Intestinal immunopotentiator, food and medicine

Abstract

The invention relates to an intestinal immunopotentiator, a food and a medicine. The purpose of the present invention is to provide an intestinal immunopotentiator capable of sufficiently increasing IgA in the intestinal tract at a low dose and maintaining the increased amount of IgA for a long period of time. An intestinal immunopotentiator containing cellulose acetate having a total degree of substitution of acetyl groups of 0.4-1.1 (inclusive).

Description

[0001] This application is a divisional application of the invention application with application date of October 23, 2018, application number 201880069752.X, and invention name “Intestinal Immunity Enhancers, Foods and Drugs”. Technical Field

[0002] The invention relates to an intestinal immunity enhancer, food and medicine. Background Art

[0003] Immunoglobulin (Ig), one of the glycoproteins, includes IgA, IgD, IgE, IgG, and IgM. Among them, IgA is found in mammals and birds. In particular, IgA contained in exocrine fluids such as saliva, tears, nasal mucus, respiratory mucus, digestive tract secretions, and breast milk is classified as secretory IgA, which functions as the first line of defense of the immune mechanism to protect the mucosal surface against antigens and microorganisms.

[0004] The digestive tract is constantly in contact with a variety of substances including antigens and microorganisms, and it is necessary to prevent these antigens and microorganisms from invading the body. In particular, IgA secreted in the intestine plays an important role in mucosal immune function, such as preventing bacteria and viruses from adhering to the mucosal surface and capturing foreign antigens and excreting them out of the body. Promoting the secretion of IgA in the intestine can be expected to enhance mucosal immune function, prevent infectious diseases, allergic diseases, etc., so it is desirable to develop foods that promote IgA secretion.

[0005] The following (key points) are recorded in non-patent document 1. "The effect of galacto-oligosaccharide, one of the indigestible oligosaccharides, on the immune system of mice was studied. BALB / c mice were raised and allowed to freely ingest feed containing galacto-oligosaccharides. As a result, the amount of total IgA contained in the feces increased significantly after 2 weeks of ingestion, and then decreased to the same level as the control group. After 4 weeks of ingestion, the total IgA in the prepared Peyer's patch cell culture fluid and colon tissue extract was dissected and showed an increasing trend in the galacto-oligosaccharide group."

[0006] Non-patent document 2 describes the following (abstract). It proposes to use lactofructooligosaccharide (4 G -β-D-galactosylsucrose (LS) was used as an oligosaccharide for increasing bifidobacteria in the intestine. Mice were fed a diet supplemented with 2% or 5% LS for 4 weeks, and the immune responsiveness of the small intestinal mucosa was investigated. As a result, the amount of IgA in feces and cecal contents increased significantly in the 2% or 5% LS ingestion group.

[0007] Patent Document 1 describes that it was found that fructo-oligosaccharide, one of the indigestible oligosaccharides, enhances the production of IgA and pIgR in the intestinal mucosa.

[0008] Patent Document 2 describes that a significant IgA secretion promoting effect is observed by oral administration of indigestible dextrin.

[0009] Patent document 3 describes a nutritional composition, a livestock feed, a lipid metabolism improving agent, a preventive and / or therapeutic agent for inflammatory bowel disease and / or immune abnormalities, a preventive and / or therapeutic agent for cancer, a preventive and / or therapeutic agent for non-alcoholic fatty hepatitis, a preventive and / or therapeutic agent for obesity and / or diabetes, and a preventive and / or therapeutic agent for hypercholesterolemia, characterized in that it contains cellulose acetate having a total acetyl substitution degree of 0.4 to 1.1.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: Japanese Patent Application Publication No. 2003-201239

[0013] Patent Document 2: Japanese Patent Application Publication No. 2014-152125

[0014] Patent Document 3: International Publication No. 2015 / 146853

[0015] Non-patent literature

[0016] Non-patent document 1: Sato et al., "Effects of galacto-oligosaccharides on the immune system of mice", Journal of the Japanese Society of Nutrition and Food, Vol. 61, No. 2, 2008, pp. 79-88

[0017] Non-Patent Document 2: Hino Keiko et al., "Effect of Dietary Lactosucrose (4G-.BETA.-D-Galactosylsucrose) on the Intestinal Immune Functions in Mice", Journal of Applied Glycoscience, 2007, Vol. 54, No. 3, pp. 169-172 Summary of the invention

[0018] Problems to be solved by the invention

[0019] Although non-patent document 1 states that total IgA tends to increase by taking feed containing galacto-oligosaccharides, mouse colon tissue extract, etc., the feed needs to contain up to 5% by weight of galacto-oligosaccharides (p.80 right column, Table 2). In addition, in the second week of taking feed containing galacto-oligosaccharides, there was a case where the total IgA in the feces of the test group that took the feed was up to about twice that of the control group, showing a significant difference, but in the third week of taking it, it dropped to the same level as the control group, showing no significant difference (p.81 right column, Figure 1 ). In addition, although it was reported that a trend of increasing total IgA was observed in the analysis of colon tissue extracts, it was clearly stated that these effects were not statistically significant (p. 81 right column, Figure 2 and 3 ).

[0020] In Non-Patent Document 2, after consuming a feed supplemented with lactofructo-oligosaccharide, the amount of IgA in the feces of mice became about twice that of the control group after 1 week, but it decreased significantly after 2 weeks and did not increase significantly thereafter ( Figure 1 ). In particular, in the case of feed supplemented with 2% lactofructo-oligosaccharide, the amount of IgA decreased significantly after 2 weeks and then hardly increased ( Figure 1 ).

[0021] Patent Document 1 also describes that mice were fed an experimental feed supplemented with up to 5% (w / w) fructooligosaccharide. The IgA antibody content in the feces of the 36-day-old mice in the test group that ingested the feed was about twice that of the control group, showing a significant difference ( Figure 5 ), but no significant difference was found when the feeding days were 28 and 42 days in the same experiment ( Figure 5 ). In addition, the IgA antibody content of the whole colon of the test group was about 1.5 times that of the control group at 44 days of age, showing a significant difference (Figure 7), but the amount of IgA antibody per unit weight of colon tissue did not show a significant difference (Figure 9). In addition, the amount of anti-cholera toxin-specific IgA against Vibrio cholerae belonging to the Proteobacteria was investigated, and as a result (Figure 14), the p value between the test group and the control group was 0.07, showing no significant difference at the 5% significance level.

[0022] Patent Document 2 also describes the use of a control feed mixed with 5% and 7.5% by mass of indigestible dextrin to feed mice. Figure 1 ) and feces ( Figure 2 ) of IgA, but the number of mice used in the experiment is not shown, and the results of the significant difference test are not shown, and error bars are not shown in some figures ( Figures 1 to 4). In addition, the error bars in the figure indicate a large error range, and it can be seen that the test results are not significantly different and no record is made. Therefore, there is no probability statistical verification of whether the technology increases IgA. It should be noted that the substrate specificity of IgA has not been investigated.

[0023] Although Patent Document 3 describes cellulose acetate having a total degree of acetyl substitution of 0.4 to 1.1, it does not describe or suggest an intestinal immune enhancer.

[0024] In the method for using galacto-oligosaccharide, lactofructo-oligosaccharide, oligofructose and indigestible dextrin etc. in the past, in order to increase the IgA in the large intestine etc., a quite high dosage is needed. If high dose is needed, when the amount of daily intake is converted to that of people, there is the hidden worries such as pain when oral intake in the form of medicine, loss of pleasure of common dining when intake in the form of food. In addition, in the method in the past, the amount of the IgA increased cannot be maintained for a long time. In addition, there is no design related to the affinity (substrate specificity) of IgA to pathogenic bacteria, and the specificity to pathogenic bacteria is low.

[0025] An object of the present invention is to provide an intestinal immunity enhancer that can sufficiently increase IgA in the intestine at a low dose and maintain the increased amount of IgA for a long period of time.

[0026] Means for solving problems

[0027] A first aspect of the present invention relates to an intestinal immunity enhancer comprising cellulose acetate having a total degree of acetyl substitution of 0.4 or more and 1.1 or less.

[0028] In the intestinal immunity enhancer, the composition distribution index (CDI) of the cellulose acetate defined by the following formula is preferably 2.0 or less.

[0029] CDI = (measured value of half-peak width of composition distribution) / (theoretical value of half-peak width of composition distribution)

[0030] Measured value of composition distribution half-peak width: the composition distribution half-peak width determined by HPLC analysis of cellulose acetate propionate obtained by propionylating all the remaining hydroxyl groups of cellulose acetate (sample);

[0031] [Mathematical formula 1]

[0032]

[0033] DS: total degree of acetyl substitution;

[0034] DPw: weight average degree of polymerization (a value determined by a GPC-light scattering method using cellulose acetate propionate obtained by propionylating all the remaining hydroxyl groups of cellulose acetate (sample)).

