention. Furthermore, after removing the insoluble matter, it may be recovered as a precipitate by cooling, thawing after freezing, salt precipitation or the like to obtain a solubilized product according to the present invention. The solubilized product has excellent emulsifying action and foaming property and can be used as an emulsifying agent. In addition, since it is also a solubilized product of pectin, it can be used as various food materials as prebiotics.SELECTED DRAWING: Figure 6

Description

  The present invention relates to a method for solubilizing plant secondary cell walls.

  Plant food materials are produced in huge quantities as agricultural products such as cereals, fruits, or strawberries, and are used in various food raw materials, fruit juices, fermentation raw materials, and feeds. However, there are many so-called unused agricultural products that are not easily processed. These are treated as hardly degradable fibers, and some of them are used as feed, but most of them have been discarded. Moreover, even the feed has many problems that the fattening effect is small because of its indegradability, that is, it is difficult to digest and the use components are difficult to extract. For example, in soybean, it is snow flower vegetables (okara), soy sauce cake, in potato it is potato pulp, and in apples and mandarin oranges, it is apple cake and mandarin orange cake. These are all so-called cocoons mainly composed of cell walls obtained by squeezing fats and oils, proteins, starches and fruit juices. In addition, a large amount of potato (fermented potato) is produced after fermentation from soybeans, sweet potatoes, apples, etc., which are widely used as fermentation raw materials, and most of them are discarded. And not only the straw but also the agricultural products themselves are not fully processed and used. On the other hand, the use of plant materials as raw materials such as bioethanol and biomass has been studied, but it is not sufficiently used in this field because it is difficult to decompose.

  By the way, solubilization is important in order to use these persistent plant materials more than ever, and if they can be easily solubilized, they should be further usable as food materials and biomass materials. These persistent plant food materials have been clarified in their tissues and constituents, and among the cell walls, the main tissue of the persistent cells is the secondary cell wall existing inside the cells. (Non-Patent Documents 1 to 3). Therefore, a method of solubilizing the secondary cell wall is desired for effective use of the hardly degradable fiber. For example, in soybean seeds, there is a soybean body structure composed of an oil body containing oil and a protein body containing protein inside the secondary cell wall inside the primary cell wall. Since the primary cell wall is mainly composed of cellulose, it can be easily solubilized and removed by cellulase if it is made into a single cell. However, since the secondary cell wall is a glycoprotein mainly composed of pectin and protein, it cannot be solubilized by cellulase or ordinary pectinase.

  Until now, as a method for solubilizing the secondary cell wall, a method using a strong alkali such as sodium hydroxide has been known (Non-patent Document 2), but these methods can be used as food materials as they are after solubilization. Can not. Further, since solubilization in the alkaline region easily generates nephrotoxic substances such as lysinoalanine, the method cannot be used. There is a method using sodium dodecyl sulfate (SDS), which is a denaturing agent, but since SDS is also toxic, it cannot be used as a food material. In addition, strong acid degradation is used for the production of monosaccharides in the biomass field. For example, when it is used in the food field, it is a viscous material, gelled material, polysaccharide or protein. Is unsuitable for low molecular weight. Although methods for solubilizing secondary cell walls with specific pectinases have been reported (Patent Document 1, Non-Patent Document 2), low-molecular digests such as monosaccharides, oligosaccharides, and peptides are used for enzymatic degradation. Although it is suitable for the production of lysozyme, it is not suitable for uses other than such low-molecular-weight digests, and it is considered not preferable that the enzyme production is expensive. Another simple and inexpensive method Is desired.

JP 2004-43584 A

Bob. B. Buchanan et al., Plant Biochemistry and Molecular Biology, Academic Publishing Center, pages 47-48 Kasai et al., Enzymatic High Digestion of Soybean Milk Residue (Okara), J. Agr & Food Chem., 2004, Vol. 52, No. 18, pp. 5709-5716 Kasai, Are Agricultural Plant Products and Residues Indigestive Fiberes? , J. Appl. Glycosci., 2008, 55, pp. 133-141 (2008).

  The present invention is to provide a new food material by decomposing a secondary cell wall under mild conditions without using an enzyme.

  The present invention relates to a method for solubilizing a secondary cell wall, comprising a step of heating in an environment of pH 2.5 to 3.5.

  According to the present invention, a new food material containing a degradation product that can be used as an emulsifier is provided by degrading and solubilizing secondary cell walls under mild conditions.

FIG. 1 is a microscopic image of soybean cells before and after heat treatment of the secondary cell wall. (A) is an image after primary cell wall decomposition, and (B) is an image after secondary cell wall decomposition. The broken line box in (A) shows the secondary cell wall. FIG. 2 is a diagram showing the effect of changes in pH on solubilization of soybean secondary cell walls. FIG. 3 is a diagram showing the influence of changes in heating time on solubilization of soybean secondary cell walls. FIG. 4 is a diagram showing the influence of changes in heating temperature on solubilization of soybean secondary cell walls. FIG. 5 is a diagram showing the relationship between the solubilized pH of the soybean secondary cell wall and the heating temperature. FIG. 6 is a microscopic image after heating a commercially available raw okara at pH 3.0. (A) is an image before heating, and (B) is an image after heating. The box in (A) shows the secondary cell wall. FIG. 7 is an image showing the state change of the liquid before and after the heat treatment of the soybean secondary cell wall. (A) shows the state before heating, and (B) shows the state after heating. FIG. 8 is an SDS-PAGE image of a solubilized product of soybean secondary cell walls. (A) shows protein staining with Bio-Safe CBB G250, (B) shows protein staining with CBB R-250, and (C) shows sugar staining with PAS staining. FIG. 9 is a chromatogram in HPLC analysis of a solubilized product of soybean secondary cell walls under various pH conditions. FIG. 10 is a chromatogram in HPLC analysis of aggregates obtained by solubilizing soybean secondary cell walls. A is the supernatant of the solution obtained by solubilization, B is the supernatant of the solution obtained by freezing and thawing it, C is the solution obtained by re-dissolving the aggregate obtained by freezing and thawing Indicates. FIG. 11 is a chromatogram in gel filtration chromatography (by Sephacryl S-100HR) of solubilized product of soybean secondary cell wall. FIG. 12 is a chromatogram in HPLC analysis of the fraction obtained by gel filtration chromatography (using Sephacryl S-100HR). FIG. 13 is an SDS-PAGE image of the fraction obtained by gel filtration chromatography (using Sephacryl S-100HR). FIG. 14 is a diagram showing the analysis results of neutral sugars in the fraction obtained by gel filtration chromatography (using Sephacryl S-100HR). FIG. 15 is a graph showing the degree of methylation of uronic acid in the fraction obtained by gel filtration chromatography (using Sephacryl S-100HR). FIG. 16 shows the results of amino acid analysis in the fraction obtained by gel filtration chromatography (using Sephacryl S-100HR). FIG. 17 is a chromatogram in HPLC analysis of a fraction (Fr. 20) obtained by gel filtration chromatography (using Sephacryl S-100HR). (A) is a chromatogram with a detection wavelength of 280 nm, and (B) is a chromatogram with RI. FIG. 18 is a chromatogram in HPLC analysis of a fraction (Fr. 20) obtained by gel filtration chromatography (using Sephacryl S-100HR). (A) is a chromatogram with a detection wavelength of 280 nm, and (B) is a chromatogram with RI. FIG. 19 is a microscopic image showing the emulsifying action of the solubilized product of soybean secondary cell walls. FIG. 20 is a graph showing the emulsion stability of a solubilized product of soybean secondary cell walls. FIG. 21 is a microscopic image showing a state after 5 hours of emulsification with a solubilized product of soybean secondary cell walls. FIG. 22 is a microscopic image showing the effect of pH change on the emulsifying action of a solubilized product of soybean secondary cell walls. FIG. 23 is an image showing foaming properties of a solubilized product of soybean secondary cell walls. (A) is an image by a solubilized product of soybean secondary cell wall, (B) is an image by casein aqueous solution, and (C) is an image by albumin aqueous solution. FIG. 24 is a diagram showing the influence of pH change on foamability. FIG. 25 is a microscopic image after heating the secondary cell wall of red beans at pH 3.0. (A) is an image before heating, and (B) is an image after heating. The arrow in the figure indicates the secondary cell wall. FIG. 26 is a microscopic image after the secondary cell wall of the mandarin orange was heated at pH 3.0. (A) is an image before heating, and (B) is an image after heating. The arrow in the figure indicates the secondary cell wall. FIG. 27 is a chromatogram in HPLC analysis of the aggregate obtained by solubilizing the secondary cell wall of apple. (A) is a chromatogram with a detection wavelength of 280 nm, and (B) is a chromatogram with RI. FIG. 28 is a chromatogram in HPLC analysis of the aggregate obtained by solubilizing the secondary cell wall of mandarin orange. (A) is a chromatogram with a detection wavelength of 280 nm, and (B) is a chromatogram with RI. FIG. 29 is an image showing the emulsifying action of the solubilized product of the apple secondary cell wall. FIG. 30 is an image showing the foamability of the lysate of the apple secondary cell wall. (A) is an image of an apple secondary cell wall lysate, (B) is an image of a casein aqueous solution, and (C) is an image of an albumin aqueous solution.

