Acidic polysaccharide and method for producing same

Abstract

The purpose of the present invention is to provide a dispersion stabilizer for suppressing precipitation and aggregation of protein in an acidic condition, particularly in a pH range of 3.6-4.4 that is more acidic than around the isoelectric point of protein, and for providing an acidic protein beverage having a low viscosity and refreshing mouthfeel.　An acidic polysaccharide having a mass average molecular weight of 2,000,000-8,000,000 and containing 60% by weight or more of galactose, 20% by weight or less of uronic acid and 4% by weight or more of glucuronic acid as constituent sugars is extracted from lupin beans at pH 4-9 and a temperature above 100°C. The acidic polysaccharide can disperse and stabilize protein particles in a wide pH range, from pH 4.0-4.4, and particularly good ones from pH 3.6-4.4.

Description

Acidic polysaccharides and method for producing same

[0001] The present invention relates to an acidic polysaccharide, a method for producing the same, a dispersion stabilizer for proteins using the acidic polysaccharide, and a method for stabilizing an acidic protein drink.

[0002] Beverages containing proteins such as milk or soy milk fermented with lactic acid bacteria, or beverages containing these proteins that have been directly added with organic acids, are called acidic protein beverages. While these acidic protein beverages are popular for their refreshingly sour taste, proteins can aggregate and even precipitate under acidic conditions, particularly at temperatures higher than the protein's isoelectric point, significantly reducing their commercial value. Therefore, dispersion stabilizers such as high-methoxyl pectin and carboxymethylcellulose have been used to stabilize protein dispersion (Patent Document 1). These dispersion stabilizers react electrostatically with protein particles dispersed in water and create a weak polysaccharide network in the beverage, imparting viscosity to the aqueous system and inhibiting protein aggregation and precipitation, which can lead to the problem of imparting a unique viscosity to the beverage (Non-Patent Document 1).

[0003] Meanwhile, water-soluble soybean polysaccharides extracted from the cotyledons of soybean seeds have the ability to inhibit protein aggregation under acidic conditions, similar to high-methoxy pectin and carboxymethylcellulose, and have been used as stabilizers for acidic protein beverages (Patent Document 2). Water-soluble soybean polysaccharides are said to inhibit aggregation by electrostatically reacting with protein particles to coat their surfaces, and to stabilize the protein particles that are finely dispersed by homogenization through Brownian motion. This prevents acidic milk beverages from becoming viscous, giving them a refreshing, light taste (Non-Patent Document 2). Polysaccharides derived from peas have also been reported as stabilizers for acidic protein beverages with a pH of 3.4 to 4.5 (Patent Documents 3 and 4).

[0004] Acidic protein beverages can be prepared at a variety of pH levels, from near the isoelectric point of the protein to more acidic levels. At pH 4.0 to 4.5, near the isoelectric point of milk protein, the protein surface is neutral to slightly positively charged, and strongly negatively charged polysaccharides exhibit good dispersion stabilization. For this reason, high-methoxy pectin, carboxymethyl cellulose, and phosphate-crosslinked polysaccharides exhibit good dispersion stability (Patent Document 5). On the other hand, under more acidic conditions below pH 4.0, the protein surface is more strongly positively charged, and the strongly negatively charged polysaccharides react strongly with the protein particles, causing agglomeration and precipitation known as depression, preventing a stable state. It is believed that weakly negatively charged water-soluble soybean polysaccharides exhibit good dispersion stability in this pH range.

[0005] Patent No. 2599477 Patent No. 3280768 WO2012 / 176852 pamphlet WO2016 / 031858 pamphlet WO2008 / 149738 pamphlet

[0006] Food Hydrocolloids, 17, 333-343, 2003J. Agric. Food Chem., 54 (17), 6241-6246, 2006

[0007] High-methoxy pectin and carboxymethyl cellulose, polysaccharides that inhibit protein aggregation at pH 4.0 to 4.5, are polysaccharides that exhibit high viscosity characteristics, resulting in high viscosity and a heavy mouthfeel. While the protein content of acidic protein beverages is typically 0.3 to 3% by weight, in recent years, the market has demanded stabilizers that maintain low viscosity and inhibit protein aggregation, even for high-protein beverages exceeding 3% by weight. Dispersion stabilizers that can maintain the stability of protein beverages under slightly acidic conditions, from near the isoelectric point of the protein, without imparting viscosity to the beverage, include the acidic polysaccharides disclosed in Patent Documents 2 and 3. However, these require a deesterification process to function. Even the invention of Patent Document 4, which claims to not require a deesterification process, requires a raw material washing process. An object of the present invention is to provide a dispersion stabilizer for preparing an acidic protein beverage that suppresses protein precipitation and aggregation under acidic conditions, particularly in the pH range of 4.0 to 4.4, which is more acidic than the isoelectric point of the protein, and is particularly excellent in the pH range of 3.6 to 4.4, and that has low viscosity and a refreshing taste. A second object of the present invention is to provide such a stabilizer without requiring either or both of a raw material washing step and a deesterification step.

[0008] As a result of extensive research into the above-mentioned problems, the inventors discovered that acidic polysaccharides extracted from lupine seeds can stabilize the dispersion of protein particles over a wide pH range from pH 3.6 to pH 4.4, and thus completed the present invention.