[0035] A second aspect of the present invention relates to a food containing the above-mentioned intestinal immunity enhancer.

[0036] The content of the cellulose acetate in the food is preferably 0.1% by weight or more and less than 5% by weight.

[0037] A third aspect of the present invention relates to a medicine comprising the above-mentioned intestinal immunity enhancer.

[0038] Effects of the Invention

[0039] According to the present invention, an intestinal immunity enhancer that can sufficiently increase IgA in the intestine at a low dose and maintain the increased amount of IgA for a long period of time can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] [ Figure 1 ] is a graph showing the quantitative results of IgA in the feces of wild-type mice.

[0041] [ Figure 2 ] is a graph showing the quantitative results of IgA-bound bacteria in the feces of wild-type mice fed for 4 weeks.

[0042] [ Figure 3 ] is a graph showing the results of quantification of IgA in various parts of the intestine of wild-type mice fed for 4 weeks.

[0043] [ Figure 4 ] is a graph showing the quantitative results of IgA-producing plasma cells in the lamina propria of the large intestine mucosa of wild-type mice fed for 4 weeks.

[0044] [ Figure 5 ] is a graph showing the quantitative results of IgA-bound bacteria in the cecum and feces of single-bacteria-colonized mice fed for 4 weeks. DETAILED DESCRIPTION

[0045] [Intestinal immune enhancer]

[0046] The intestinal immunopotentiator of the present disclosure contains cellulose acetate having a total degree of substitution of acetyl groups of 0.4 or more and 1.1 or less. Here, the intestinal tract refers to the small intestine including the duodenum, jejunum, ileum, etc., and the large intestine including the cecum, proximal colon, distal colon, etc. In addition, as an immunopotentiator, an increase in IgA can be cited, and the increase in IgA includes an increase in IgA due to an increase in plasma cells producing IgA.

[0047] By taking or administering (especially orally) the intestinal immune enhancer of the present disclosure, the following effects can be obtained: an increase in plasma cells producing immunoglobulin A (IgA) in the intestinal tract (especially the mucosal lamina propria of the large intestine), an increase in IgA in the intestine, the ability to maintain the increased amount of IgA for a long time, and / or a decrease in Proteobacteria bacteria in the intestinal mucosa due to an increase in the antigen-antibody reaction specificity of IgA to Proteobacteria bacteria, etc. Proteobacteria is a phylum that includes a variety of pathogenic bacteria such as pathogenic Escherichia coli, Salmonella, and Vibrio. By reducing the above-mentioned bacteria in the large intestinal mucosa, the intestine can be protected from pathogenic bacteria. In addition, the intestinal immune enhancer of the present disclosure can still achieve this effect even if the dosage is lower than before.

[0048] (Total degree of acetyl substitution of cellulose acetate)

[0049] The total degree of substitution (average degree of substitution) of acetyl groups of the cellulose acetate of the present disclosure is 0.4 or more and 1.1 or less. If the total degree of substitution of acetyl groups is within this range, the solubility in water is excellent, and if it is outside this range, the solubility in water tends to decrease. It is believed that cellulose acetate is decomposed into acetic acid and cellulose by intestinal bacteria in the digestive tract, and cellulose is further decomposed into short-chain fatty acids such as acetic acid via oligosaccharides and monosaccharides. It is believed that the metabolic products such as acetic acid produced in such a process and the intestinal bacteria that assimilate cellulose acetate in such a process are related to the increase in IgA-producing cells and the increase in IgA. It is believed that extracellular enzymes cause cellulose acetate to decompose into acetic acid and cellulose, and therefore, cellulose acetate with high solubility in water is more easily decomposed, and the effect of promoting the increase in IgA-producing cells and the increase in IgA is high. In addition, it is also related to improving the intestinal immune enhancement effect. From such a viewpoint, the preferred range of the above-mentioned total degree of substitution of acetyl groups is 0.5 or more and 1.0 or less, and the further preferred range is 0.6 or more and 0.95 or less.

[0050] The total degree of acetyl substitution can be measured by a known titration method of dissolving cellulose acetate in water to calculate the degree of substitution of cellulose acetate.

[0051] The total degree of acetyl substitution can be calculated by converting the degree of acetylation obtained by the method for measuring the degree of acetylation in ASTM: D-817-91 (Testing methods for cellulose acetate, etc.) according to the following formula. This is the most common method for calculating the degree of substitution of cellulose acetate.

[0052] DS=162.14×AV×0.01 / (60.052-42.037×AV×0.01)

[0053] DS: total degree of acetyl substitution

[0054] AV: Acetylation degree (%)

[0055] First, 500 mg of dried cellulose acetate (sample) was accurately weighed and dissolved in 50 ml of a mixed solvent of ultrapure water and acetone (volume ratio of 4:1), and then 50 ml of a 0.2N-sodium hydroxide aqueous solution was added and saponified at 25°C for 2 hours. Next, 50 ml of 0.2N-hydrochloric acid was added, and the amount of acetic acid released was titrated with a 0.2N-sodium hydroxide aqueous solution (0.2N-sodium hydroxide equivalent solution) using phenolphthalein as an indicator. In addition, a blank test (a test without a sample) was performed in the same manner. Then, AV (acetylation degree) (%) was calculated according to the following formula.

[0056] AV(%)=(AB)×F×1.201 / sample weight(g)

[0057] A: Titration of 0.2N sodium hydroxide equivalent solution (ml)

[0058] B: Titration of 0.2N-sodium hydroxide equivalent solution in blank test (ml)

[0059] F: Factor of 0.2N-sodium hydroxide equivalent solution

[0060] In addition to the above, the total degree of acetyl substitution can also be measured by dissolving the hydroxyl groups of cellulose acetate in deuterated chloroform after propionylation. The propionylation of the hydroxyl groups of cellulose acetate can be carried out by the method of complete derivatization of cellulose acetate described below, that is, in a pyridine / N,N-dimethylacetamide mixed solvent, using N,N-dimethylaminopyridine as a catalyst and allowing propionic anhydride to act.

[0061] (Composition Distribution Index (CDI) of Cellulose Acetate)

[0062] In the present disclosure, the composition distribution index (CDI) of the cellulose acetate is not particularly limited. The composition distribution index (CDI) may be, for example, 1.0 or more and 3.0 or less. The composition distribution index (CDI) is preferably 2.0 or less, more preferably 1.0 or more and 2.0 or less, further preferably 1.0 or more and 1.8 or less, further preferably 1.0 or more and 1.6 or less, and particularly preferably 1.0 or more and 1.5 or less.

[0063] Although the lower limit of the composition distribution index (CDI) is 0, this refers to the case where it is achieved by a special synthesis technology, such as acetylation of only the 6-position of the glucose residue with 100% selectivity and no acetylation of other positions, and such a synthesis technology is not yet known. In the case where all the hydroxyl groups of the glucose residue are acetylated and deacetylated with the same probability, the CDI is 1.0, but in the actual reaction of cellulose, it takes considerable effort to approach such an ideal state. The smaller the above-mentioned composition distribution index (CDI), the more uniform the composition distribution (intermolecular substitution distribution). When the composition distribution is uniform, it has the following advantages: the total degree of substitution of acetyl groups can ensure water solubility in a wider range than usual, achieve uniform dissolution, and will not show structural viscosity, so it is easy to ingest or administer, it is easy to be decomposed, and it is easy to show the effect of enhancing intestinal immunity.

[0064] Here, the compositional distribution index (CDI) is defined as: the ratio of the measured value of the half-peak width of the composition distribution to the theoretical value [(measured value of the half-peak width of the composition distribution) / (theoretical value of the half-peak width of the composition distribution)]. The half-peak width of the composition distribution is also called the "half-peak width of the distribution of the degree of substitution between molecules", or simply "half-peak width of the distribution of the degree of substitution".

[0065] In order to evaluate the uniformity of the total degree of substitution of acetyl groups in cellulose acetate, the half-width (also referred to as "half-peak width") of the maximum peak of the intermolecular substitution distribution curve of cellulose acetate can be used as an index. It should be noted that the half-peak width is the width of the curve at half the height of the peak of the curve when the total degree of substitution of acetyl groups is the horizontal axis (x-axis) and the amount of the substitution is the vertical axis (y-axis), and is an index that characterizes the degree of dispersion of the distribution. The half-peak width of the substitution distribution can be obtained by high-performance liquid chromatography (HPLC) analysis. It should be noted that the method of converting the horizontal axis (elution time) of the elution curve of cellulose ester in HPLC into the degree of substitution (0 to 3) is described in Japanese Patent Application Laid-Open No. 2003-201301 (paragraphs 0037 to 0040).

[0066] (Theoretical value of half peak width of composition distribution)

[0067] The composition distribution half-peak width (substitution degree distribution half-peak width) can be calculated as a theoretical value by probability theory. That is, the theoretical value of the composition distribution half-peak width can be obtained by the following formula (1).