  The solubilization method in the present invention is a method of solubilizing a secondary cell wall by heating in an environment of pH 2.5 or more and 3.5 or less. This selectively solubilizes the secondary cell wall. Here, “selective” means that only the secondary cell wall is decomposed and solubilized, and the secondary cell wall is selectively solubilized so that the secondary cell wall is removed from the cells from which the primary cell wall has been removed or crushed. Is solubilized, leaving a so-called body part. Selective means that the chemical components of the body part are hardly decomposed, and preferably the body structure remains without destroying the structure of the body structure. However, when used as feed, it may be preferable that the body structure is structurally broken in order to improve digestion, and the body structure may be broken. In the solubilization of the present invention, the body structure may be destroyed, and the chemical composition of the body part may be decomposed and eluted as a part of the solubilized product. The proportion of cells eluted in the lysate by the chemical composition is preferably less than 10%, more preferably less than 5%, desirably less than 1% of the number of cells having secondary cell walls before heating. is there. Further, the secondary cell wall is selectively solubilized so that the secondary cell wall is solubilized from the cells from which the primary cell wall has not been removed, and if the primary cell wall is later solubilized and removed by cellulase, the body structure remains. In addition, selective solubilization means the solubilization rate of the secondary cell wall, that is, the ratio of the number of cells that have lost the secondary cell wall after heating to the number of cells that had the secondary cell wall before heating. Is at least 30%, preferably 70% or more, more desirably 90% or more, and more desirably 95% or more.

  The solubilized plant in the present invention is not particularly limited as long as it has a secondary cell wall, and may preferably be a plant cell that can be a food material. Plant food materials are beans such as soybeans and red beans, potatoes such as potatoes and sweet potatoes, cereals such as rice, wheat and corn, fruits such as bananas, tangerines and apples, and sesame seeds. And other plant seeds such as sunflower, spinach and Japanese mustard leaf. The target of solubilization may be the food material itself, but is preferably various squeezed rice cakes such as okara, soy sauce cake, apple cake, and defatted soybean, which are residues after being processed into food. In addition to these food materials, oil seeds such as camellia seeds can also be preferred targets.

  In the present invention, a step of making a plant cell into a single cell before solubilization is necessary. The method of unicellularization is not particularly limited as long as the plant cell can be converted into a single cell. For example, the method described in Patent Document 1 or Japanese Patent Application Laid-Open No. 2002-256281, that is, a method of immersing in hot water or an autoclave is used. A method of heating is shown. Although it is desirable that all cells are unicellular after being unicellularized, some of the cells may be in contact with other cells via an intercellular adhesive substance. Preferably, the number of unicellular cells is 60% or more, more preferably 80% or more of the total number of cells. In addition, in the case of a plant having a large amount of soluble pectin, it is preferable to remove the soluble pectin without using a single cell, or after using a single cell, using hot water or the like.

  In the present invention, in order to obtain a lysate of the secondary cell wall, it is preferable to remove the primary cell wall of the plant cell before solubilization. As a method for removing the primary cell wall, for example, a method using cellulase, specifically, for example, a method described in Patent Document 1 is exemplified. Further, the primary cell wall may be crushed or broken without removing the primary cell wall. For example, a method of removing lipid or drying using an extruder or solvent extraction is exemplified. For example, it can not be said that primary cell walls have been removed in defatted soybeans, but the primary cell walls are crushed (or destroyed) by depressurization and extraction (degreasing) of fats and oils, and so-called pores are formed in the primary cell walls. It is in a state. If the defatted soybean in such a state is heated in an acidic environment, the secondary cell wall is solubilized. Therefore, in the defatted soybean, such an operation of removing the primary cell wall is not necessary, but the secondary cell wall is easily solubilized by further removing the primary cell wall.

  Plant cells are heated in an environment having a pH of 2.5 to 3.5, preferably 3.0 to 3.5. Under acidic conditions where the pH is less than 2.5, not only the secondary cell walls but also proteins such as so-called body structures from proteins other than the secondary cell walls are solubilized or extracted, and the molecular weight is reduced by acid. The amount of released material increases. In addition, when the pH is higher than 3.5, the secondary cell wall cannot be sufficiently solubilized.

  The heating temperature is 80 ° C. or higher and 125 ° C. or lower, preferably 100 ° C. or higher and 125 ° C. or lower. The heating time is 10 minutes or more and 30 minutes or less, preferably about 20 minutes. If it exceeds 30 minutes, the amount of protein or the like leaking from the body part may increase, or the secondary cell wall may be further reduced in molecular weight. Further, at a temperature lower than 80 ° C., heating for 30 minutes or more is required, and the secondary cell wall may not be sufficiently solubilized.

  Plant cells may be placed in an environment of the above pH during heating, and may be in a wet state in a paste form. Moreover, in order to isolate | separate the obtained solubilized material, Preferably it heats in the liquid adjusted to the said pH range. The liquid is preferably water. The amount of water to be added may be any amount that allows the plant cells to wet in a paste form, and is preferably 1/10 or more, more preferably twice or more, and more preferably about 10 times the amount of cells and mass.