[0009] That is, the present invention provides: (1) an acidic polysaccharide having a mass-average molecular weight of 2,000,000 to 8,000,000, containing, as constituent sugars, 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid; (2) the acidic polysaccharide according to (1), in which the degree of methyl esterification of uronic acid is 60% or more; (3) the acidic polysaccharide according to (1) or (2), which is derived from beans; (4) the acidic polysaccharide according to (3), which is derived from lupine bean seeds; (5) the acidic polysaccharide according to claim 1 or 2, characterized in that the single-molecule structure observed with an atomic force microscope has a linear structure; and (6) the acidic polysaccharide according to (1), characterized in that the degree of methyl esterification of uronic acid is 60% or more, which is derived from lupine bean seeds, and which is characterized in that the single-molecule structure observed with an atomic force microscope has a linear structure. (7) A method for producing an acidic polysaccharide, comprising using lupine seeds or their fiber fraction as a raw material and subjecting them to thermal extraction in an aqueous system having a pH of 4 to 9 at a temperature exceeding 100°C. (8) A method for producing an acidic polysaccharide according to (7), characterized in that neither a deesterification step nor a raw material washing step is performed. (9) A method for producing an acidic polysaccharide according to (7), wherein the obtained acidic polysaccharide contains, as constituent sugars, 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid, and has a molecular weight of 2,000,000 to 8,000,000. (10) A method for producing an acidic polysaccharide according to (8), wherein the obtained acidic polysaccharide contains, as constituent sugars, 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid, and has a molecular weight of 2,000,000 to 8,000,000. (11) A dispersion stabilizer for protein, comprising as an active ingredient an acidic polysaccharide having a molecular weight of 2 million to 8 million and containing, as constituent sugars, 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid. (12) The dispersion stabilizer for protein according to (11), wherein the acidic polysaccharide is derived from beans. (13) The dispersion stabilizer according to (11) or (12), which is for use in an acidic protein beverage. (14) The dispersion stabilizer according to (13), which is for use in an acidic protein beverage having a protein content of 1% by weight or more. (15) The dispersion stabilizer for protein according to (11), wherein the acidic polysaccharide is derived from beans and is for use in an acidic protein beverage having a protein content of 1% by weight or more.(16) A method for stabilizing dispersion of an acidic protein drink using an acidic polysaccharide having a molecular weight of 2 million to 8 million, containing as constituent sugars 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid. (17) A method for stabilizing dispersion of an acidic protein drink according to (16), wherein the acidic polysaccharide is derived from beans. (18) A method for stabilizing dispersion of an acidic protein drink according to (16), wherein the drink has a protein content of 1% by weight or more. (19) A method for stabilizing dispersion of an acidic protein drink according to (17), wherein the drink has a protein content of 1% by weight or more. The present invention also relates to: (1) An acidic polysaccharide having a mass-average molecular weight of 2 million to 8 million, containing as constituent sugars 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid. (2) The acidic polysaccharide according to (1), having a degree of methyl esterification of uronic acid of 60% or more. (3) The acidic polysaccharide according to (1) or (2), which is derived from beans. (4) The acidic polysaccharide according to (3), which is derived from lupine bean seeds. (5) The acidic polysaccharide according to (1) or (2), characterized in that the monomolecular structure observed with an atomic force microscope has a linear structure. (6) A method for producing an acidic polysaccharide, which comprises using lupine bean seeds or their fiber fraction as a raw material and subjecting them to heat extraction at a temperature exceeding 100°C in an aqueous system having a pH of 4 to 9. (7) A method for producing the acidic polysaccharide according to (6), which is characterized in that neither a deesterification step nor a raw material washing step is performed. (8) The method for producing an acidic polysaccharide according to (6) or (7), wherein the obtained acidic polysaccharide contains, as constituent sugars, 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid, and has a molecular weight of 2,000,000 to 8,000,000. (9) A dispersion stabilizer for protein, comprising, as an active ingredient, an acidic polysaccharide having a molecular weight of 2,000,000 to 8,000,000, and containing, as constituent sugars, 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid. (10) The dispersion stabilizer for protein according to (9), wherein the acidic polysaccharide is derived from beans. (11) The dispersion stabilizer according to (9) or (10), which is for use in an acidic protein beverage. (12) The dispersion stabilizer according to (11), which is for use in an acidic protein beverage having a protein content of 1% by weight or more.(13) A method for stabilizing dispersion of an acidic protein drink, using an acidic polysaccharide having a molecular weight of 2 million to 8 million and containing, as constituent sugars, 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid. (14) A method for stabilizing dispersion of an acidic protein drink according to (13), wherein the acidic polysaccharide is derived from beans. (15) A method for stabilizing dispersion of an acidic protein drink according to (13) or (14), wherein the protein content in the drink is 1% by weight or more.