[0068] [Mathematical formula 2]

[0069]

[0070] m: the total number of hydroxyl groups and acetyl groups in one cellulose acetate molecule

[0071] p: The probability that the hydroxyl group in one cellulose acetate molecule is replaced by an acetyl group

[0072] q=1-p

[0073] DPw: weight average degree of polymerization (based on GPC-light scattering method)

[0074] In addition, the measuring method of the weight average degree of polymerization (DPw) is as described later.

[0075] Formula (1) is the half-peak width of the composition distribution that is bound to occur when all hydroxyl groups of cellulose are acetylated and deacetylated with the same probability, and is derived according to the so-called binomial theorem. When the theoretical value of the half-peak width of the composition distribution is further expressed by the degree of substitution and the degree of polymerization, it can be expressed as follows. The following formula (2) is used as the definition formula for calculating the theoretical value of the half-peak width of the composition distribution.

[0076] [Mathematical formula 3]

[0077]

[0078] DS: total degree of acetyl substitution

[0079] DPw: weight average degree of polymerization (based on GPC-light scattering method)

[0080] In addition, the measuring method of the weight average degree of polymerization (DPw) is as described later.

[0081] In addition, in formula (1) and formula (2), it is more rigorous to consider the distribution of the degree of polymerization. In this case, "DPw" in formula (1) and formula (2) should be replaced by the degree of polymerization distribution function, and the entire formula should be integrated from the degree of polymerization of 0 to infinity. However, as long as DPw is used, formula (1) and formula (2) can approximately give a theoretical value with sufficient accuracy. Since the influence of the degree of polymerization distribution cannot be ignored if DPn (number average degree of polymerization) is used, DPw should be used.

[0082] (Measured value of half peak width of composition distribution)

[0083] In the present disclosure, the measured value of the half-peak width of the composition distribution refers to the half-peak width of the composition distribution obtained by HPLC analysis of cellulose acetate propionate obtained by propionylating all the remaining hydroxyl groups (unsubstituted hydroxyl groups) of cellulose acetate (sample).

[0084] Generally, for cellulose acetate having a total degree of acetyl substitution of 2 to 3, the composition distribution half-peak width can be determined by high performance liquid chromatography (HPLC) analysis without pretreatment. For example, Japanese Patent Application Laid-Open No. 2011-158664 describes a composition distribution analysis method for cellulose acetate having a degree of substitution of 2.27 to 2.56.

[0085] On the other hand, the measured value of the half-peak width of the composition distribution (half-peak width of the substitution degree distribution) of the cellulose acetate of the present disclosure is obtained by derivatizing the residual hydroxyl groups in the cellulose acetate molecule as a pretreatment before HPLC analysis, and then performing HPLC analysis. The purpose of this pretreatment is to convert the low-substituted cellulose acetate (e.g., cellulose acetate with a total acetyl substitution degree of 1.1 or less) into a derivative that is easily soluble in an organic solvent so that it can be analyzed by HPLC. That is, the residual hydroxyl groups in the molecule are completely propionylated, and the completely derivatized cellulose acetate propionate (CAP) is subjected to HPLC analysis to obtain the half-peak width of the composition distribution (measured value). Here, the derivatization must be completed so that there are no residual hydroxyl groups in the molecule, and only acetyl and propionyl groups are present. That is, the sum of the total acetyl substitution degree (DSac) and the total propionyl substitution degree (DSpr) is 3. The reason for this is that the relationship: DSac+DSpr=3 is used to prepare a calibration curve for converting the horizontal axis (elution time) of the HPLC elution curve of CAP into the total degree of acetyl substitution (0 to 3).

[0086] The complete derivatization of cellulose acetate can be carried out by reacting propionic anhydride in a pyridine / N,N-dimethylacetamide mixed solvent using N,N-dimethylaminopyridine as a catalyst. More specifically, 20 parts by weight of a mixed solvent [pyridine / N,N-dimethylacetamide = 1 / 1 (v / v)] as a solvent, 6.0 to 7.5 equivalents of propionic anhydride as a propionylating agent to the hydroxyl groups of the cellulose acetate, and 6.5 to 8.0 mol% of N,N-dimethylaminopyridine as a catalyst to the hydroxyl groups of the cellulose acetate are used to carry out propionylation at a temperature of 100°C and a reaction time of 1.5 to 3.0 hours. After the reaction, the mixture is precipitated using methanol as a precipitation solvent to obtain completely derivatized cellulose acetate propionate. More specifically, for example, 1 part by weight of the reaction mixture is added to 10 parts by weight of methanol at room temperature to precipitate the mixture, and the precipitate is washed five times with methanol and vacuum dried at 60° C. for 3 hours to obtain completely derivatized cellulose acetate propionate (CAP). It should be noted that the dispersity (polydispersity, Mw / Mn) and weight average degree of polymerization (DPw) described later are also obtained by converting cellulose acetate (sample) into completely derivatized cellulose acetate propionate (CAP) and measuring the same using this method.

[0087] In the above HPLC analysis, a plurality of cellulose acetate propionates having different total acetyl substitution degrees can be used as standard samples, and HPLC analysis can be performed using a predetermined measuring device and measuring conditions. The half-peak width (actually measured value) of the composition distribution of cellulose acetate (sample) can be obtained from a calibration curve [a curve showing the relationship between the elution time of cellulose acetate propionate and the total acetyl substitution degree (0 to 3), usually a cubic curve] prepared using the analysis values ​​of these standard samples. The relationship between the elution time and the distribution of the total acetyl substitution degree of cellulose acetate propionate is obtained by HPLC analysis. Since this is the relationship between the elution time of a substance in which all the residual hydroxyl groups in the sample molecule are converted into propionyloxy groups and the distribution of the total acetyl substitution degree, what is obtained is still the distribution of the total acetyl substitution degree of cellulose acetate of the present disclosure.

[0088] The conditions for the above HPLC analysis are as follows.

[0089] Device: Agilent 1100 Series

[0090] Column: Waters Nova-Pak phenyl 4μm (150mm×3.9mmΦ) + guard column

[0091] Column temperature: 30°C

[0092] Detection: Varian 380-LC

[0093] Injection volume: 5.0 μL (sample concentration: 0.1% (wt / vol))

[0094] Eluent: Solution A: MeOH / H2O=8 / 1 (v / v), Solution B: CHCl3 / MeOH=8 / 1 (v / v)

[0095] Gradient: A / B = 80 / 20 → 0 / 100 (28 minutes); Flow rate: 0.7 mL / min

[0096] In the substitution degree distribution curve [the substitution degree distribution curve of cellulose acetate propionate with the amount of cellulose acetate propionate present as the vertical axis and the total degree of acetyl substitution as the horizontal axis] (also referred to as "intermolecular substitution degree distribution curve") obtained from the calibration curve, the half-peak width of the substitution degree distribution is obtained as follows for the maximum peak (E) corresponding to the average substitution degree. A baseline (AB) is drawn that touches the base (A) on the low-substitution side and the base (B) on the high-substitution side of the peak (E), and a perpendicular line is drawn from the maximum peak (E) to the horizontal axis relative to the baseline. The intersection point (C) of the perpendicular line and the baseline (AB) is determined, and the midpoint (D) between the maximum peak (E) and the intersection point (C) is obtained. A straight line is drawn that passes through the midpoint (D) and is parallel to the baseline (AB), and two intersection points (A', B') with the intermolecular substitution degree distribution curve are obtained. A perpendicular line was drawn from the two intersection points (A', B') to the horizontal axis, and the width between the two intersection points on the horizontal axis was taken as the half-peak width of the maximum peak (ie, half-peak width of the substitution degree distribution).

[0097] As for the half-peak width of the substitution degree distribution, the molecular chain of cellulose acetate propionate in the sample is reflected in the retention time (retention time) according to the degree to which the hydroxyl groups of the glucose rings constituting each polymer chain of the molecular chain are acetylated. Therefore, ideally, the width of the retention time represents the width of the composition distribution (in substitution degree units). However, in HPLC, there are tube parts (such as a guard column for protecting the chromatographic column) that do not contribute to the distribution. Therefore, due to the structure of the measuring device, the width of the retention time that is not caused by the width of the composition distribution is often included as an error. As described above, this error is affected by the length and inner diameter of the chromatographic column, the length from the chromatographic column to the detector, the connection, etc., and varies depending on the device structure. Therefore, the half-peak width of the substitution degree distribution of cellulose acetate propionate can usually be calculated as a correction value Z based on the correction formula represented by the following formula. When such a correction formula is used, even if the measuring device (and the measuring conditions) are different, a more accurate half-peak width of the substitution degree distribution (actually measured value) can be calculated with the same (approximately the same) value.

