AU3611999A - Use of arabinoxylans for preparing a composition - Google Patents

Description

WO 99161036 PCTFR9901146 1 USE OF ARABINOXYLANES FOR THE PREPARATION OF A COMPOSITION The present invention pertains to the use of soluble fibers extracted from corn bran, the arabinoxylanes, characterized by physicochemical and metabolic properties allowing applications in the dietary and pharmaceutical fields. In the industrialized countries, changes in alimentary habits and the use of increasingly refined products in the food industry have led to a decrease in the ingestion of fiber to approximately 15 to 20 g/day whereas the WHO recommends an intake of 37 g/day. Numerous studies (Kritchevsky and Klurfeld, Dietary Fiber and Cancer in Human Nutrition "A comprehensive treaty" Alfin - Slater KB Kritchevsky Editors, pp. 211-220, Plenum Press, 1991; Hubbard et al., Nutr. Res., 14 pp., 1853-1895, 1994) have demonstrated the protective role of dietary fiber against the leading western diseases such as gastrointestinal diseases (constipation, diverticulosis and inflammatory diseases of the colon, colon cancer) and metabolic diseases such as diabetes and cardiovascular diseases. The food industry has become involved in the population's fiber deficiency and introduced numerous products that are rich in fiber, most especially in the realm of breakfast cereals, by promoting the value of these products for the good functioning of intestinal transit.