[0010] The acidic polysaccharides prepared according to the present invention suppress protein aggregation and precipitation in the pH range of 4.0 to 4.4, particularly 3.6 to 4.4, where protein aggregation or precipitation typically occurs. For example, acidic protein beverages with low viscosity and a clean taste can be produced in systems with a nonfat milk solids content of 3% by weight or more (1% by weight of protein in the system). This enables the preparation of acidic protein beverages that maintain lactic acid bacteria growth in the pH range of 4.0 to 4.4, thereby suppressing the formation of protein aggregates over time due to overfermentation. Furthermore, when preparing sterilized acidic protein foods in the pH range of 3.6 to 4.0, protein denaturation and the formation of aggregates due to heat sterilization are suppressed, providing acidic protein beverages of stable quality. Furthermore, the acidic polysaccharides can be prepared without the need for either a deesterification step or a raw material washing step.

[0011] 1 shows atomic force microscope images of an acidic polysaccharide molecule (A) having a linear structure obtained from lupine seeds and a water-soluble soybean polysaccharide molecule (B) having a hyperbranched structure obtained from soybean seeds. This shows an example of the molecular weight distribution obtained by analyzing lupine polysaccharide A by gel filtration chromatography. The mass average molecular weight in this invention refers to the mass average molecular weight of the main component of peak 1 obtained by gel filtration chromatography analysis.

[0012] (Molecular Weight) The present invention is specifically described below. The molecular weight of the acidic polysaccharide of the present invention can be analyzed by gel filtration chromatography using HPLC under the following conditions. The mass-average molecular weight is 2,000,000 to 8,000,000, preferably 2,000,000 to 7,000,000, and more preferably 4,500,000 to 6,500,000. The mass-average molecular weight is calculated by gel filtration chromatography (Size Exclusion Chromatography-Multiangle Light Scattering: SEC-MALS) using a TSK-gel G-6000PWXL (φ7.8 mm × 300 mm; Tosoh) as the separation column and an RI detector and a multi-angle light scattering detector calibrated with toluene as the detector. Separation conditions are as follows: eluent: 50 mM sodium acetate aqueous solution (pH 5.0), flow rate: 0.6 mL / min.

[0013] (Constituent Sugars) The acidic polysaccharides of the present invention are characterized by containing, as constituent sugars, 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid. Furthermore, those containing galactose, arabinose, glucose, rhamnose, xylose, fucose, galacturonic acid, and glucuronic acid are preferred. The galactose content is preferably 65% ​​by weight or more, more preferably 70% by weight or more. The uronic acid content, which is the sum of the galacturonic acid content and the glucuronic acid content, is preferably 6% to 15% by weight. In the present invention, those containing 5% by weight or more of glucuronic acid are preferred, more preferably 5.5% by weight or more. Furthermore, those containing 1% to 6% by weight of galacturonic acid are preferred, more preferably 3% to 5% by weight. Furthermore, the weight ratio of galactose to uronic acid is preferably 6.8% or more, more preferably 7.6% or less. When the constituent sugars fall within this range, protein precipitation and aggregation can be suppressed in the pH range of 4.0 to 4.4, and especially in the pH range of 3.6 to 4.4, and an acidic polysaccharide with low viscosity can be obtained.

[0014] Galacturonic acid and glucuronic acid were analyzed by sulfuric acid hydrolysis followed by ion exclusion chromatography using a Shodex Sugar-SH1821 column (φ8.0 mm × 300 mm; Showa Denko) and an RI detector. Neutral sugar composition was analyzed by sulfuric acid hydrolysis followed by ion chromatography using a CarboPac PA-1 column (φ2.0 mm × 250 mm; Thermo) and an electrochemical detector.

[0015] (Degree of Esterification) The degree of esterification is the proportion of methyl esters of uronic acids constituting a polysaccharide, expressed as a molecular ratio. The acidic polysaccharide of the present invention functions as a dispersion stabilizer without actively adjusting the degree of esterification of uronic acids. The degree of esterification of the acidic polysaccharide of the present invention is 60% or more, preferably 70% or more. Even if the degree of esterification is less than 60%, it still functions as a dispersion stabilizer, but requires the active adjustment of the degree of esterification, making the process complicated. For ordinary pectin, the degree of esterification is adjusted by alkaline heating, preferably at pH 12 or higher, in a so-called deesterification step. In the present invention, heating at a weak alkaline temperature, such as pH 9, when extracting acidic polysaccharides has only a slight effect on the degree of esterification and is therefore not included in the adjustment step for the degree of esterification. The deesterification step, which is not particularly required in the present invention, refers to heating in an alkaline environment at pH 10 or higher. The heating temperature in this case is generally 40 to 60°C, which provides high reactivity and little coloration. The degree of methyl esterification is determined by quantifying uronic acid and methyl esterified uronic acid by the Doesburg titration method and calculating the degree of methyl esterified uronic acid by dividing the total amount of uronic acid by 100 (mol %).

[0016] (Structure) The acidic polysaccharide of the present invention preferably has a linear structure as a single molecule observed with an atomic force microscope. In the present invention, the linear structure refers to a structure in which almost no side chains branching from the linear chain of the polysaccharide are observed. For example, the molecular structure of the acidic polysaccharide obtained from lupine seeds, which is an example of the present invention, can be observed by preparing a 0.1 wt% aqueous solution of the polysaccharide, diluting it with pure water to 1 ppm of acidic polysaccharide, dropping 0.5 μl onto mica, allowing it to dry naturally, and observing the structure with a 20 N / m cantilever in DFM mode on an atomic force microscope (SPI3800N, SPA300HV; SII Corporation).