[0098] Z=(X 2 -Y 2 ) 1 / 2

[0099] [Wherein, X is the half-peak width of the substitution distribution (uncorrected value) determined using a specified measuring device and measuring conditions. Y = (ab)x / 3 + b (0≤x≤3). Here, a is the half-peak width of the apparent substitution distribution of cellulose acetate with a total substitution degree of 3 determined using the same measuring device and measuring conditions as those for X above (in reality, the total substitution degree is 3, so there is no substitution distribution), and b is the half-peak width of the apparent substitution distribution of cellulose propionate with a total substitution degree of 3 determined using the same measuring device and measuring conditions as those for X above. x is the total acetyl substitution degree of the measurement sample (0≤x≤3)]

[0100] It should be noted that the cellulose acetate (or cellulose propionate) having a total substitution degree of 3 is a cellulose ester obtained by esterifying all the hydroxyl groups of cellulose, and is actually (ideally) a cellulose ester having no half-value width of the substitution degree distribution (i.e., the half-value width of the substitution degree distribution is 0).

[0101] The measured value of the half-peak width of the composition distribution (half-peak width of the substitution degree distribution) of the cellulose acetate is preferably 0.12 to 0.34, more preferably 0.13 to 0.31, and even more preferably 0.13 to 0.25.

[0102] The above-described substitution degree distribution theoretical formula is a probabilistically calculated value assuming that all acetylation and deacetylation proceed independently and equally. That is, it is a calculated value following binomial distribution. Such an ideal situation is impossible to occur in reality. Without a special design to make the hydrolysis reaction of cellulose acetate close to an ideal random reaction and / or to make the composition graded in terms of post-reaction post-treatment, the substitution degree distribution of cellulose ester is much wider than the substitution degree distribution determined probabilistically according to binomial distribution.

[0103] As one of the special designs of the reaction, it is possible to consider maintaining the system under conditions where deacetylation and acetylation are balanced. However, in this case, the decomposition of cellulose will proceed due to the acid catalyst, so it is not preferred. As a special design of other reactions, reaction conditions that slow down the deacetylation rate for low-degree-of-substitution products can be adopted. However, such a specific method is still unknown in the prior art. That is, the special design of the reaction that controls the degree of substitution distribution of cellulose ester to follow the binomial distribution in the manner of reaction probability theory is still unknown. In addition, various conditions such as the unevenness of the acetylation process (acetylation process of cellulose), the localization caused by the staged addition of water in the aging process (hydrolysis process of cellulose acetate), and the occurrence of temporary precipitation will cause the degree of substitution distribution to develop in a direction wider than the binomial distribution, and it is impossible to avoid all of these and achieve ideal conditions in reality. This is similar to the fact that ideal gases are ideal products after all, and the behavior of actually existing gases will be more or less different from them.

[0104] In the synthesis and post-treatment of low-substituted cellulose acetate in the past, little attention has been paid to the issue of substitution distribution, and no measurement, verification, or investigation of substitution distribution has been conducted. For example, according to the literature (Journal of the Fiber Society, 42, p25 (1986)), it is stated that the solubility of low-substituted cellulose acetate is determined by the distribution of acetyl groups at the 2nd, 3rd, and 6th positions of the glucose residues, but the composition distribution is not considered at all.

[0105] According to the research of the inventors of the present application, surprisingly, it was found that the degree of substitution distribution of cellulose acetate can be controlled by designing the post-treatment conditions after the hydrolysis step of cellulose acetate, as described later. According to the literature (CiBment, L. and Rivibre, C., Bull. Soc. Chim., (5) 1, 1075 (1934), Sookne, AM, Rutherford, HA, Mark, H. and Harris, MJ Research Natl. Bur. Standards, 29, 123 (1942), AJ Rosenthal, BB White Ind. Eng. Chem., 1952, 44 (11), pp2693-2696.), it is believed that in the fractional precipitation of cellulose acetate having a total degree of substitution of 2.3, a fractionation depending on the molecular weight and a slight fractionation due to the degree of substitution (chemical composition) occur, but there is no report that such a significant fractionation due to the degree of substitution (chemical composition) as found by the inventors of the present application can occur. Furthermore, regarding low-substituted cellulose acetate, it has not been verified that the degree of substitution distribution (chemical composition) can be controlled by fractional dissolution or fractional precipitation.

[0106] Another design for narrowing the substitution degree distribution found by the inventors of the present application is the hydrolysis reaction (ripening reaction) of cellulose acetate at a high temperature of 90°C or higher (or higher). Conventionally, although the degree of polymerization of the product obtained by the high temperature reaction has not been analyzed and examined in detail, it is believed that the decomposition of cellulose occurs preferentially in the high temperature reaction of 90°C or higher. It can be said that such a consideration is a knowledge (old knowledge) obtained only based on the examination of viscosity. The inventors of the present application have found that when cellulose acetate is hydrolyzed to obtain low-substituted cellulose acetate, when the reaction is carried out at a high temperature of 90°C or higher (or higher), preferably in the presence of a strong acid such as sulfuric acid, and in a large amount of acetic acid, a decrease in the degree of polymerization is not observed, but a decrease in viscosity occurs with a decrease in CDI. That is, it is clarified that the decrease in viscosity accompanying the high temperature reaction is not caused by a decrease in the degree of polymerization, but is based on a decrease in structural viscosity caused by a narrowing of the substitution degree distribution. When cellulose acetate is hydrolyzed under the above conditions, not only the forward reaction but also the reverse reaction occurs, so the CDI of the product (low-substituted cellulose acetate) becomes extremely small and the solubility in water is significantly improved. On the other hand, when cellulose acetate is hydrolyzed under conditions where the reverse reaction is not easy to occur, the substitution degree distribution becomes broad for various reasons, and the content of cellulose acetate having a total acetyl substitution degree of less than 0.4 and cellulose acetate having a total acetyl substitution degree of more than 1.1, which are poorly soluble in water, increases, and the solubility in water decreases as a whole.

[0107] (Standard deviation of substitution degree at positions 2, 3, and 6)

[0108] The total degree of substitution of acetyl groups at the 2nd, 3rd, and 6th positions of the glucose ring of the cellulose acetate can be measured by the NMR method according to the method of Tezuka (Carbonydr. Res. 273, 83 (1995)). That is, the free hydroxyl groups of the cellulose acetate sample are propionylated with propionic anhydride in pyridine. The obtained sample is dissolved in deuterated chloroform and the NMR is measured. 13 C-NMR spectrum. The carbon signal of the acetyl group appears in the order of position 2, position 3, and position 6 from the high magnetic field in the range of 169ppm to 171ppm. In addition, the signal of the carbonyl carbon of the propionyl group appears in the same order in the range of 172ppm to 174ppm. According to the existence ratio of the acetyl group to the propionyl group at the corresponding positions, the degree of acetyl substitution at positions 2, 3, and 6 of the glucose ring in the original cellulose acetate can be calculated. It should be noted that the sum of the degrees of acetyl substitution at positions 2, 3, and 6 calculated in this way is the total degree of acetyl substitution, and the total degree of acetyl substitution can also be calculated by this method. It should be noted that, except for 13 In addition to C-NMR, 1 H-NMR was used to analyze the total degree of acetyl substitution.

[0109] The standard deviation σ of the substitution degree at the 2-, 3-, and 6-positions is defined by the following formula.

[0110] [Formula 4]

[0111]

[0112] σ: standard deviation

[0113] n=3

[0114] x: x1 is the degree of substitution at position 2, x2 is the degree of substitution at position 3, and x3 is the degree of substitution at position 6

[0115] Total degree of acetyl substitution / 3

[0116] The standard deviation of the degree of acetyl substitution at the 2nd, 3rd and 6th positions of the glucose ring of cellulose acetate is preferably 0.08 or less (0 to 0.08). Cellulose acetate having a standard deviation of 0.08 or less has evenly substituted positions at the 2nd, 3rd and 6th positions of the glucose ring and has excellent solubility in water.

[0117] (Dispersity (polydispersity, Mw / Mn))

[0118] The dispersion degree (polydispersity, Mw / Mn) of the molecular weight distribution (polymerization degree distribution) is a value obtained by a GPC-light scattering method using cellulose acetate propionate obtained by propionylating all the remaining hydroxyl groups of cellulose acetate (sample).

[0119] The dispersion degree (polydispersity, Mw / Mn) of the cellulose acetate of the present disclosure is preferably in the range of 1.2 to 2.5. The molecular size of cellulose acetate having a dispersion degree Mw / Mn within the above range is uniform and has excellent solubility in water. It is believed that cellulose acetate is decomposed into acetic acid and cellulose by intestinal bacteria in the digestive tract, and cellulose is further decomposed into short-chain fatty acids such as acetic acid via oligosaccharides and monosaccharides. It is believed that the metabolic products such as acetic acid produced in such a process and the intestinal bacteria that assimilate cellulose acetate in such a process are related to the increase in IgA-producing cells and the increase in IgA. It is believed that the decomposition of cellulose acetate into acetic acid and cellulose is caused by extracellular enzymes, and therefore, cellulose acetate with high solubility in water is more easily decomposed, and the effect of promoting the increase in IgA-producing cells and the increase in IgA is high. In addition, it is also related to improving the intestinal immune enhancement effect.