  The solubilized product according to the present invention is a decomposed product of the secondary cell wall obtained by removing insolubles from a solution obtained by heating the plant cell after removing or crushing the primary cell wall under the above conditions. The decomposition product may be a mixture of decomposition products or may be a substance (solubilized substance) obtained by separation from the mixture. When the solubilized product according to the present invention is obtained as a mixture, it is also a solution from which insoluble materials that have not been solubilized after heating under the above conditions are removed, and the body and intercellular tissue remaining without being solubilized by heating are removed. It can also be a contained solution. Further, it may be a concentrate obtained by concentrating these solutions.

  The solubilized substance is also an aggregate recovered by aggregating from the liquid excluding the body part and intercellular tissue remaining unsolubilized under the above conditions, and from the liquid excluding the body part and intercellular tissue. Or a fraction collected by a fractionation operation such as gel filtration chromatography (a fraction containing a substance capable of forming the aggregate). This aggregate is obtained by cooling a solution from which the body part or intercellular tissue has been removed (for example, a supernatant of centrifugation), a method of thawing after freezing, or a method of concentrating on a separation membrane having a molecular weight of 5000 or more (in this case, If necessary, a method such as freeze-drying is added after concentration), a method of adding an organic solvent such as ethanol or acetone, a method of adding calcium ions to gel, a method of freeze-drying, and the like. The solubilized product recovered as an aggregate is easily resolubilized with an acid, an alkali, or a chelating agent.

  The aggregate (solubilized substance) is a substance that appears as a polymer peak H under the HPLC conditions described in the Examples, and includes a pectin composed of a polymer polymerized with galacturonic acid and a protein composed therewith. It is a degradation product of the secondary cell wall containing. This degradation product is a glycoprotein having a molecular weight of 4500-75000 when gel filtration chromatography (by Sephacryl S-100HR) is performed, and has a characteristic that the molecular weight is associated with 500,000 or more by applying pressure of 2 MPa or more. Have. Moreover, the methylation degree of the glycoprotein is 25 to 35%, and isoleucine (Ile) is 12 to 25 mol% in all amino acids. The degree of methylation here is the molar ratio (MtOH / Gal A) of methanol produced by saponifying the glycoprotein unit to the total galacturonic acid in the glycoprotein unit. The above-described characteristics are the minimum characteristics required to the extent that they can be specified as the solubilizing substance. Further, the solubilizing substance according to the present invention is a polymer substance in which a protein is bound to a sugar chain centered on galacturonic acid, and it is impossible to directly specify the structure of the solubilizing substance. It can be said that it is not practical to specify exactly. An example of the profile of neutral sugars and constituent amino acids constituting the solubilizing substance is as follows. For example, in soybean, aspartic acid and asparagine (Asx) are 9 to 10 mol% and glutamic acid is present in soybean. And glutamine (Glx) 8-20 mol%, lysine (Lys) 3-8 mol%, alanine (Ala) 2-10 mol%, arginine (Arg) 3-6 mol%, valine (Val) 3 to 8 mol%. In addition, the composition ratio of neutral sugars that appear characteristically is, for example, soybean, 20-30 mol% arabinose (Ara), 30-70% galactose, 0-30% rhamnose, and 10% glucose. It is as follows. The solubilized product according to the present invention is obtained from a plant secondary cell wall, and is different from that obtained from a middle lamella which is a plant primary cell wall gap.

  Since the solubilized product according to the present invention exhibits an emulsifying action and is excellent in foaming properties, it can be used as an emulsifier or a foaming agent. Further, since it is obtained under weak acidity of pH 2.5 or more and 3.5 or less, the solution obtained by removing the body part and intercellular tissue after solubilizing the secondary cell wall can be used, for example, as a food additive or the like without neutralization. It can be used as feed and fertilizer. But you may decide to use the solution after neutralizing the said solution, and can also use the solubilization substance collect | recovered from the said solution by the said means as an emulsifier or a foaming agent. Without being neutralized, it can be used as it is, for example, as a food additive, feed or fertilizer. The amount used as an emulsifier or a foaming agent can be appropriately determined by those skilled in the art according to the purpose of use and the emulsification target. Furthermore, since the pectin degradation product grows Bifidobacteria well, a prebiotic effect is expected. Therefore, the solubilized product according to the present invention can be used as a food material having a prebiotic effect.

  Next, the present invention will be described in more detail based on the following examples, but it goes without saying that the present invention is not limited to the following examples.

(Preparation of primary cell wall degrading soybean (2CW paste))
100 ml of water (10 times the weight of soybean) was added to 10 g of US-grown soybean for essential oil (oil content: 20.68% by Soxhlet extraction method), and autoclaved at 121 ° C. for 20 minutes. After removing the extract, it was made into a paste. To this soybean paste, 100 ml of 20 mM acetate buffer (pH 5.0) and 5% of cellulase (GODO-TCF: Godo Sakesei Co., Ltd.) were added and reacted at 37 ° C. overnight. After removing the supernatant by centrifugation (2000 rpm, 10 minutes), suspended by adding 100 ml of 20 mM acetate buffer (pH 5.0), and then centrifuging again and discarding the supernatant (repeated 3 times) . Thereafter, an equal volume of 20 mM acetate buffer (pH 5.0) was added to the residue and mixed to obtain soybean cells after degrading the primary cell wall (hereinafter referred to as “2CW paste”). Soybean was unicellularized and cells with expanded secondary cell walls were observed under a phase contrast microscope.

[Solubilization of secondary cell wall (2CW) by acid heat treatment]
(1) Effect of pH
80 μL of 2CW paste was added to 800 μL of 0.1 M acetate buffer or CH ₃ COONa / HCl buffer (pH 4 to 1.5). After stirring, 400 μL was collected and heated at 100 ° C. for 20 minutes, and then the supernatant was collected by centrifugation (10000 rpm, 10 minutes, 4 ° C.). The total sugar amount, protein amount, and total uronic acid amount before and after heating were measured for the supernatant, and the amount of increase in total sugar amount, protein amount, and total uronic acid amount due to heating was calculated. Further, the solubilization rate of 2CW was measured with a phase contrast microscope. The total sugar amount, protein amount, and total uronic acid amount were determined by the following methods. These measurements were performed in the same manner below. The amount of increase when the same heating was performed using distilled water (pH 5.3) was subtracted from each increase before and after heating as a blank to obtain the increase before and after heating.