[0017] (Raw Material) Beans are preferably used as the raw material for the acidic polysaccharides of the present invention. Examples of beans include soybeans, peas, mung beans, broad beans, lupine beans, chickpeas, kidney beans, lentil beans, and cowpeas. Among these beans, lupine seeds are particularly preferred. When lupine beans are used, it is preferable to use the cotyledons after removing the inner skin. Although lupine beans for animal feed can also be used, edible lupine beans with reduced or removed alkaloids are more preferred. An example of an industrial method involves extracting and separating water-soluble fractions, such as protein fractions, from lupine beans in an aqueous system once or multiple times. Using the residual fiber fraction as a raw material, acidic polysaccharides can be extracted by heating under the conditions described below. The fiber fraction separated as the residue does not require further washing as a raw material, and washing is preferably not performed. By using beans as a raw material, acidic polysaccharides having the desired constituent sugars and protein dispersibility can be easily obtained.

[0018] (Heat Extraction) The pH during extraction is preferably between pH 4 and 9, with pH 6 and 8 being preferred, because acidic conditions below pH 4 tend to accelerate hydrolysis of polysaccharides, while alkaline conditions above pH 9 tend to accelerate decomposition of polysaccharides. After adding water to the raw material, an acid or alkali is added to adjust the pH to between pH 4 and 9. The acidic polysaccharides are then extracted under pressure at temperatures above 100°C, preferably 150°C or less, and more preferably 105°C to 135°C. Temperatures below 100°C result in poor extraction efficiency and are therefore unrealistic. Temperatures above 150°C may hydrolyze the acidic polysaccharides during the extraction process, resulting in the loss of their linear structure. The extraction time is generally 0.5 to 3 hours, but can be adjusted as desired depending on the raw material condition, temperature, and other factors. There are no particular limitations on the acid or alkali used. Acids such as hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, and formic acid, and alkalis such as sodium hydroxide, calcium hydroxide, sodium bicarbonate, sodium carbonate, and ammonia can be used. Also, high-purity cellulase, hemicellulase, and pectinase that do not hydrolyze the target acidic polysaccharides can be used alone or in combination for enzymatic reaction treatment before or after extraction.

[0019] (Purification) The extracted acidic polysaccharides can be obtained by separating and removing insoluble fiber and drying. However, to enhance their functionality, it is desirable to perform purification steps such as protein removal, desalting, and removal of pigment components. Protein removal can be achieved by adjusting the pH to cause protein aggregation and precipitation using methods such as isoelectric precipitation, followed by physical separation methods such as filtration, centrifugation, and membrane separation. Alternatively, proteins can be hydrolyzed using any proteolytic enzyme, and the hydrolyzed products can be adsorbed and removed using dialysis membranes, activated carbon, ion exchange, or hydrophobic resins. Any method capable of removing these components can be used for desalting, such as electrodialysis, ion exchange resins, or UF membrane separation. Pigment removal can be achieved by methods that decompose pigment components, such as ozone treatment or UV irradiation, as well as partitioning with hydrophilic polar solvents such as ethanol or isopropanol. It is preferable to use one or more of these methods in combination. The purified acidic polysaccharides can be subjected to an optional sterilization treatment, followed by freeze-drying, spray-drying, or hot-air drying of ethanol precipitates to obtain a dried product.

[0020] (Destarching) When starch is contained in the raw material for the acidic polysaccharide of the present invention, it is possible to obtain the acidic polysaccharide as is. However, when used in an acidic protein beverage, the starch may cause thickening or precipitation. Therefore, it is preferable to remove starch at the stage of the bean raw material, the stage of the fiber separated from the bean raw material, the stage of extracting the acidic polysaccharide, or the stage after extraction. Starch can be removed by one or a combination of two or more methods: hydrolysis with amylase, cooling precipitation, and coagulation precipitation with an emulsifier. While dry fractionation is possible at the raw material stage, wet fractionation is preferred. Starch granules can be separated by adding water to the crushed raw material, heating to a temperature at which the starch does not gelatinize, and centrifugal filtration. Alternatively, the hydrolyzed raw material can be degraded and removed by heating above the starch gelatinization temperature and treating with amylase. At the stage of fibrous material separated from the bean raw material, the fiber can be degraded and removed by dispersing the fiber in water, heating to a temperature at which the starch gelatinizes, and treating with amylase. Examples of methods for destarching during or after the extraction of acidic polysaccharides include adding amylase to the hydrated raw material before extraction, adding amylase to the slurry before solid-liquid separation after extraction, and adding amylase to the filtrate after solid-liquid separation.

[0021] Amylase is a general term for enzymes that hydrolyze starch, and examples include β-amylase, α-amylase, glucoamylase, and pullulanase. For the present purpose, these highly purified amylases may be used, but commercially available amylase preparations containing one or more of these may also be used. It is possible to degrade and remove starch from raw materials, fibers, or before or after extraction of acidic polysaccharides by chemical methods such as acid hydrolysis or alkaline elimination hydrolysis. However, because there is a possibility that the acidic polysaccharides of the present invention may be degraded at the same time, destarch treatment by enzyme treatment is preferred.