WO 99161036 PCT/FR99/01146 2 The fibers are named according to a complex nomenclature but they can be classified into two categories: - the insoluble fibers: cellulose, lignin and certain hemicelluloses, and - the soluble fibers: pectin, gums and several hemicelluloses. This distinction is important because it affects the physicochemical properties and the nutritional effects of the different fibers. The insoluble fibers are essentially cereal fibers. They are characterized by their ability to retain water, they have a low capability to ferment and they are only slightly degraded by the bacteria of the colon. They seem to also play a role in the regulation of intestinal transit. In contrast, the soluble fibers have a high capability to ferment and they are almost entirely degraded by the bacteria of the colon, which promotes the growth of the bacterial population and liberates volatile fatty acids which are absorbed by the intestinal mucosa. The soluble/insoluble fiber proportions varies among the various fibers and plant species (Englyst H. N. et al., J. Human Nutrition & Dietetics, 1988, 1, 247 286 and 2, 253-271). Raw plant products generally contain from 10 to 30% of dietary fibers with respect to dry matter, with the exception of products that are rich in starch. In contrast to the insoluble fibers, especially of cereal brans, few soluble fibers are available on the market. Nevertheless, one can mention fruit and vegetable pectins and the gums (arabic gum, guar gum or carob-seed gum). But use as alimentary ingredients can not be claimed for these compounds since their WO 99161036 PCT/FR99/01146 3 physicochemical characteristics do not allow incorporation of large quantities in commonly consumed food products without modification of their texture or organoleptic properties. They are therefore presently employed as additives. Thus, the pectins and the gums, because of the viscous and/or gelling texture that they provide to the product in which they are incorporated, can barely be used as soluble fibers but rather for their fermentability. Inulin is the sole soluble fiber available on the market that is sold for its nutritional properties and credited in declarations of ingredients. Inulin is a linear polymer of fructose linked at p1-2 and associated with an initial glucose molecule. Inulin represents the form of glucose reserve of plants that do not accumulate starch (Jerusalem artichoke, artichoke, onion). This oligosaccharide can not be digested in the small intestine and thus exhibits interesting nutritional effects. It is prebiotic and has effects on decreasing the blood triglyceride level. In contrast, its effect of decreasing the blood cholesterol level has only rarely been described in the literature and the sensations of intestinal discomfort (flatulence) linked to its highly fermentable character contraindicate its use at high concentrations in food products. Thus, the soluble fibers that are presently available do not satisfy the combination of "metabolic effects - physicochemical characters" that the food industry searches for in the incorporation of a product at a useful dose that also respects the original quality of the product. Research on soluble fibers has revealed their particular effects on digestion and lipid metabolism. It has been shown that corn hemicellulose exhibits a WO 99/61036 PCT/FR99/01146 4 beneficial effect on the accumulation of lipids in the liver in rats fed with diets supplemented by orotic acid (Miyazaki et al., Biosci. Biotech. Biochem., 56, 157 158, 1992). This effect is attributed to the short-chain fatty acids which are the products stemming from the fermentation of the corn hemicelluloses in the colon. Nevertheless, attribution of these effects solely to the soluble fibers can not be definite because of the systematic addition of orotic acid as inducer. Thus, to date, it has not been possible to identify an alimentary fiber constituent that combines all of the effects described above and which could be used on an industrial scale for alimentary applications. In effect, such a constituent must also advantageously be responsive to physicochemical criteria (stability with respect to pH, viscosity) and industrial criteria that would enable the preparation of alimentary and/or pharmaceutical products. The present invention has precisely as its goal the preparation and the use of soluble fibers extracted from corn bran, rich in arabinoxylanes, which exhibit physicochemical and metabolic properties that are remarkable for alimentary and pharmaceutical industrial applications. In fact, the beneficial role of the arabinoxylanes on the accumulation of lipids in the liver has not been reported to date. In addition, the in vitro results obtained by Sugawara et al. (Agree. Biol. Chem. 55, 565-567, 1991) on the weak fermentability of the hemicelluloses by human intestinal bacteria would not suggest in the least any possible correspondence between the effects of the hemicelluloses and the arabinoxylanes, such as was demonstrated in the research WO 99161036 PCTIFR99/01146 5 carried out in the context of the present invention and reported in the experimental part below. Thus, the research performed in the context of the present invention made it possible to demonstrate that the arabinoxylanes, especially the arabinoxylanes from corn bran, exhibit properties that are very advantageous on intestinal fermentation, the bioavailability of minerals and the metabolism of cholesterol. In addition, their stability, viscosity and fermentability characteristics make them especially useful agents in the alimentary, dietary and pharmaceutical industries. Thus, the object of the invention is the use of arabinoxylanes or a substance rich in arabinoxylanes for the preparation of a composition intended for the prevention or treatment of disorders or diseases linked to metabolic dysfunction in humans or animals, and more particularly hypercholesterolemia. The invention pertains more particularly to the use of soluble corn bran fibers containing at least 60%, preferably at least 80%, arabinoxylanes and presenting a molecular weight between 150 and 300 kD, preferably on the order of 240 kD. The arabinoxylanes and the substances containing them, such as the aforementioned soluble corn bran fibers, present remarkable physicochemical and metabolic properties, notably a good viscosity and resistance to acid pH and a slow fermentability with a good distribution in the mammal colon, enabling compositions containing them to exhibit metabolic effects on the digestion, mineral balance and lipid metabolism in mammals after ingestion.

WO 99/61036 PCT/FR99/01 146 6 These effects concern especially acidification of the medium, production of short-chain fatty acids, augmentation of the solubility and absorption of minerals, diminution of the plasma and hepatic cholesterol, correlated with increased excretion of bile acids. Advantageously, the arabinoxylanes or a substance rich in arabinoxylanes such as soluble corn bran fibers are, according to the invention, useful for the preparation of alimentary, dietary or pharmaceutical compositions intended to prevent or treat disorders or diseases linked to metabolic dysfunctions in humans or animals, and more particularly hypercholesterolemia. Such compositions can contain arabinoxylanes in variable quantities depending on the type of alimentary or therapeutic product and the desired effect, for example between 2 and 20% and more, preferably between 5 and 10%, particularly 5%. The invention thus also pertains to a product comprising plant fibers, such as a food product or a drug, characterized in that it contains at least approximately 5% of arabinoxylanes or a substance rich in arabinoxylanes such as the aforementioned corn bran fibers. Such a product advantageously comprises a mixture of approximately 5% of arabinoxylanes or a substance rich in arabinoxylanes such as the aforementioned corn bran fibers and other rapidly fermentable soluble and/or insoluble fibers at an adequate dose and not damaging the qualities of said products conferred by the arabinoxylanes, WO 99/61036 PCTIFR99/01146 7 For example, the other soluble fibers are selected from among inulin and the polysaccharides. A product of the invention, combining the soluble slow arabinoxylane and rapid inulin or polysaccharide fibers, for example, makes it possible to optimize the mechanisms of fermentation. Such products, in accordance with the compositions of the invention, are intended to prevent or treat the disorders or diseases linked to metabolic dysfunctions of the lipid, mineral or digestive type, especially hypercholesterolemia. As examples of such food products, one can cite breakfast cereals, cereal bars, biscuits, health-food beverages, fruit juices, cooked dishes, candies and dairy products. Other characteristics and advantages of the invention will become apparent from the examples below concerning the extraction of arabinoxylanes from corn bran, and the demonstration of their metabolic and bifidogenic effects, and which refer to the attached drawings in which: - Figure 1 shows the Brookfield viscosity at 30*C in relation to the arabinoxylane concentration and the pH. - Figure 2 shows the evolution of the weight of the cecum and its pH during treatment for the conditions of control diet and diet supplemented with arabinoxylane.