  Images of soybean cells before and after the heat treatment (pH 3.0) under a phase contrast microscope are shown in FIG. The results are shown in FIG. 2CW became solubilized around pH 3.5, and the minimum acidic condition necessary to solubilize almost 100% of 2CW was pH 3.0. At pH 2.0 and below, the increase in TP, TS, and TU increased further, indicating that 2CW could be further reduced in molecular weight and soy body solubilization.

a) Measurement of total sugar (TS) Total sugar was determined by the phenol-sulfuric acid method. 5% phenol and concentrated sulfuric acid were added to the sample, the absorbance at 492 nm was measured, and the total amount of sugar in the sample was determined from a calibration curve prepared in advance.
b) Measurement of protein amount (TP: Total Protein) It was determined by the Bradford method. A protein assay staining solution was added to the sample, mixed, and allowed to stand at room temperature for 5 minutes. Then, the absorbance at 600 nm was measured, and the protein concentration in the sample was determined from a calibration curve prepared in advance.
c) Measurement of total uronic acid (TU)
Obtained by the 3-phenylphenol method. Add Na ₂ B ₄ O ₇ / H ₂ SO ₄ to the sample, mix well, heat at 100 ° C, cool with running water, add 3-phenylphenol, mix well, and then measure the absorbance at 546 nm. The protein concentration in the sample was determined from a calibration curve prepared in advance.
d) Calculation of solubilization rate The solubilization rate was calculated | required by the following formula | equation (Equation 1). The percentage (%) of cells that do not already have 2CW before heating was blank.

(2) Effect of heating time
80 μL of 2CW paste was added to 80 μL of 0.1 M acetate buffer. After stirring, 400 μL was collected and heated at 100 ° C. for 3 to 20 minutes, and then the supernatant was collected by centrifugation (10000 rpm, 10 minutes, 4 ° C.). The total sugar amount, protein amount, and total uronic acid amount in the supernatant were measured. Further, the 2CW solubilization rate was measured with a phase contrast microscope. The result is shown in FIG. The results show that 2CW can be almost solubilized by heating for at least 10 minutes at pH 3.0 and 100 ° C.

(3) Influence of heating temperature
80 μL of 2CW paste was added to 800 μL of 0.1 M acetate buffer. After stirring, 400 μL was collected and heated at 60 to 100 ° C. and autoclave 121 ° C. for 20 minutes, and then the supernatant was collected by centrifugation (10000 rpm, 10 minutes, 4 ° C.). The total sugar amount, protein amount, and total uronic acid amount in the supernatant were measured. Further, the 2CW solubilization rate was measured with a phase contrast microscope. The result is shown in FIG. It was found that 2CW can be almost solubilized by heating at least 100 ° C under the conditions of pH 3.0 and 20 minutes. It was also observed that the increase in TP, TS, and TU increased when the temperature was 121 ° C or higher. The possibility of solubilization of soybean body as well as 2CW was also considered.

(4) Determination of minimum conditions for solubilized pH and heating temperature
0.1M acetate buffer solutions having pH of 2, 2.5, 3, and 3.5 were prepared, and 80 μL of 2CW paste was added to 800 μL each. After stirring, 400 μL of the sample is collected, heated at 20-110 ° C and autoclave 121 ° C for 20 minutes, and then measured for 2CW solubilization rate with a phase-contrast microscope. The temperature conditions when solubilized were determined. The result is shown in FIG. As a result, it was found that heating at 100 ° C. was necessary at pH 3.0. In addition, 2CW could be solubilized by autoclaving even at pH 3.5. When the pH was lowered to 2.0, solubilization was possible even at 60 ° C.

(5) Solubilization of raw okara Ten times the amount of distilled water was added to commercially available raw okara, 5% of cellulase (GODO-TCF) was added to the total amount, and the mixture was reacted at 37 ° C. overnight. After removing the supernatant by centrifugation (2000 rpm, 10 minutes), the same amount of water was added to prepare an Okara 2CW paste. Both were mixed at a ratio of 3 mL of 0.1 M pH 3.0 acetate buffer and 600 μL of Okara 2 CW paste, and heated at 100 ° C. for 20 minutes to solubilize the secondary cell wall. Then, it was centrifuged (3500 rpm, 5 min).

  When observed with a phase-contrast microscope before and after heating, 2CW in the form of a transparent film was observed before heating, but 2CW disappeared after boiling, and soy body and intercellular tissues were observed (see FIG. 6). ). When the paste solution (14 mL) before and after heating was centrifuged, the precipitate after heating was reduced to 18% with respect to the precipitate before heating (see FIG. 7). Thus, it was confirmed that the secondary cell wall was solubilized from okara which is soybean meal.

[Characterization of secondary cell wall (2CW) lysate by acid heat treatment 1]
The characteristics of the solubilizate obtained by heating under acidic conditions were investigated.
(1) SDS-PAGE
10 mL of the 2CW paste prepared in Example 1 was added to 40 mL of ion-exchanged water, and solutions of pH 5 to 1 were prepared using 6M HCl. 3 mL of each was transferred to a test tube and heated at 60 ° C., 80 ° C., 100 ° C., and an aluminum block thermostatic bath 121 ° C. for 20 minutes. The supernatant was collected by centrifugation (4000 rpm, 10 minutes), and further filtered with a membrane filter (0.2 μm), and 1 mL of the supernatant was lyophilized. 100 μL of ion exchange water was added to this to prepare a sample. These samples and the sample treatment solution were mixed at a ratio of 4: 1, heated for 10 minutes, and then allowed to cool. Using an electrophoresis apparatus and 15% gel (Ato) and 15% Tricine gel (Ato), electrophoresis was performed at 20 mA for 75 minutes, and Bio-Safe CBB G250 and CBB R-250 (Quick Start (each (Trade name) Bradford 1x Dye Reagent (Bio-Rad Laboratories) was used for protein staining and PAS staining for sugar.

  Sample treatment solutions include 3.75 g sodium dodecyl sulfate (SDS), 2.50 mL 2-mercaptoethanol, 8.38 mL 1 M Tris-HCl buffer (pH 6.8), 6.25 mL glycerin, 2.50 mg BPB (bromophenol blue) was dissolved in ion-exchanged water to make 500 mL. In addition, 1.5 g (25 mM) of trishydroxymethylaminomethane (Tris), 0.5 g (0.1%) of SDS, and 7.2 g (192 mM) of glycine were added to ion-exchanged water in the electrophoresis buffer (10-fold solution). A solution that was dissolved to a total volume of 500 mL was used.

  The SDS-PAGE image is shown in FIG. In CBB G250 staining and CBB R-250 staining, an average molecular weight of about 6.6 kDa (molecular weight was calculated from the central value in the vicinity of the strong color appearance) was generated by solubilization of 2 CW when heated at pH 3.0 and 100 ° C. It was found that there was a band considered to be a material. Even in PAS staining, light staining was observed around the same molecular weight, suggesting that the substance may be a compound in which a sugar is bound to a protein. Similarly, bands were observed around 6.6 kDa at pH 3.5 / 121 ° C, pH 2.5 / 80 ° C, and pH 2.0 / 60 ° C. Further, in the heat-treated extract having a pH of less than 3.0, a staining band was observed even at a molecular weight of 50 kDa or more, suggesting that many substances other than the substance were solubilized.