[0022] (Dispersion Stabilizer) The acidic polysaccharide of the present invention functions as a dispersion stabilizer that inhibits aggregation of protein particles and maintains a stable dispersion. Its pH range is extremely wide, from pH 4.0 to pH 4.4, with particularly excellent examples ranging from pH 3.6 to pH 4.4. It is suitable for acidic protein beverages, such as fermented milk-based yogurt drinks and acidic milk beverages prepared by direct acid addition. Conventional dispersion stabilizers, such as pectin, carboxymethylcellulose, and water-soluble soybean polysaccharides, exhibit reduced stability in beverages containing 3% or more nonfat milk solids by weight (protein concentration of 1% or more by weight), preferably 6% or more by weight (protein concentration of 2% or more by weight). For example, in a beverage containing 8.4% nonfat milk solids by weight (protein concentration of 2.8% by weight), clear crystals and precipitation occur after two weeks of storage. However, the acidic polysaccharide of the present invention exhibits almost no clear crystals or precipitation even under the same storage conditions.

[0023] The dispersion stabilizer of the present invention can provide beverages with a low viscosity that are less sticky than conventionally used pectins, have a less cloying flavor, and have a strong sour and milky taste. Furthermore, because of its low viscosity, it is possible to adjust the viscosity and drinking experience to any desired level by blending gums, thickeners, proteins, or their hydrolysates, as needed. Examples of substances that can be used in combination include starch, modified starch, various celluloses, agar, carrageenan, phaselan, guar gum, locust bean gum, fenogreek gum, konjac mannan, tamarind seed polysaccharides, tara gum, gum arabic, tragacanth gum, karaya gum, pectin, xanthan gum, pullulan, gellan gum, and other polysaccharides, as well as proteins such as gelatin and collagen. The low-viscosity acidic protein beverage of the present invention can be, for example, an acidic milk beverage with a non-fat milk solids content of 8.4% having a viscosity of 100 mPa·s or less, preferably 50 mPa·s or less, and more preferably 30 mPa·s or less.

[0024] (Acidic Protein Beverage) The acidic protein beverage of the present invention refers to an acidic protein beverage containing animal- or plant-derived protein, including concentrated products such as drink bases. Those containing animal-derived protein primarily refer to animal milks such as cow's milk and goat's milk, specifically cow's milk, skim milk, whole milk powder, skim milk powder, whey powder, buttermilk, buttermilk powder, sweetened milk, condensed milk, concentrated milk, processed milk or fermented milk enriched with minerals such as calcium or vitamins. Those containing plant-derived protein include so-called plant milks, such as soy milks obtained from soybeans. Specific examples include soy milk, skim soy milk, powdered soy milk, powdered skim soy milk, isolated soy protein, or fermented products. Other plant milks include peas, mung beans, lentils, almonds, peanuts, rice, oats, etc. Acidic protein beverages are beverages made by combining one or more of these animal milk or plant beverages and fermenting them with microorganisms such as lactic acid bacteria, or by adding fruit juice, or organic acids such as lactic acid or citric acid, or inorganic acids such as phosphoric acid to adjust the pH to an acidic level. Specific examples include pasteurized or live-bacterial lactic acid bacteria beverages, yogurt drinks, kefir, etc. Among these acidic protein beverages, those made from the above-mentioned animal milks are acidic milk beverages, and are particularly preferred for use in the present invention.

[0025] The dispersion stabilizer comprising the acidic polysaccharide of the present invention effectively functions in acidic protein beverages containing 3% or more nonfat milk solids by weight, i.e., 1% or more protein, when made with milk, suppressing protein aggregation and precipitation and maintaining a stable dispersion state for a long period of time. This function is even more pronounced in acidic protein beverages containing 6% or more nonfat milk solids by weight (2% protein). The acidic polysaccharide of the present invention, when added to an acidic protein beverage at a concentration of preferably 0.1 to 1.2% by weight, more preferably 0.2 to 1% by weight, can stabilize the dispersion of protein particles in a pH range of 4.0 to 4.4, and particularly in a pH range of 3.6 to 4.4. While dispersion stabilizers can adversely affect the taste of beverages when incorporated at high concentrations, the acidic polysaccharide of the present invention, even when incorporated at 1% by weight, has minimal effect on the flavor of acidic protein beverages, unlike citrus pectin or water-soluble soybean polysaccharides.

[0026] The acidic protein beverage prepared according to the present invention is preferably homogenized to improve the dispersion stability of protein particles. There are no particular limitations on the homogenization equipment, but examples include a high-pressure homogenizer, a homomixer, Clearmix (M Technique), a Nanomizer (Nanomizer), and an ultrasonic emulsifier. The homogenization pressure cannot be determined uniquely because the fluidity of the acidic protein beverage varies depending on the solids concentration, but a pressure of 50 kgf / cm^2 to 500 kgf / cm^2 is preferred. Homogenization can be performed at any stage in the production process of the acidic protein beverage, but is preferably performed after dispersing the protein in water, after fermenting the protein or adding an acid, or after adding the dispersion stabilizer of the present invention.

[0027] The present invention will be explained below by describing examples, but the technical concept of the present invention is not limited to these examples. In the examples, % and parts are by weight unless otherwise specified.