[0120] The number average molecular weight (Mn), weight average molecular weight (Mw) and dispersion degree (polydispersity, Mw / Mn) of cellulose acetate can be determined by a known method using HPLC. The dispersion degree (polydispersity, Mw / Mn) of cellulose acetate can be determined by making the measurement sample soluble in an organic solvent, by the same method as in the case of calculating the measured value of the half peak width of the composition distribution, cellulose acetate (sample) is completely derivatized into cellulose acetate propionate (CAP), and then size exclusion chromatography analysis (GPC-light scattering method) is performed under the following conditions.

[0121] Equipment: Shodex GPC "SYSTEM-21H"

[0122] Solvent: Acetone

[0123] Chromatographic columns: 2 GMHxl (Tosoh), guard column (TSKgel guardcolumn HXL-H manufactured by Tosoh)

[0124] Flow rate: 0.8ml / min

[0125] Temperature: 29℃

[0126] Sample concentration: 0.25% (wt / vol)

[0127] Injection volume: 100 μl

[0128] Detection: MALLS (Multi-angle Light Scattering Detector) (manufactured by Wyatt, “DAWN-EOS”)

[0129] MALLS calibration standard material: PMMA (molecular weight 27600)

[0130] (Weight average degree of polymerization (DPw))

[0131] The weight average degree of polymerization (DPw) is a value determined by a GPC-light scattering method using cellulose acetate propionate obtained by propionylating all the remaining hydroxyl groups of cellulose acetate (sample).

[0132] The weight average degree of polymerization (DPw) of the cellulose acetate of the present disclosure is preferably in the range of 50 to 800. When the weight average degree of polymerization (DPw) is too high, the solubility in water tends to deteriorate. The above-mentioned weight average degree of polymerization (DPw) is preferably 55 to 700, and more preferably 60 to 600. It is believed that cellulose acetate is decomposed into acetic acid and cellulose by intestinal bacteria in the digestive tract, and cellulose is further decomposed into short-chain fatty acids such as acetic acid via oligosaccharides and monosaccharides. It is believed that the metabolic products such as acetic acid produced in such a process and the intestinal bacteria that assimilate cellulose acetate in such a process are related to the increase in IgA-producing cells and the increase in IgA. It is believed that extracellular enzymes cause cellulose acetate to decompose into acetic acid and cellulose. Therefore, cellulose acetate with high solubility in water is more easily decomposed, and the effect of promoting the increase in IgA-producing cells and the increase in IgA is high. In addition, it is also related to improving the intestinal immune enhancement effect.

[0133] The weight average degree of polymerization (DPw) can be determined similarly to the above-mentioned degree of dispersion (polydispersity, Mw / Mn) by subjecting cellulose acetate (sample) to completely derivatized cellulose acetate propionate (CAP) and then subjecting it to size exclusion chromatography analysis (GPC-light scattering method) in the same manner as in the case of determining the measured value of the half-peak width of the composition distribution.

[0134] As described above, the molecular weight (polymerization degree) and dispersion (polydispersity, Mw / Mn) of cellulose acetate can be measured using the GPC-light scattering method (GPC-MALLS, GPC-LALLS, etc.). It should be noted that, in general, it is difficult to perform light scattering detection in an aqueous solvent. The reason is that aqueous solvents usually have a lot of foreign matter, and secondary contamination is prone to occur even if they are temporarily purified. In addition, in aqueous solvents, due to the influence of trace amounts of ionic dissociated groups, the extension of the molecular chain is sometimes unstable. If a water-soluble inorganic salt (such as sodium chloride) is added to suppress this, the dissolved state sometimes becomes unstable and an association is formed in the aqueous solution. One of the effective methods for avoiding this problem is to derivatize cellulose acetate, dissolve it in an organic solvent with less foreign matter and less prone to secondary contamination, and perform GPC-light scattering measurement in the organic solvent. As a derivatization of cellulose acetate for this purpose, propionylation is effective, and the specific reaction conditions and post-treatment are as described in the description of the measured value of the half-peak width of the above-mentioned composition distribution.

[0135] (Manufacture of cellulose acetate)

[0136] The cellulose acetate (cellulose acetate) can be produced, for example, by (A) a hydrolysis step (ripening step) of cellulose acetate having a medium to high degree of substitution, (B) a precipitation step, and (C) a washing and neutralization step, which are carried out as required. The total degree of substitution of acetyl groups in the cellulose acetate having a medium to high degree of substitution is, for example, 1.5 or more and 3 or less, preferably 2 or more and 3 or less.

[0137] [(A) Hydrolysis step (aging step)]

[0138] The hydrolysis reaction can be carried out by reacting the raw material cellulose acetate with water in an organic solvent in the presence of a catalyst (ripening catalyst). Examples of the organic solvent include acetic acid, acetone, alcohol (methanol, etc.), and mixed solvents thereof. Among them, a solvent containing at least acetic acid is preferred. As the catalyst, a catalyst generally used as a deacetylation catalyst can be used. As the catalyst, sulfuric acid is particularly preferred.

[0139] The amount of the organic solvent (eg, acetic acid) used is, for example, 0.5 to 50 parts by weight, preferably 1 to 20 parts by weight, and more preferably 3 to 10 parts by weight, based on 1 part by weight of the raw material cellulose acetate.

[0140] The amount of the catalyst (e.g., sulfuric acid) used is, for example, 0.005 to 1 part by weight, preferably 0.01 to 0.5 part by weight, and more preferably 0.02 to 0.3 part by weight, relative to 1 part by weight of the raw material cellulose acetate. If the amount of the catalyst is too small, the hydrolysis time becomes too long, and the molecular weight of the cellulose acetate may be reduced. On the other hand, if the amount of the catalyst is too large, the degree of change of the depolymerization rate with respect to the hydrolysis temperature increases, and even if the hydrolysis temperature is lowered to a certain extent, the depolymerization rate becomes high, and it is difficult to obtain cellulose acetate with a molecular weight large to a certain extent.

[0141] The amount of water in the hydrolysis step is, for example, 0.5 to 20 parts by weight, preferably 1 to 10 parts by weight, and more preferably 2 to 7 parts by weight, relative to 1 part by weight of the raw material cellulose acetate. Moreover, the amount of water is, for example, 0.1 to 5 parts by weight, preferably 0.3 to 2 parts by weight, and more preferably 0.5 to 1.5 parts by weight, relative to 1 part by weight of the organic solvent (e.g., acetic acid). The entire amount of water may be present in the system at the start of the reaction, but in order to prevent precipitation of cellulose acetate, a portion of the water to be used may be present in the system at the start of the reaction, and the remaining water may be added to the system in one or more portions.

[0142] The reaction temperature in the hydrolysis step is, for example, 40 to 130° C., preferably 50 to 120° C., and more preferably 60 to 110° C. In particular, when the reaction temperature is 90° C. or higher (or a temperature higher than 90° C.), the equilibrium of the reaction tends to tilt in the direction of increasing the rate of the reverse reaction (acetylation reaction) relative to the forward reaction (hydrolysis reaction), and as a result, the degree of substitution distribution becomes narrower, and even without particularly designing the post-treatment conditions, cellulose acetate with an extremely small composition distribution index CDI can be obtained. In this case, it is preferred to use a strong acid such as sulfuric acid as a catalyst, and it is preferred to use an excess of acetic acid as a reaction solvent. In addition, even when the reaction temperature is 90° C. or lower, as described later, by using a mixed solvent containing two or more solvents as a precipitation solvent in the precipitation step to precipitate, or by performing fractional precipitation and / or fractional dissolution, cellulose acetate with an extremely small composition distribution index CDI can be obtained.

[0143] [(B) Precipitation step]

[0144] In this step, after the hydrolysis reaction is completed, the temperature of the reaction system is cooled to room temperature, and a precipitation solvent is added to precipitate cellulose acetate. As the precipitation solvent, an organic solvent miscible with water or an organic solvent having a high solubility in water can be used. Examples thereof include ketones such as acetone and methyl ethyl ketone; alcohols such as methanol, ethanol, and isopropanol; esters such as ethyl acetate; nitrogen-containing compounds such as acetonitrile; ethers such as tetrahydrofuran; mixed solvents thereof; and the like.

[0145] When a mixed solvent containing two or more solvents is used as a precipitation solvent, the same effect as the fractional precipitation described below can be obtained, and cellulose acetate having a narrow composition distribution (molecular substitution degree distribution) and a small composition distribution index (CDI) can be obtained. Preferred mixed solvents include, for example, a mixed solvent of acetone and methanol, a mixed solvent of isopropyl alcohol and methanol, and the like.

[0146] Furthermore, by further subjecting the precipitated cellulose acetate to fractional precipitation (precipitation fractionation) and / or fractional dissolution (dissolution fractionation), cellulose acetate having a narrow composition distribution (intermolecular substitution degree distribution) and a very small composition distribution index CDI can be obtained.