(2) HPLC analysis 10 mL of the 2CW paste prepared in Example 1 was added to 40 mL of ion-exchanged water, and solutions of pH 5 to 1 were prepared using 6M HCl. Transfer 3 mL of each to a test tube and heat at 100 ° C (pH 2, 2.5, and 3.5 were also heated at 60 ° C, 80 ° C, and 121 ° C, respectively), then centrifuge (4000 rpm, 10 minutes) Was filtered through a membrane filter (0.20 μm) to prepare a sample for analysis. The obtained sample was subjected to HPLC analysis. For HPLC analysis, pump DP-8020 (manufactured by Tosoh Corporation), UV / visible spectrophotometer UV-8020 (manufactured by Tosoh Corporation), differential refractive index (RI) detector RID-6A (Shimadzu Corporation), HPLC column TSK-GEL G3000 SWXL (manufactured by Tosoh Corporation) was used. Analysis was performed at a flow rate of 1.0 mL / min using distilled water as an eluent.

  The result is shown in FIG. When the pH is lowered, it can be seen that a polymer peak H (about 3.5 min) appears in UV280 and RI at pH 3.5 to 2.5, and this polymer peak H is generated by solubilization of the secondary cell wall. It was suggested that the main component of When the pH is further lowered below 2.5, the polymer peak H becomes larger and the appearance of other peaks (about 12 min and about 13 min) gradually begins to appear. ), It is assumed that other than 2CW is also solubilized.

(3) Formation of aggregates by freezing 10 mL of the 2CW paste prepared in Example 1 was added to 40 mL of ion-exchanged water, and a 2CW mixed solution having a pH of 3.0 was prepared using 6M HCl. 1600 μL of this solution was heated at 100 ° C. for 20 minutes. After centrifugation (13200 rpm, 4 ° C., 10 minutes), the supernatant was collected (this solution was designated as A), and 1200 μL thereof was frozen. The resulting supernatant was collected by centrifuging (13200 rpm, 4 ° C., 10 minutes) to give a solution B. The remaining aggregate was washed by adding ion-exchanged water, and the supernatant was discarded by centrifugation (13200 rpm, 4 ° C., 10 minutes). This operation was repeated twice. An aqueous solution of HCl adjusted to pH 3.0 in the same amount as the supernatant collected after thawing was added to the washed aggregate and stirred, and then heated at 100 ° C. for 20 minutes to give solution C. The prepared solutions A, B, and C were measured for total sugar amount, protein amount, and total uronic acid amount, and each solution subjected to filter filtration was subjected to HPLC analysis under the same conditions as in Example 2 (2). It was. The result is shown in FIG.

  In the solution C in which the precipitate was redissolved, it was found that the polymer peak H solubilized at pH 3.0 / 100 ° C. shown in FIG. 9 appeared as the main peak. Further, in the remaining solution B excluding aggregates after freezing, the polymer peak H disappeared. Table 1 shows the TS, TP, and TU concentrations of solutions A, B, and C. Solution C was found to contain about 60% uronic acid in Solution A. In addition, TU / TS = 0.26 of solution A, while TU / TS = 0.42 of solution C, the ratio of uronic acid to the total amount of sugar in the solution was high. From this result, the aggregate formed by freezing is the main substance of the solubilized product produced by heating at pH 3.0, and is a substance with a high uronic acid content in which a high molecular peak H appears by HPLC analysis, and also contains protein. It was thought to do.

[Characteristics of secondary cell wall (2CW) lysate by acid heat treatment 2]
Gel filtration chromatography (Sephacryl S-100HR) was performed on 2CW solubilized product solubilized by heating at pH 3.0, and the characteristics of the solubilized product were further investigated.
(1) Fractionation by gel filtration chromatography (Sephacryl S-100HR)
Adjust the pH to 3.0 by adding a small amount of 6M HCl to 60 mL of 2CW paste, boil at 100 ° C for 10 minutes, and confirm that 2CW is solubilized by microscopic observation, then centrifuge (4000 rpm, 5 minutes) The supernatant was recovered at Further, the oil was removed by repeating centrifugation (10000 rpm, 10 minutes) twice to obtain a solubilized solution. This solution was passed through a filter at the fitting portion of the column, and it was confirmed that the solution passed through the filter to prepare a sample.

  18 mL of a sample (TP: 43540 μg, TS: 32400 μg) was fractionated by an open column (2.5 × 94 cm) packed with Sephacryl S-100HR (GE Healthcare Japan), and 10 mL each was collected by a fraction collector. Elution was performed with ion exchange water at a flow rate of 0.3 mL / min. The total sugar amount, protein amount, and total uronic acid amount of the collected fraction were measured. The chromatogram obtained by gel filtration chromatography is shown in FIG.

  TP peaks are obtained at Fr.20, 24, 29, 36, and 46. Among them, Fr.20, 29, and 36 have TS peaks that are solubilized in a state where sugar and protein are bound. It was considered. In addition, Fr. 20 and 29 showed a TU peak, which also contained uronic acid. This gel filtration chromatography was performed to isolate the polymer peak H (see Example 2 (2)) obtained by heating at pH 3.0 / 100 ° C., but Sephacryl S-100HR has a relatively low molecular weight Fr. The first peak was obtained at .20 (elution volume 200 mL). When the molecular weight was calculated from the ratio of the elution amount to the column volume, the molecular weights corresponding to Fr. 20 and 29 were about 46 kDa and 7.4 kDa, respectively.

(2) HPLC analysis of each fraction The recovered Fr. 20, 29, 36, 46 was filtered through a membrane filter, and HPLC analysis was performed under the same conditions as in Example 2 (2). The results are shown in FIG. In Fr. 20 and 29, the polymer peak H observed in the solubilizate of Example 2 appeared mainly. Some peaks were also observed in Fr. The molecular peak H obtained by HPLC analysis was considered to be higher than the molecular weight because the exclusion limit of the column (TSK gel G3000 SWXL) was 500 kDa or more.

(3) SDS-PAGE of each fraction
Centrifugation (13200 rpm) using a 10 kcut spin column (VIVASPIN 500 (molecular weight fraction 10 kDa: sartorius stedim)) Fr.20 is 1.5 times, 3.0 times, 7.0 times, Fr.24 is 7.0 times, Fr.29 is Fr.36 was concentrated to 7.0 times by centrifugation (13200 rpm) using a 3kcut spin column (VIVASPIN 500 (molecular weight fraction 3 kDa: sartorius stedim)). SDS-PAGE was performed according to the method described in 2 (1), and Bio-Safe Coomassie G250 was used for protein staining, and the results are shown in FIG.

  In the case of using a 15% gel, in Fr. 20, 24, and 29, in both CBB G250 and PAS staining, bands that seem to be solubilized substances obtained by heating were observed as in FIG. 8 (15 When the% gel was used, the PAS staining was low in the concentration of the staining solution or sample, so that only a trace of the band was seen (FIG. 13A). From this, it was found that the solubilized product that appears as the polymer peak H in the HPLC analysis appears as a relatively low molecule in the gel filtration chromatography using Sephacryl S-100HR. Further, in the 15% tricine gel, a band considered to be a solubilized substance was also observed in Fr.36 by staining with CBB G250 (FIG. 13 (B)). However, no band was observed in PAS staining, and Fr.36 was considered to be a solubilizate different from Fr.20, 24, and 29.