[0028] (Production Example 1) 50 kg of lupin seeds (Lupinus luteus) were dehulled and then soaked in five times the volume of water for 24 hours. The seeds were crushed using a homomixer (5,000 rpm, 30 minutes) to extract protein and starch. The aqueous phase containing dispersed and dissolved protein and starch was removed using a centrifugal filter at 2,000 × g for 20 minutes, and the remaining fiber was recovered. 80 parts of lupin fiber were dispersed in 920 parts of water, adjusted to pH 8 with sodium hydroxide, and heated at 120°C for 90 minutes to extract acidic polysaccharides. Insoluble fiber was removed by centrifugation (5,000 × g, 30 minutes), and the supernatant was recovered. The pH was adjusted to 7 and heated to 60°C. The starch was hydrolyzed for 1 hour by adding amylase (BAN480L; Novozyme) equivalent to 0.1% by weight of the solids. The pH was adjusted to 5 with hydrochloric acid, and ethanol was added to a concentration of 60% by weight to precipitate acidic polysaccharides. The precipitate was washed with 90% by weight aqueous ethanol, and air-dried to obtain lupine bean polysaccharide A.

[0029] (Production Example 2) Comparison of pH during extraction Lupine bean polysaccharides B, C, D, E, F, G, and H were obtained in exactly the same manner as in the production of lupine bean polysaccharide A, except that the pH during extraction was changed to 3, 4, 5, 6, 7, 9, and 10. Note that the deesterification step was not carried out in the extractions at pH 4 to 9. The extractions at pH 3 and pH 10 were comparative production examples, in which the deesterification reaction had substantially progressed.

[0030] Example 1: Measurement of Molecular Structure and Mass-Average Molecular Weight. Lupine polysaccharides A to H and a comparative water-soluble soybean polysaccharide (SOYAFIVE-S-DA100: Fuji Oil) were dried on mica, and their molecular structures were observed using an atomic force microscope. The mass-average molecular weights of lupine polysaccharides A to H were also measured using gel filtration chromatography (SEC-MALS). The analytical conditions were as follows: A 1 wt% aqueous solution of lupine polysaccharide (50 mM sodium acetate solution, pH 5.0) was prepared, filtered through a 0.8 μm filter, and the filtrate was subjected to analysis. 30 μL of the solution was loaded onto a TSK-gel G-6000PWXL column (φ7.8 mm × 300 mm; Tosoh) previously equilibrated at 40°C in 50 mM sodium acetate solution, pH 5.0, and then separated at a flow rate of 0.6 mL / min. The separated polysaccharides were detected using an RI detector and a multi-angle light scattering analyzer, and the mass-average molecular weight was calculated. The results are summarized in Table 1.

[0031] (Table 1) Mass average molecular weight of each extracted polysaccharide

[0032] Figure 1(A) shows the molecular structure of lupine bean polysaccharide A observed with an atomic force microscope. It was confirmed to have a linear structure, significantly different from the water-soluble soybean polysaccharide shown in Figure 1(B), which has a hyperbranched structure. Lupine bean polysaccharides C, D, E, F, and G also had a similar linear structure. Analysis of the mass-average molecular weight by SEC-MALS revealed that all of the above lupine-derived polysaccharides were composed of components with molecular weights of less than 7 million (Figure 2). Lupine bean polysaccharides B and H, which were comparative samples, did not exhibit a linear structure due to accelerated decomposition during extraction, and their mass-average molecular weights were small, less than 2 million. In contrast, the mass-average molecular weights of lupine bean polysaccharides other than B and H were estimated to be between 2.4 million and 6.3 million.

[0033] (Example 2) Sugar Composition The sugar compositions of lupine bean polysaccharides A to H were measured and the results are summarized in Table 2. DE (%) indicates the degree of esterification of uronic acid.

[0034] (Table 2) Sugar composition

[0035] Regardless of the extraction conditions, acidic polysaccharides containing galacturonic acid and glucuronic acid were obtained, but the galactose content was reduced to less than 50% by weight in the strong acid extraction at pH 3 (lupine bean polysaccharide B) and the alkaline extraction at pH 10 (lupine bean polysaccharide H). Furthermore, lupine bean polysaccharides E, F, and A extracted at pH 6, 7, and 8 had galacturonic acid contents exceeding 4% by weight.

[0036] (Production Example 3) Lupine bean polysaccharides I, J, K, L, M and N were obtained in the same manner as in Production Example 1, except that the extraction temperatures were 80°C, 90°C, 100°C, 110°C, 130°C and 140°C.

[0037] (Recovery Rate of Acidic Polysaccharides from Raw Fiber and Uronic Acid Ratio to Total Sugars) The recovery rate of lupine bean polysaccharides A, I, J, K, L, M, and N from the lupine bean fiber used as the raw material was calculated using the formula: recovered lupine bean polysaccharides (g) / lupine bean fiber used (g) * 100 (wt%). The total sugar content of the lupine bean polysaccharides was quantified using the phenol-sulfuric acid method, and the uronic acid content (wt%) relative to the total sugar content was calculated from the amount of uronic acid quantified using the Blumenkrantz method. Uronic acid is a component that contributes to the dispersion stabilization of the protein of the present invention, and a higher uronic acid content indicates a more functional lupine bean polysaccharide. The results are summarized in Table 3.