[0147] Fractional precipitation can be performed, for example, by dissolving the cellulose acetate (solid) obtained by precipitation in water to prepare an aqueous solution of a suitable concentration (e.g., 2 to 10% by weight, preferably 3 to 8% by weight), adding a poor solvent to the aqueous solution (or adding the aqueous solution to the poor solvent), maintaining the temperature at a suitable temperature (e.g., 30° C. or lower, preferably 20° C. or lower), precipitating the cellulose acetate, and recovering the precipitate. Examples of the poor solvent include alcohols such as methanol and ketones such as acetone. The amount of the poor solvent used is, for example, 1 to 10 parts by weight, preferably 2 to 7 parts by weight, relative to 1 part by weight of the aqueous solution.

[0148] The fractional dissolution can be performed, for example, by adding a mixed solvent of water and an organic solvent (e.g., ketones such as acetone, alcohols such as ethanol, etc.) to the cellulose acetate (solid) obtained by the above precipitation or the cellulose acetate (solid) obtained by the above fractional precipitation, stirring at an appropriate temperature (e.g., 20 to 80° C., preferably 25 to 60° C.), separating into a concentrated phase and a dilute phase by centrifugal separation, adding a precipitation solvent (e.g., ketones such as acetone, alcohols such as methanol, etc.) to the dilute phase, and recovering the precipitate (solid). The concentration of the organic solvent in the mixed solvent of water and the organic solvent is, for example, 5 to 50% by weight, preferably 10 to 40% by weight.

[0149] [(C) Washing and neutralization process]

[0150] The precipitate (solid) obtained in the precipitation step (B) is preferably washed with an organic solvent (poor solvent) such as an alcohol such as methanol or a ketone such as acetone. In addition, it is also preferably washed and neutralized with an organic solvent containing an alkaline substance (e.g., an alcohol such as methanol, a ketone such as acetone, etc.). It should be noted that the neutralization step may also be provided immediately after the hydrolysis step, in which case, it is preferred to add an alkaline substance or an aqueous solution thereof to the hydrolysis reaction bath.

[0151] As the alkaline substance, for example, alkali metal compounds (e.g., alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal carbonates such as sodium carbonate and potassium carbonate; alkali metal bicarbonates such as sodium bicarbonate; alkali metal carboxylates such as sodium acetate and potassium acetate; sodium alkoxides such as sodium methoxide and sodium ethoxide; etc.), alkaline earth metal compounds (e.g., alkaline earth metal hydroxides such as magnesium hydroxide and calcium hydroxide; alkaline earth metal carbonates such as magnesium carbonate and calcium carbonate; alkaline earth metal carboxylates such as magnesium acetate and calcium acetate; alkaline earth metal alkoxides such as magnesium ethoxide; etc.) can be used. Among them, alkali metal compounds such as potassium acetate are particularly preferred.

[0152] By washing and neutralization, impurities such as the catalyst (sulfuric acid, etc.) used in the hydrolysis step can be removed efficiently.

[0153] The cellulose acetate thus obtained may be pulverized, sieved or granulated as necessary to adjust the particle size to a specific range.

[0154] [Food, medicine]

[0155] The intestinal immune enhancer of the present disclosure can be included in food or medicine. Only cellulose acetate having a total degree of acetyl substitution of 0.4 to 1.1 can be made into food or medicine, or can be used as a constituent element of various foods or medicines as described below.

[0156] As a method of taking or administering the food or drug, oral intake or administration can be particularly mentioned. Various forms can be selected. For example, conventional drug forms such as powders, granules, tablets, sugar-coated tablets, capsules, syrups, pills, suspensions, liquid preparations, and emulsions can be used, as well as beverages; candy products such as chewing gum, chocolate, candy, yokan, and jelly; noodles; baked foods such as bread, cakes, and biscuits; canned foods; retort pouch foods; meat products; aquatic gruel foods; edible oil compositions such as margarine, sauces, and mayonnaise; nutritional supplements; milk products such as butter, ice cream, and yogurt; and other conventional food forms can be used.

[0157] The food or medicine of the present disclosure can be applied not only to humans, but also to animals such as livestock (cows, pigs, horses, sheep, etc.), poultry (chickens, ducks, etc.), and pets (dogs, cats, monkeys, mice, rats, guinea pigs, etc.). The dosage for achieving the effect in humans is preferably 1 to 10 g per day in terms of the intake of cellulose acetate. Sugar-coated tablets, noodles, baked goods such as biscuits, etc. are preferred from the perspective of being able to ingest a large amount. In addition, the intestinal immune enhancer of the present disclosure can also be added to the form of medicine or food as a thickener.

[0158] The content of cellulose acetate having a total degree of acetyl substitution of 0.4 or more and 1.1 or less in the food of the present disclosure is not particularly limited, but is preferably 0.1% by weight or more in the food. This is because an effective amount of cellulose acetate can be ingested by ingesting the food for one day. From the viewpoint of not impairing the taste and mouthfeel of the food, sufficiently increasing IgA in the intestine, and maintaining the increased amount of IgA for a long period of time (e.g., 2 to 4 weeks, or more than 4 weeks), it is preferably 0.1% by weight or more and less than 5% by weight, and more preferably 1.5% by weight or more and less than 3% by weight.

[0159] Specific examples of target diseases that can be prevented and / or treated (mitigation or prevention of adverse effects) when the intestinal immunopotentiator of the present disclosure is contained in food or medicine include infections caused by pathogenic bacteria belonging to the Proteobacteria, such as Salmonella infection, cholera, Vibrio enteritis, etc. By increasing the number of IgA-producing plasma cells in the intestinal tract, especially in the lamina propria of the large intestine, effects such as increasing IgA, strengthening mucosal immune function, and preventing infections and allergic diseases can be expected.

[0160] The amount of the intestinal immune enhancer of the present disclosure is taken or administered as long as it is sufficient for bringing the desired intestinal immune enhancement effect. Specifically, the conditions related to the individual, such as the age, weight, sex, health status, and the state of the stomach, small intestine, and large intestine, etc., the method of taking or administering, and the form of the preparation can be determined based on experience. The amount of each intake or administration can be, for example, 5mg / kg body weight to 60mg / kg body weight, or 10mg / kg body weight to 40mg / kg body weight. In addition, the number of times an individual takes or administers to an individual can be 1 time, or more than 1 time. In the case of more than 1 time, it can be administered regularly, irregularly, or as needed. With regard to the appropriate number of times, it can be determined based on experience by considering the conditions related to the individual, the method of taking or administering, and the form of the preparation as well as the amount taken or administered.

[0161] Example

[0162] Hereinafter, the present invention will be specifically described by way of examples, but the technical scope of the present invention is not limited to these examples.

[0163] [Preparation of Cellulose Acetate]

[0164] 4.4 parts by weight of acetic acid and 1.9 parts by weight of water were added to 1 part by weight of cellulose acetate (manufactured by Daicel Corporation, trade name "L-70", total degree of acetyl substitution: 2.43, 6% viscosity: 145 mPa·s), and the mixture was stirred for 3 hours to dissolve the cellulose acetate. A mixture of 0.58 parts by weight of acetic acid and 0.13 parts by weight of sulfuric acid was added to the solution, and the resulting solution was kept at 70° C. to perform hydrolysis. During the hydrolysis, water was added to the system twice in order to prevent precipitation of cellulose acetate. Specifically, 0.65 parts by weight of water was added to the system over 5 minutes after 1 hour from the start of the reaction. Furthermore, 1.29 parts by weight of water was added to the system over 10 minutes after 2 hours, and the reaction was further performed for 4 hours. The total hydrolysis time was 7 hours. It should be noted that the period from the start of the reaction to the first addition of water is referred to as the first hydrolysis step (first aging step), the period from the first addition of water to the second addition of water is referred to as the second hydrolysis step (second aging step), and the period from the second addition of water to the end of the reaction is referred to as the third hydrolysis step (third aging step).

[0165] After the hydrolysis, a 24% aqueous solution of magnesium acetate containing 1.1 equivalents of magnesium acetate relative to sulfuric acid was added to the reaction mixture to stop the reaction. The reaction mixture was added dropwise to 3.6 times the weight of acetone relative to the reaction mixture under stirring for 60 minutes to form a precipitate. The precipitate was recovered by filtration as a wet cake having a solid content of 15% by weight. 16 parts by weight of a mixed solvent of acetone / water (acetone concentration of 20% by weight) was added to 1 part by weight of the solid content of the obtained precipitate, and the mixture was stirred at 40°C for 8 hours, and the solid matter was recovered by filtration as a wet cake having a solid content of 15% by weight. 16 parts by weight of methanol was added to 1 part by weight of the solid content of the obtained wet cake, and the mixture was stirred at 25°C for 1 hour, and the solid matter was recovered by filtration as a wet cake having a solid content of 15% by weight. The above operation was repeated 5 times, and the mixture was dried to obtain low-substituted cellulose acetate.