(4) Neutral sugar analysis of each fraction The collected Fr. 20, 29, and 36 were analyzed for neutral sugars by gas chromatography (alditol acetate derivatization method). 1 mL of each sample was taken, 181 μL of TFA and 20 μL of 1% inositol were added, mixed and sealed. After heating at 121 ° C. for 2 hours, TFA was removed by distillation under reduced pressure. Further, ethanol was added, and TFA was removed again by distillation under reduced pressure, followed by hydrolysis. To this, 500 μL of NaBH ₄ solution was added and reduced at room temperature for 60 minutes. Thereafter, glacial acetic acid was slowly added dropwise until the foam disappeared. 500 μL of acidic methanol was added, evaporation to dryness was repeated 3 times or more, 250 μL of methanol was added, and evaporation to dryness was repeated 5 times or more to obtain a sugar alcohol (dryness). Next, 500 μL of anhydrous pyridine was added, 400 μL of acetic anhydride was immediately added, mixed, and acetylated at 70 ° C. for 20 minutes. 4 mL of distilled water was added to this and stirred, followed by addition of 800 μL of chloroform and well stirred. The aqueous layer was removed, the chloroform layer was washed with water several times, and this chloroform solution was used as a sample. Gas chromatography uses a silica capillary column (inner diameter 0.25 mm, length 30 m, film thickness 0.25 μm, DB-225 manufactured by J & W), column temperature 180 ° C.-230 ° C. (increases 5 ° C. every minute), The carrier gas, N ₂ gas, was allowed to flow through the column at 100 mL / min. FID was used for the detector. The results are shown in FIG.

  Fr. 20, 29, and 36 all contained galactose and arabinose, each having the largest amount of galactose followed by arabinose. This suggests that this solubilized substance is rich in arabinan, galactan, and arabinogalactan (Fr. 20, 29, and 36 are Ara: Gal = 1: 1.3, 1: 2.7, 1: 1.6, respectively). ). Fr.20 contained rhamnose and xylose, and Rha: Gal A = 1: 1.2. From this, it was considered that Fr.20 has a structure in which arabinogalactan is bound with rhamnogalacturonan or xylogalacturonan as the main chain. Fr.29 accounted for 90% of arabinose and galactose, and was thought to contain mainly arabinogalactan. About 50% of Fr.36 was glucose, followed by galactose, arabinose, and rhamnose. As a result of HPLC analysis, the polymer peak H, which is a solubilized substance produced by heating, was the main peak in Fr. 20 and 29, and only a small amount of polymer peak H was observed in Fr.

(5) Measurement of methylation degree of fractions Fr. 20, 24, 29 in which uronic acid was detected were measured for methylation degree in uronic acid. 5 μL of 2M NaOH was added to 500 μL of each of Fr. 20, 24, and 29, and left at room temperature for 1 h. Thereafter, 10 μL of the product neutralized with 0.5 M HCl was subjected to gas chromatography to detect methanol. In the gas chromatography, a glass column (inner diameter: 3.4 mm, column length: 1 m) packed with Uniport HP (60 / 80mesh) containing 5% PEG20M as a column carrier is used, the column temperature is 70 ° C., and N _{2 as a} carrier gas. The gas flowed through the column at 110 mL / min. FID was used for the detector. The degree of methylation was determined by the following formula.
Degree of methylation = (methanol mol / Gal A mol) x 100
The results are shown in FIG. The methylation degree was about 30% in all the Fr. 20, 24, and 29 fractions.

(6) Amino acid analysis of fractions
Fr. 20, 29, and 36 were subjected to amino acid analysis (phenyl isothiocyanate (PTC) derivatization method). 1 mL of concentrated hydrochloric acid was added to the sample to form a 6N hydrochloric acid solution, which was hydrolyzed at 110 ° C. for 24 hours under reduced pressure. After hydrolysis, it was quickly evaporated to dryness with a rotary evaporator. 20 μL of an ethanol / water / TEA mixed solution (2: 2: 1) was added thereto, and the mixture was dried under reduced pressure. 50 μL of an ethanol / water / TEA / PITC mixed solution (7: 1: 1: 1) was added and allowed to stand at room temperature for 20 minutes, and then dried under reduced pressure to remove excess reagents. What was dissolved in 1 mL of the HPLC eluate was further diluted 100 times, filtered through a 0.45 μm HPLC filter (IWAKI), and then HPLC analysis was performed on 50 μL of the sample.

  For HPLC, gradient (flow rate: 1.5 mL / min) using the following eluate was performed using a TSK-GEL Super-ODS column (TOSOH). Eluent A (acetonitrile / 50 mM acetate buffer (pH 6.0) = 3/97) and eluent B (acetonitrile / 50 mM acetate buffer = 60/40), eluent B is 5 → 70 v / eluent B is 70v / v% for v%, 12 → 16min, eluent B is 70 → 100v / v% for 16 → 20min, eluent B is 100v / v% for 20 → 25min, eluent is for 25 → 30min The column was passed through the column at 40 ° C. so that B was 100 → 3 v / v%. Detection was performed at UV 254 nm. The results are shown in FIG. 16 and Table 3 indicate the amino acid analysis value of a general soybean protein.

  All fractions had similar composition and were characterized by a high content of Asx, Glx, and Ile. However, Fr.20 had less Glx (10.88%) and Lys (3.69%) and more Ala (8.22%) and Leu (9.14%) than Fr.29,36. In Fr. 29, the ratio of Thr (5.46%) was relatively high and Ile (13.6%) was low compared to other fractions.

  From the above analysis results, Fr. 20 to 29 are fractions containing a solubilizing substance appearing as a polymer peak H, and the solubilizing substance is a random sugar chain in the sugar chain containing galactose and arabinose as main constituent sugars. While it was a bound substance, Fr.36 was considered to be a solubilizate different from the main substance produced by this method. Further, the protein contained in the solubilized substance contains a lot of isoleucine as a constituent amino acid, and then contains a lot of glutamic acid and glutamine (Glx), aspartic acid and asparagine (Asx), and leucine.

[Characteristics of secondary cell wall (2CW) lysate by acid heat treatment 3]
Further characterization of the selectively solubilized polymer peak H as revealed in Example 2 was performed.
(1) Molecular weight estimation by HPLC analysis Using Fr.20 (fraction obtained by Sephacryl S-100HR) obtained in Example 2, the molecular weight of polymer peak H is estimated from comparison with blue dextran having a molecular weight of 2 million. did. The obtained Fr. 20 solution was diluted 10 times with distilled water to prepare a sample. 10 mg of powder of blue dextran (manufactured by sigma) was dissolved in 1 mL of distilled water and diluted to prepare a 0.08% solution. Each was filtered with a filter for HPLC, and 20 μL was subjected to HPLC. Furthermore, 40 μL of the same amount was prepared for each mixed solution and subjected to HPLC analysis. HPLC analysis was performed by the method described in Example 2 (2). Further, the HPLC analysis was performed with the flow rates in the HPLC analysis being 1/2 and 1/4.