[0038] (Table 3) Recovery rate and uronic acid content

[0039] Extraction at 80°C showed low values ​​for recovery (6.3 wt%) and uronic acid content (7.7 wt%), indicating insufficient extraction of lupine bean polysaccharides. Extraction at 90°C showed a high uronic acid content (9.6 wt%), but a low recovery (9.9 wt%), indicating insufficient extraction of lupine bean polysaccharides. Extraction at 140°C showed a high recovery (51.9 wt%), but a low uronic acid content (7.0 wt%). This is thought to be due to the fact that lupine bean polysaccharides were sufficiently extracted, but at the same time, hydrolysis of the polysaccharides occurred. Based on these results, extraction at 110-130°C was preferable.

[0040] Example 3: Preparation of an acidic milk beverage and evaluation of its stability (protein concentration 2.8 wt %, stabilizer 0.4 wt %) Preparation of fermented milk An aqueous solution containing 21 wt % skim milk powder (manufactured by Yotsuba Dairy Products Co., Ltd.) was prepared and sterilized by heating at 95°C while stirring. After cooling, commercially available plain yogurt was inoculated and fermented in an incubator at 40°C until the pH reached 4.7. The fermented yogurt was homogenized through a high-pressure homogenizer (EnonizerLabo-01; Sanmaru Machinery Industry Co., Ltd.) at a pressure of 15 MPa.

[0041] Preparation of Acidic Milk Beverage: 20 parts of a 2 wt% aqueous solution of lupine polysaccharides A through H, 14 parts of a 50 wt% aqueous granulated sugar solution, and 26 parts of water were mixed and cooled to 4°C. 40 parts of fermented milk, also cooled to 4°C, was added to the mixture while stirring on ice, and the pH was adjusted to a desired value between 4.6 and 3.4 with a 50 wt% lactic acid solution. The resulting mixture was homogenized twice through a high-pressure homogenizer (15 MPa), transferred to a glass bottle, sealed, and sterilized by heating in an 80°C water bath for 20 minutes. The acidic milk beverage prepared with the above formulation had a non-fat milk solids content of 8.4 wt% and a stabilizer content of 0.4 wt%. Acidic milk beverages were also prepared using a water-soluble soybean polysaccharide (SOYAFIVE-S-DA100: Fuji Oil) and HM-pectin (GENU-PECTIN type USP-H: CP Kelco) as controls.

[0042] Evaluation of Stability of Acidic Milk Drinks The stability of the prepared acidic milk drinks was evaluated based on a comprehensive evaluation of viscosity, sedimentation rate, and clearness. The results are shown in Table 4 below. The measurement and evaluation methods for each were described below. [Viscosity] After storage at 4°C for 1 day and 2 weeks, the viscosity of the prepared acidic milk drinks at 4°C was measured using a cone-plate viscometer (DV2TCP; Brookfield) with a cone spindle (CPA-52Z; Brookfield) at a shear rate of 120 / sec, and the results were expressed in units of mPa·s. [Sedimentation Rate] After storage at 4°C for 1 day and 2 weeks, 20 g of the acidic milk drinks were weighed into centrifuge tubes and centrifuged at 2,000 rpm for 20 minutes in a Kokusan centrifuge. The supernatant was decanted, and the weight of the sediment was measured. The sedimentation rate was calculated using the following formula: Sedimentation rate (wt%) = (weight of sediment) / (weight of acidic milk beverage dispensed) × 100. The sedimentation rate was indicated as follows: a sedimentation rate of less than 2 wt% was indicated as ◎; a sedimentation rate of 2 wt% or more but less than 3 wt% was indicated as ○; a sedimentation rate of 3 wt% or more but less than 5 wt% was indicated as △; and a sedimentation rate of 5 wt% or more was indicated as ×. [Clearance] The presence or absence of clearness on the top surface of the solution after standing for 1 day and 2 weeks was judged by visual observation. Presence of clearness was indicated as +, and absence of clearness was indicated as -. [Overall evaluation] The above stability evaluation items were summarized, and the stability was indicated as very good with ◎, good with ○, somewhat good with △, and poor with ×. Specifically, the results after either 1 day or 2 weeks of preparation were poor, with a sedimentation rate of ◎ and clearness - overall indicated as ◎; a sedimentation rate of ○ and clearness - overall indicated as ○; a sedimentation rate of △ overall indicated as △; and a sedimentation rate of × overall indicated as ×.

[0043] (Table 4) Stability of acidic milk drinks (4-1) (4-2)

[0044] Lupine polysaccharides C, D, and G have a linear structure, a mass-average molecular weight of 2.4 million to 5 million, a uronic acid content (the sum of glucuronic acid and galacturonic acid) of 8% or more by weight, and a galactose content of 70% or more by weight. These lupine polysaccharides were able to stabilize proteins in a high-protein system with a protein concentration of 2.8% by weight (non-fat milk solids of 8.4% by weight) in the pH range of 4.0 to 4.4. On the other hand, lupine bean polysaccharides A, E, and F extracted in the pH range of 6 to 8 had a mass-average molecular weight of 5.2 to 6.3 million, a uronic acid content (the sum of glucuronic acid and galacturonic acid) of 9% by weight or more, a galactose content of 70% by weight or more, and a galactose to uronic acid weight ratio of 6.9 to 7.4, and were able to stabilize the protein in an acidic milk beverage at pH 3.6 to 4.4 under the same conditions. Furthermore, the acidic milk beverages that were confirmed to be stable had a low viscosity of 30 mPa·s or less and a refreshing taste.