[0166] The total degree of acetyl substitution, weight average degree of polymerization (DPw), dispersity (polydispersity, Mw / Mn), measured value of half-peak width of composition distribution, and composition distribution index (CDI) of the obtained cellulose acetate were measured by the above-mentioned method. As a result, the total degree of acetyl substitution of cellulose acetate was 0.78, the weight average degree of polymerization (DPw) was 124, the dispersity (polydispersity, Mw / Mn) was 2.0, the measured value of half-peak width of composition distribution was 0.305, and the composition distribution index (CDI) was 1.90.

[0167] [Evaluation of intestinal immune enhancers]

[0168] <Preparation of feed>

[0169] Refined feed AIN-93G (REEVE et al., Journal of Nutrition, 123, 1939-1951 (1993)), and when AIN-93G contains 5% by weight of cellulose, a feed obtained by replacing 2% by weight of cellulose with the cellulose acetate obtained above (hereinafter sometimes referred to as AIN-93G-acetate) was used.

[0170] <Feeding experiment>

[0171] (Experimental Animals)

[0172] Wild-type mice

[0173] Seven-week-old male mice of the C57BL / 6J strain were used.

[0174] Single bacterial colonization of mice

[0175] Escherichia coli K-12 strain (E. coli) was administered to male C57BL / 6N germ-free mice in an isolator and the mice were raised to prepare mice colonized with E. coli alone. In addition, mice colonized with Bacteroides thetaiotaomicron were prepared in the same manner.

[0176] AID KO mice

[0177] AID KO mice (in other words, mice in which activation-induced cytidine deaminase is deficient (knockout)) were prepared by the method of Muramatsu et al. (Cell, 102, 553-563 (2000)). It should be noted that AID KO mice do not produce IgA.

[0178] (Raising method)

[0179] The mice used in the experiments described below were raised by the following method. AIN-93G was fed to 8 mice and raised for 1 week. The mice were allowed to freely ingest feed. Then, the mice were divided into 2 groups, 4 in each group, and further raised for 4 weeks. During the 4-week raising, AIN-93G was fed to 1 group, which was used as the control group. AIN-93G-acetate was fed to the remaining 1 group, which was used as the cellulose acetate (Acetate) group. However, the number of AID KO mice and E. coli single bacteria colonized mice was different from that of other mice, and 10 mice were divided into 2 groups, 5 in each group, and raised.

[0180] <Quantification of IgA in feces>

[0181] Feces were collected on day 0, week 1, week 2, week 3, and week 4 from the start of administration of AIN-93G or AIN-93G-acetate, and the fecal IgA concentration (μg / mL) was quantified. The quantification was performed using a Mouse IgA ELISA Quantitation Set (manufactured by Bethyl Laboratories Inc., USA). The results are shown in Figure 1 .

[0182] After the first week from the start of administration of AIN-93G or AIN-93G-acetate, the cellulose acetate group showed higher values ​​than the control group, reached a plateau in the second week, and a significant increase in IgA concentration was observed until the fourth week.

[0183] <Quantification of IgA-coated bacteria>

[0184] The bacterial community of the feces of wild-type mice that had been fed for 4 weeks since the administration of AIN-93G or AIN-93G-acetate was stained with PE-labeled anti-IgA antibody (rat anti-mouse IgA, clone 11-44-2, Southern Biotech) and 4,6-diamino-2-phenylindole (DAPI), and analyzed by flow cytometry to obtain flow cytometry primary data. Then, a gate was set on DAPI positive cells stained with DAPI, and the IgA positive part was quantified as IgA-bound bacteria. Flow cytometry was performed using FACS AriaII, and the data was analyzed using FlowJo software (TreeStar Inc.). The results are shown in Figure 2 . Figure 2 (a) represents the primary data of flow cytometry, Figure 2 (b) shows the ratio (%) of the number of IgA-bound bacteria in DAPI-positive cells (% DAPI-positive cells).

[0185] The ratio (%) of the number of IgA-bound bacteria in DAPI-positive cells (%DAPI-positive cells) showed a higher value in the cellulose acetate group than in the control group. In other words, in the cellulose acetate group, the increase in IgA-bound bacteria resulted from the increase in IgA having affinity for more bacteria. By ingesting cellulose acetate of the present disclosure, an immunoenhancing effect such as protection of the intestine from specific bacteria can be expected.

[0186] <Bacterial community analysis of IgA-binding bacteria in feces>

[0187] The feces of wild-type mice that had been fed for 4 weeks since the administration of AIN-93G or AIN-93G-acetate were suspended and centrifuged with bacterial community phosphate buffered saline (PBS) to obtain a supernatant. The bacteria contained in the supernatant were stained with IgA-PE antibody (rat anti-mouse IgA, clone 11-44-2, SouthernBiotech) and DAPI, and magnetic cell sorting (Miltenyi Biotec) was performed. After collecting IgA-negative bacteria, FACS Aria II was used to collect IgA-positive bacteria. Using these samples, bacteria were identified by 16S rRNA gene analysis. The results of the bacterial community analysis of the feces of the wild-type mouse cellulose acetate group that had been fed for 4 weeks are shown in Table 1, and the results of the bacterial community analysis of the feces of the wild-type mouse control group that had been fed for 4 weeks are shown in Table 2.

[0188] [Table 1]

[0189] Results of fecal bacterial community analysis of wild-type mice fed with cellulose acetate for 4 weeks

[0190] (Fecal microbiota at phylum level of mice fed with Acetate diet for4weeks.)

[0191]

[0192] ＊ There is a significant difference (significant difference in the same row (Mann-Whitney U test, p < 0.05)).

[0193] As shown in Table 1, in the feces of the cellulose acetate group of wild-type mice fed for 4 weeks, among the Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, Tenericutes, and Verrucomicrobia, Proteobacteria bacteria showed significantly positive IgA compared with other classifications of bacteria, and had a high affinity for IgA. It should be noted that Proteobacteria include bacteria that may be the cause of the disease, such as Escherichia coli, Salmonella, Vibrio, and Helicobacter.

[0194] [Table 2]

[0195] Results of fecal bacterial community analysis of wild-type mice control group fed for 4 weeks

[0196] (Fecal microbiota at phylum level of mice fed with Control diet for 4 weeks.)

[0197]

[0198] ＊ There is a significant difference (significant difference in the same row (Mann-Whitney U test, p<0.05)).

[0199] As shown in Table 2, in the feces of the wild-type mouse control group that was fed for 4 weeks, among the Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, Tenericutes, and Verrucomicrobia, especially the bacteria of the Proteobacteria, did not show IgA positivity compared with bacteria of other classifications, and their affinity with IgA was not high.

[0200] <Quantification of IgA Concentration in Various Parts of the Intestinal Tract>

[0201] In order to investigate where in the intestine the IgA concentration increases, the IgA concentration is quantified at each site. The method is as follows.

[0202] For wild-type mice that have been raised for 4 weeks since the administration of AIN-93G or AIN-93G-acetate, the duodenum, jejunum, ileum, cecum, proximal colon, and distal colon tissues were collected and stored in PBS supplemented with cOmplete EDTA-free protease inhibitor mixed tablets (Roche), and then broken with stainless steel beads. The protein concentration was determined using the BCA protein quantitative analysis kit (BCA Protein Assay) (ThermoFisher Scientific), and after the concentration of each tissue was unified, the IgA concentration was quantified. Quantification was carried out using the Mouse IgA ELISA Quantitation Set (Mouse IgA ELISA Quantitation Set, made by Bethyl Laboratories Inc., USA). The results are shown in Figure 3 (a) and (b).

[0203] The tissue IgA concentration in the cellulose acetate group showed higher values ​​than that in the control group in the duodenum, cecum, proximal colon, and distal colon. In particular, a significant increase was observed from the cecum to the colon.

[0204] <Quantification of IgA-producing plasma cells in the lamina propria of the large intestine>

[0205] In order to separate lymphocytes from the colon mucosal lamina propria of wild-type mice that have been fed for 4 weeks since the administration of AIN-93G or AIN-93G-acetate, the colon was collected, cut in the length direction, washed and feces were removed. Then, the washed colon was shaken for 30 minutes at 37°C in HBSS containing 20mM EDTA. After removing epithelial cells and adipose tissue, the intestinal tissue was finely shredded into small slices, RPMI 1640 culture medium (containing 2% fetal bovine serum (FBS), 400 units / ml (Roche Diagnostics KK) collagenase D, 0.25 units / ml dispase (Corning), and 0.1mg / ml DNaseI (Wako Pure Chemical Industries, Ltd.)) was added, and shaken in a 37°C water bath for 30 hours. The digested tissue was washed with RPMI 1640 medium (containing 2% fetal bovine serum (FBS)), resuspended in 10 ml 35% Percoll (GE Healthcare), and layered on 2.5 ml 70% Percoll in a 15 ml Falcon tube. Then, the cells were centrifuged at 2000 rpm for 20 minutes at room temperature and separated using a Percoll density gradient. The cells at the interface were recovered and used as mucosal lamina propria lymphocytes.