  The results are shown in FIGS. The peaks of Fr.20 (polymer peak H) and blue dextran in gel filtration chromatography using Sephacryl S-100HR appeared at approximately the same time by HPLC analysis. The polymer peak H was considered to have appeared at almost the same time because the molecular weight was at the exclusion limit. Since the molecular weight of blue dextran was 2000 kDa and the exclusion limit of TSK-GEL G3000SWXL, the column used, was 500 kDa, the molecular weight of the polymer peak H was considered to be 500 kDa or more. When the flow rate was reduced to 1/2 or 1/4, the appearance time of the lysate polymer peak H was slowed to 2 or 4 times, and the peak shape changed. When the flow rate was changed from 1.0 mL / min to 0.5 mL / min, the two peaks for both UV280 and RI changed to one on the low molecule side, and when changed from 0.5 mL / min to 0.25 L / min, The peak at about 14 minutes when the polymer peak H appears was small, and a large peak appeared at about 25 minutes with UV280. From this, it is considered that when the flow rate is slowed, the shape of the peak changes, such as the solubilized substance showing the polymer peak H is reduced in molecular weight. In the elution profile in gel filtration chromatography in Example 3, the solubilized substance appeared on the low molecular side. This means that the open column used in the gel filtration chromatography has a lower flow rate than the HPLC and the pressure is very small, so it appeared on the low molecule side. That is, it was shown that the solubilized substance (polymer peak H) has the property of associating under a pressure of 2 MPa or more from the pressure condition in HPLC analysis (about 2 MPa).

[Emulsification of secondary cell wall (2CW) solubilizate by acid heat treatment]
(1) Presence or absence of emulsifying action Suspension of distilled water and soybean 2CW paste prepared in Example 1 at a ratio of 10: 1 was adjusted to pH 3.0 with 6M HCl, boiled at 100 ° C for 20 minutes, and centrifuged After (4000 rpm, 1 minute), the supernatant filtered through a filter was used as the 2CW solubilized product. To 495 μL of this 2CW solubilizate, 5 μL of soybean oil was added and stirred vigorously for 1 minute. Immediately after that, 100 μL of the solution was taken from the bottom, 200 μL of the solution diluted 4 times by adding to 300 μL of distilled water was added to the microplate, and the absorbance at a wavelength of 630 nm was measured. In addition, the absorbance at a wavelength of 630 nm was measured using as a control the same operation using distilled water instead of 2CW solubilized product. Absorbance obtained by adding 200 μL of distilled water alone to the microplate was used as a blank. Furthermore, soybean oil was added and immediately after stirring, the size of soybean oil particles was observed using a microscope to examine the presence or absence of an emulsifying action.

  The results are shown in FIG. Observation with a microscope confirmed that the particle diameter of soybean oil was reduced in a solution containing 2CW solubilized product and soybean oil. In the absorbance measurement, the value was higher than that of a solution obtained by mixing soybean oil with distilled water. From these results, emulsifying action was observed in the 2CW solubilized product.

(2) Emulsification stability over time 8 μL of soybean oil was added to 792 μL of the 2CW solubilized product prepared in Example 5 (1) and stirred vigorously for 1 minute. Then, 60 μL of the solution is taken from the bottom every 0, 1, 2, 3, 4, and 5 hours, 200 μL is added to 180 μL of distilled water and diluted 4-fold, and the absorbance at a wavelength of 630 nm is measured. did. The control was performed in the same manner using distilled water instead of 2CW solubilized product.

  The results are shown in FIGS. As a result of the absorbance measurement, the absorbance slightly decreases with time (see FIG. 20), but in the microscopic observation, many of the soybean oil particles after 5 hours remain small as they were immediately after stirring (FIG. 21). It was confirmed that the emulsifying action was well maintained.

(3) Emulsification stability in pH change 0.1-0.5M NaOH was added little by little to the prepared 2CW solubilized product to prepare 2CW solubilized products having pH of 3, 5, 7, 9 (At this time, the same amount of distilled water was added so that dilution of the lysate by adding NaOH was constant.) Using these 2CW solubilizates, the effect of pH on emulsification was investigated. The emulsifying action was performed by the method described in Example 6 (1).

  The results are shown in FIG. 22 and Table 5 (pH 3.0 was omitted because it was almost the same as pH 9.0). In both the observation with a microscope and the measurement of absorbance, many of the soybean oil particles were larger at pH 5.0 and 7.0 than at pH 3.0 and 9.0, and the absorbance was also low. From these results, it was confirmed that the 2CW solubilized product by acidic heat treatment has a characteristic that the emulsifying action is high when the pH is 3.0 or 9.0, and is weak near neutral.

(4) Foamability 2 mL each of the 2CW solubilized product prepared in Example 6 (1), 1% casein (solution dissolved by neutralization with 0.01M NaOH), and 480 μg / mL ovalbumin each. Placed in test tube and stirred vigorously for 1 minute. The height (h) from the liquid level of the foam generated after stirring to the top of the foam was considered to be foaming. As shown in FIG. 23, it was considered that the foamability of the 2CW solubilized product was very high as compared with ovalbumin. Further, even after 400 minutes, bubbles of about 53% height remained immediately after stirring (not shown), and the stability was considered high.

(5) Foamability in pH change Using the 2CW solubilized product having pH of 3, 5, 7, 9 prepared in Example 5 (3), the method described in Example 5 (4) above. The effect on foaming properties was investigated. As shown in FIG. 24, the decrease in foam was the fastest at pH 5.0 and the slowest at pH 3.0. Immediately after stirring, the height (h) from the liquid level to the top of the foam was slightly increased at pH 9.0.

[Solubilization of secondary cell wall (2CW) by acid heat treatment in other plants]
Other plants used were red beans, apples, mandarin oranges, and potatoes. Upon solubilization, secondary cell walls were prepared by the following method.

(1) Preparation of 2CW paste
(a) Red beans 70 ml of water was added to 7 g of red beans and autoclaved at 121 ° C. for 20 minutes. After removing the extract, it was peeled and lightly crushed with a spatula to make a paste. To this red bean paste, 70 ml of 20 mM acetate buffer (pH 5.0) and cellulase (GODO-TCF) were added to 1% of the total amount, and the mixture was reacted at 37 ° C. overnight. After removing the supernatant by centrifugation (2000 rpm, 10 minutes), 100 ml of 20 mM acetate buffer (pH 5.0) was added and centrifuged again, and the supernatant was discarded. After repeating this operation three times, an equivalent amount of 20 mM acetic acid buffer (pH 5.0) was added to the residue to obtain a red beans 2CW paste.

(b) Apple After peeling off the apple (Fuji) and removing the core, fruit juice and squeezed koji were prepared with a home juicer. The apple cake was washed and then collected by centrifugation at 4000 rpm for 10 minutes. To 23.2 g of apple koji, 2 mL of 0.5 M acetate buffer was added, 2.5 mL of cellulase manufactured by Godo Sake Seisha Co., and water were added to make the total volume 30 mL, followed by stirring. Then, it was left overnight at 45 ° C. It was confirmed with a phase contrast microscope that the primary cell wall was solubilized and removed by this operation, and an apple 2CW paste was obtained.
(c) Tangerine

  200 mL of 20 mM acetate buffer (pH 5.0) and 1% of cellulase (GODO-TCF) were added to 20 g of the pulp part excluding the mandarin peel and reacted at 37 ° C. overnight. After removing the supernatant by centrifugation (10000 rpm, 10 minutes), 100 ml of 20 mM acetate buffer (pH 5.0) was added and centrifuged again, and the supernatant was discarded. After repeating this operation three times, the same amount of 20 mM acetate buffer (pH 5.0) as the residue was added to obtain a mandarin 2 CW paste.