[0045] (Example 4) Preparation of acidic milk beverage and evaluation of stability (system with protein concentration of 1.0 wt %, stabilizer 0.1 to 1.0 wt %) Acidic milk beverages were prepared and their stability evaluated in exactly the same manner as in Example 3, except that the protein concentration was 1.0 wt % (non-fat milk solids content 3.0 wt %) and the amount of lupine bean polysaccharide A used as a stabilizer was 0.1, 0.2, 0.4, 0.6, or 1.0 wt %.

[0046] (Table 5) Stability of acidic milk drinks (5-1) (5-2) (5-3)

[0047] Lupin bean polysaccharide A was able to disperse and stabilize milk proteins over a wide pH range, from 3.6 to 4.4, at concentrations ranging from 0.2% to 1.0% by weight. When 1.0% by weight of lupin bean polysaccharide A was added to an acidic milk beverage with a protein concentration of 1.0% by weight (3.0% nonfat milk solids), no change in flavor was observed, and the viscosity remained below 30 mPa·s, maintaining the low viscosity and clean taste characteristic of lupin bean polysaccharides. On the other hand, under the same conditions, an acidic milk beverage stabilized with HM-pectin exhibited a strong sticky texture, while an acidic milk beverage stabilized with water-soluble soy polysaccharides had a strong soy flavor.

[0048] The present invention provides a dispersion stabilizer for acidic protein beverages, i.e., a dispersion stabilizer that suppresses protein precipitation and aggregation under acidic conditions. Use of the dispersion stabilizer of the present invention makes it possible to provide acidic protein beverages with low viscosity and a refreshing taste over a wide range of pH from 4.0 to 4.4, with particularly excellent pH values ​​ranging from 3.6 to 4.4. Furthermore, the beverages are easy to produce, and do not require a raw material washing step or a deesterification step.

Claims

1. An acidic polysaccharide having a mass average molecular weight of 2 to 8 million and containing as its constituent sugars 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid.

2. The acidic polysaccharide according to claim 1, wherein the degree of methyl esterification of uronic acid is 60% or more.

3. The acidic polysaccharide according to claim 1 or 2, which is derived from beans.

4. The acid polysaccharide according to claim 3, which is derived from the seeds of lupine beans.

5. The acidic polysaccharide according to claim 1 or 2, characterized in that the single molecule structure observed with an atomic force microscope has a linear structure.

6. The acidic polysaccharide according to claim 1, characterized in that the degree of methyl esterification of uronic acid is 60% or more, that it is derived from lupine seeds, and that the monomolecular structure observed with an atomic force microscope has a linear structure.

7. A method for producing acidic polysaccharides, which comprises using lupine seeds or their fiber fraction as a raw material and heating and extracting them in an aqueous system having a pH of 4 to 9 at a temperature exceeding 100°C.

8. A method for producing an acidic polysaccharide according to claim 7, characterized in that neither a deesterification step nor a raw material washing step is carried out.

9. A method for producing an acidic polysaccharide according to claim 7, wherein the obtained acidic polysaccharide contains, as constituent sugars, 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid, and has a molecular weight of 2 million to 8 million.

10. A method for producing an acidic polysaccharide according to claim 8, wherein the obtained acidic polysaccharide contains, as constituent sugars, 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid, and has a molecular weight of 2 million to 8 million.

11. A protein dispersion stabilizer having as its active ingredient an acidic polysaccharide with a molecular weight of 2 to 8 million and containing as its constituent sugars 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid.

12. A protein dispersion stabilizer according to claim 11, wherein the acidic polysaccharide is derived from beans.

13. The dispersion stabilizer according to claim 11 or 12, which is for use in an acidic protein beverage.

14. The dispersion stabilizer according to claim 13, which is for use in an acidic protein beverage having a protein content of 1% by weight or more.

15. A protein dispersion stabilizer according to claim 11, wherein the acidic polysaccharide is derived from beans and is for an acidic protein beverage having a protein content of 1% by weight or more.

16. A method for stabilizing the dispersion of an acidic protein beverage using an acidic polysaccharide having a molecular weight of 2 to 8 million and containing, as its constituent sugars, 60% by weight or more of galactose, 20% by weight or less of uronic acid, and 4% by weight or more of glucuronic acid.

17. A method for stabilizing dispersion of an acidic protein beverage according to claim 16, wherein the acidic polysaccharide is derived from beans.

18. A method for stabilizing dispersion of an acidic protein beverage according to claim 16, wherein the beverage is an acidic protein beverage having a protein content of 1% by weight or more.

19. A method for stabilizing dispersion of an acidic protein beverage according to claim 17, wherein the beverage is an acidic protein beverage having a protein content of 1% by weight or more.

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