[0206] The separated lamina propria lymphocytes were suspended in staining buffer (PBS, 2% FBS) and stained with IgA-PE antibody (clone: ​​11-44-1, Southern Biotech) and B220-APC antibody (clone: ​​RA3-6B2, BioLegend).

[0207] The mucosal lamina propria lymphocytes after the above treatment were analyzed by flow cytometry. A gate was set, and cells stained by IgA-PE antibody but not stained by B220-APC antibody were quantified as plasma cells (IgA+ plasma cells) producing IgA. In addition, cells stained by both IgA-PE antibody and B220-APC antibody were quantified as B cells (IgA+ B cells).

[0208] Here, B cells are a type of lymphocytes that become plasma cells that specialize in producing antibodies after maturation and differentiation. Since one B cell can only produce one type of antibody, only when an antigen or bacterial antigen that matches the antibody type appears will the B cell that can produce that antibody be activated and begin to produce antibodies.

[0209] Flow cytometry was performed using FACScant II, and the data were analyzed using FlowJo software (TreeStar Inc.). The results are shown in Figure 4 . Figure 4 (a) represents the primary data of flow cytometry, Figure 4 (b) shows the number of each bacteria (absolute cell number, absolute cell number) (×10 4 ).

[0210] The number of IgA-producing plasma cells in the colon mucosal lamina propria of the cellulose acetate group was significantly increased compared with the control group. Therefore, an increase in IgA can be expected by taking cellulose acetate of the present disclosure.

[0211] <Quantification of IgA-binding bacteria in cecal contents and feces>

[0212] Using PE-labeled anti-IgA antibody (rat anti-mouse IgA, clone 11-44-2, SouthernBiotech) and 4,6-diamino-2-phenylindole (DAPI), the cecal contents and fecal bacterial communities of Bacteroides thetaiotaomicron colonized mice and E.coli colonized mice that were fed for 4 weeks since the administration of AIN-93G or AIN-93G-acetate were stained and analyzed by flow cytometry to obtain flow cytometry data. Then, a gate was set for DAPI-stained DAPI-positive cells, and the IgA-positive part was quantified as IgA-bound bacteria. Flow cytometry was performed using FACS AriaII, and the data were analyzed using FlowJo software (TreeStar Inc.).

[0213] The results are shown in Figure 5 . Figure 5 (a) is the ratio (%) of the number of IgA-binding bacteria in the DAPI-positive cells of the cecum (left: cecum) and feces (right: feces) of mice colonized with Bacteroides thetaiotaomicron (%DAPI-positive cells). Figure 5(b) shows the ratio (%) of the number of IgA-binding bacteria in DAPI-positive cells in the cecum (left: cecum) and feces (right: feces) of mice colonized with E. coli belonging to the phylum Proteobacteria (% DAPI-positive cells).

[0214] like Figure 5 As shown in (a), compared with the control group, the proportion of IgA-bound bacteria in the cecum and feces of mice colonized with Bacteroides thetaiotaomicron in the cellulose acetate group was reduced, while Figure 5 As shown in (b), the proportion of IgA-bound bacteria in the cecum and feces of mice colonized with E. coli increased. That is, the affinity of IgA in the cellulose acetate group for E. coli K-12 strain belonging to the Proteobacteria was increased. As mentioned above, the Proteobacteria include bacteria such as E. coli, Salmonella, Vibrio, and Helicobacter that may be the cause of the disease, and therefore, the use of IgA induced by the cellulose acetate of the present disclosure can be expected to protect the intestines from these bacteria (intestinal protective effect).

[0215] <Evaluation of bacterial communities in the intestinal luminal contents and intestinal mucus layer>

[0216] (Bacterial community analysis)

[0217] Using the method of Gong et al. (FEMS Microbiol Ecol. 2007 Jan; 59(1): 147-57.), the bacterial communities in the colon luminal contents and the colon mucus layer were collected from wild-type mice fed for 4 weeks after the administration of AIN-93G or AIN-93G-acetate, and AID KO mice fed for 4 weeks after the administration of AIN-93G or AIN-93G-acetate, and then the bacteria were identified by 16S rRNA gene analysis.

[0218] The results of the bacterial community analysis of the lumen contents and mucus layer of the large intestine of the cellulose acetate group of wild-type mice fed for 4 weeks are shown in Table 3, and the results of the bacterial community analysis of the lumen contents and mucus layer of the wild-type mouse control group fed for 4 weeks are shown in Table 4. In addition, the results of the bacterial community analysis of the lumen contents and mucus layer of the large intestine of the cellulose acetate group of AID KO mice fed for 4 weeks are shown in Table 5, and the results of the bacterial community analysis of the lumen contents and mucus layer of the large intestine of the AID KO mouse control group fed for 4 weeks are shown in Table 6.

[0219] [Table 3]

[0220] Results of bacterial community analysis of the luminal contents and colon mucus layer of wild-type mice fed with cellulose acetate for 4 weeks

[0221] (Lumical and mucosal microbiota at phylum level of mice fed with Acetate diet for 4 weeks.)

[0222]

[0223] ＊ There are significant differences in the same row (there are significant differences in the same row (Mann-Whitney U test, p<0.05)).

[0224] [Table 4]

[0225] Results of bacterial community analysis of the luminal contents and colon mucus layer of wild-type mice fed for 4 weeks

[0226] (Lumical and mucosal microbiota at phylum level of mice fed with Acetate diet for 4 weeks.)

[0227]

[0228] ＊ There are significant differences in the same row (there are significant differences in the same row (Mann-Whitney U test, p<0.05)).

[0229] As shown in Table 3, for the wild-type mouse cellulose acetate group, the bacteria of the phylum Proteobacteria were 2.1% in the lumen contents and 0.7% in the mucus layer, and were less likely to colonize in the mucus layer than in the lumen. In contrast, as shown in Table 4, for the wild-type mouse control group, the bacteria of the phylum Proteobacteria were 0.0% in the lumen contents and 0.0% in the mucus layer, and were not less likely to colonize in the mucus layer than in the lumen.

[0230] [Table 5]

[0231] Results of bacterial community analysis of the luminal contents and colon mucus layer of the AID KO mice fed with cellulose acetate for 4 weeks

[0232] (Luminal and mucosal microbiota at phylum level of AKD KO mice fed with Acetate diet for4 weeks.)

[0233]

[0234] ＊ There are significant differences in the same row (there are significant differences in the same row (Mann-Whitney U test, p<0.05)).

[0235] [Table 6]

[0236] Analysis of bacterial communities in the luminal contents and colon mucus layer of the AID KO mouse control group fed for 4 weeks

[0237] (Luminal and mucosal microbiota at phylum level of AKD Ko mice fed with Acetatediet for 4 weeks.)

[0238]

[0239] ＊ There are significant differences in the same row (there are significant differences in the same row (Mann-Whitney U test, p<0.05)).

[0240] For AID KO mice that do not produce IgA, even in the cellulose acetate group, as shown in Table 5, the bacteria of the Proteobacteria phylum were 1.8% in the lumen contents and 32.3% in the mucus layer, which are very easy to colonize in the mucus layer compared to the lumen. In addition, even in the AID KO mouse control group, as shown in Table 6, the bacteria of the Proteobacteria phylum were 1.4% in the lumen contents and 22.2% in the mucus layer, which are very easy to colonize in the mucus layer compared to the lumen. Based on the above results, it can be considered that the increase in IgA leads to a decrease in the Proteobacteria phylum bacteria in the mucus layer of the large intestine, thereby contributing to intestinal protection.

Claims

1. An intestinal immune enhancer comprising cellulose acetate having a total degree of acetyl substitution of 0.4 or more and 1.1 or less.

2. The intestinal immune enhancer according to claim 1, wherein The composition distribution index (CDI) of the cellulose acetate defined by the following formula is 2.0 or less, CDI = (measured value of half-peak width of composition distribution) / (theoretical value of half-peak width of composition distribution) Measured value of composition distribution half-peak width: the composition distribution half-peak width determined by HPLC analysis of cellulose acetate propionate obtained by propionylating all the remaining hydroxyl groups of cellulose acetate (sample); [Mathematical formula 1] DS: total degree of acetyl substitution; DPw: weight average degree of polymerization (a value determined by a GPC-light scattering method using cellulose acetate propionate obtained by propionylating all the remaining hydroxyl groups of cellulose acetate (sample)).

3. Food containing the intestinal immunity enhancer according to claim 1 or 2.

4. The food according to claim 3, wherein The content of the cellulose acetate in the food is 0.1% by weight or more and less than 5% by weight.

5. A medicine comprising the intestinal immunity enhancer according to claim 1 or 2.

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