(d) Potato The baron was peeled and ground with grater. This was washed several times with water to remove the starch. An equal volume of 0.1 M acetate buffer was added to about 50 mL of potato solution, 1% of cellulase (GODO-TCF) was added, and the mixture was allowed to react overnight at 37 ° C. with stirring. Centrifugation (4,000 rpm, 10 minutes) was performed, and the precipitate was washed three times with ion-exchanged water. This was diluted with about 15 times water to obtain a potato 2CW paste.

(2) Solubilization of secondary cell wall (2CW paste)
160 μL of the 2CW paste derived from each plant prepared above was added to 800 μL of 0.1 M acetate buffer (pH 3.0). After stirring, 400 μL was collected, heat-treated at 100 ° C. for 20 minutes, and allowed to stand overnight, and the volume in the Eben tube was compared with that before the heat treatment. The supernatant was recovered by centrifugation (10000 rpm, 10 minutes, 4 ° C.), and 2CW solubilization was observed with a phase contrast microscope. In the observation with a microscope, a blank (pH 5.3) was prepared by adding 800 μL of distilled water instead of 0.1 M acetate buffer (pH 3.0) and stirring.

  The microscopic images of red beans and mandarin oranges are shown in FIGS. 25 and 26, respectively. In each plant, the transparent secondary cell wall indicated by an arrow in each figure disappeared compared to before heating. Further, since the volume in the tube was reduced (not shown), it was confirmed that the secondary cell wall was selectively solubilized.

(3) HPLC analysis of the solubilized product HPLC analysis of the solubilized product obtained by subjecting the prepared apple and tangerine 2CW pastes to an acid heat treatment was performed to examine the appearance of the polymer peak H. HPLC analysis was performed by the method described in Example 2 (2).
(a) Apple
200 μL of apple 2CW paste was added to 800 μL of an aqueous HCl solution adjusted to pH 3.0, and an aqueous HCl solution adjusted to pH 2.0 was further added to adjust the pH to 3.0. 400 μL was taken and heated at 100 ° C. for 20 minutes. Then, after centrifugation (1000 rpm, 5 minutes), the supernatant was filtered through a filter to prepare a sample.

(b) Tangerine Tangerine 2CW 8 μL of HCl aqueous solution adjusted to pH 1.0 was added to 800 μL to adjust pH to 3.0. 400 μL was taken and heated at 100 ° C. for 20 minutes. Then, after centrifugation (1000 rpm, 5 minutes), the supernatant was filtered through a filter to prepare a sample.

  FIG. 27 and FIG. 28 show the results. Polymer peak H appeared in both apple and mandarin by heat treatment. Thereby, also in plants other than soybeans, the polymer peak H is confirmed by the heat treatment at pH 3.0, and it can be said that 2CW solubilized substance (polymer peak H) is produced by the treatment.

(4) Emulsifying action and foaming property of solubilized product The emulsifying effect and foaming property of the solubilized product of apple secondary cell wall obtained in Example 6 (2) were examined. The emulsifying action and foaming property were examined according to the methods described in Example 5 (1) and (4). The results are shown in FIGS. 29 and 30, respectively. Microscopic observation revealed that many of the soybean oil particles after 5 hours remained small as they were immediately after stirring (see FIG. 29), and the emulsifying action was well maintained. Moreover, it had the foaming property substantially the same as casein and albumin (refer FIG. 30).

  According to the present invention, the secondary cell wall can be solubilized under mild conditions, and a new food material can be provided.

Claims (15)

  A method for solubilizing plant secondary cell walls, wherein the plant is heated in an environment having a pH of 2.5 or more and 3.5 or less.   The solubilization method according to claim 1, wherein the plant cell having a primary cell wall and / or the plant cell from which the primary cell wall has been removed or crushed is heated.   A method of preparing a lysate of a secondary cell wall by heating plant cells from which the primary cell wall has been removed or crushed in an environment of pH 2.5 or more and 3.5 or less.   The method according to claim 3, wherein the insoluble matter is removed after the heating to prepare a lysate of the secondary cell wall. A step of generating an aggregate from the processed product obtained by removing the insoluble matter;
The method according to claim 4, wherein the aggregate is collected to prepare a lysate of the secondary cell wall.   The step of generating the aggregate is any one of a cooling step, a method of thawing after freezing, a step of concentrating with a separation membrane having a molecular weight of 5000 or more, a step of salting out, and a step of gelling by adding calcium ions. The method of claim 5.   A plant cell obtained by heating a plant cell from which the primary cell wall has been removed or crushed in an environment of pH 2.5 or more and 3.5 or less and removing the insoluble matter from the treated product obtained by the heating. Next cell wall lysate. Heating the plant cell from which the primary cell wall has been removed or crushed in an environment of pH 2.5 to 3.5,
Removing insoluble matter from the processed product obtained by heating,
Producing aggregates from the treated product from which the insoluble matter has been removed;
Collecting the resulting aggregates;
Solubilized product of plant secondary cell wall obtained by Heating the plant cell from which the primary cell wall has been removed or crushed in an environment of pH 2.5 to 3.5,
Removing insoluble matter from the processed product obtained by heating,
Fractionating a fraction having a molecular weight of 4500-75000 by gel filtration chromatography from the treated product from which the insoluble matter has been removed;
Solubilized product of plant secondary cell wall obtained by A glycoprotein having a molecular weight of 4500-75000 as determined by gel filtration chromatography,
With a pressure of 2 MPa or more, the molecular weight is associated with 500,000 or more,
A pectin-protein lysate derived from a plant secondary cell wall, wherein the glycoprotein units each have a methylation degree of 25 to 35%.   The solubilized product of pectin-protein derived from plant secondary cell walls according to claim 11, wherein isoleucine is 18 to 25 mol% of all amino acids. Heating the plant cell from which the primary cell wall has been removed or crushed in an environment of pH 2.5 to 3.5,
Removing the insoluble matter from the treated product obtained by heating, and forming an aggregate from the treated product from which the insoluble matter has been removed;
The method for preparing a solubilized protein-pectin according to claim 10 or 11, wherein Heating the plant cell from which the primary cell wall has been removed or crushed in an environment of pH 2.5 to 3.5,
A step of removing insoluble matter from the treated product obtained by heating, a step of fractionating the treated product from which the insoluble matter has been removed by molecular weight sieving,
The method for preparing a solubilized protein-pectin according to claim 10 or 11, wherein   Use of the solubilizate obtained by the method according to any one of claims 3 to 6 or the solubilizate according to any one of claims 7 to 11 as a food material, feed or fertilizer.   Use of the solubilized product obtained by the method according to any one of claims 3 to 6 or the solubilized product according to any one of claims 7 to 11 as an emulsifier.