WO 99/61036 PCTIFR99/01146 8 - Figure 3 shows the variation in the production of short-chain fatty acids in the cecum during treatment for the conditions of control diet and diet supplemented with arabinoxylane. - Figure 4 shows the variation in the total Ca concentration in the cecum. Example 1: Extraction of arabinoxylanes from corn bran. The plant material used is constituted by corn bran exhibiting the following characteristics: 90% dry matter, 38% arabinoxylanes, 70% TDF (Total Dietary Fibers determined by the standard method) and 21% starch. The extraction was performed according to the protocol described by Chanliaud et al. (Journal of Cereal Science 21, pp. 195-203, 1995). The product obtained according to this protocol exhibits the following characteristics: 97% dry matter, 80% arabinoxylanes, 5% cellulose, 84% TDF, 1.9% ash and 1.7% protein. Its molecular weight is circa 240 kD but can vary between 150 and 300 kD. Example 2: Physicochemical characteristics of the arabinoxylanes of example The viscosity characteristics were measured with a Brookfield viscometer. The moderate viscosity developed by the arabinoxylanes allows their use in the alimentary field. In addition, as shown in figure 1, the developed viscosity remains stable at acid pH. This property of the arabinoxylanes allows their incorporation in certain food products of an acid character such as dairy products WO 99161036 PCTIFR99/01146 9 or fruit juices; in contrast, inulin can not be used in these products because of its sensitivity to acid pH. In addition, in contrast to the gums which develop a very high viscosity, the arabinoxylanes only introduce slight modifications of texture which allows their use at meaningful doses in terms of the concept of fiber enrichment. For example, they can be introduced at the dose of 5.55 g in a yogurt of 120 g so as to cover 15% of the recommended daily intake without modifying the taste or the texture of the yogurt. The doses at which the arabinoxylanes are used varies according to the products in which they will be incorporated. Nevertheless, they are incorporated in the ingredient category and can therefore be incorporated at a dose which is useful in terms of enrichment in fiber. Example 3: Demonstration of the metabolic effects of arabinoxylanes. 1) Supplementation of the diet of rats with arabinoxylanes. Male Wistar rats aged 7 weeks kept under suitable temperature and light conditions were first fed with a diet without fibers (control) for 6 days. After this period, 8 rats were sacrificed on DO (Day 0) and the other rats were distributed into four groups: - group (a): 16 rats fed with the diet described above (control group), - group (b): 8 rats fed with a cholesterol diet without fiber, - group (c): 24 rats fed with an arabinoxylane fiber diet, - group (d): 8 rats fed with an arabinoxylane fiber and cholesterol diet.

WO 99161036 PCTIFR99101146 10 On D+3 and D+ 11, 8 rats receiving the arabinoxylane diet were tested and sacrificed. The remaining rats were tested on D+20. Samples were collected in the morning between 8:00 and 9:00 a.m., the time at which the intestinal fermentations are still very active. The daily feed consumption and the body weights were recorded twice weekly. The feces were collected on 5 consecutive days for the quantitative determination of nitrogen, minerals and sterols. Blood samples were taken from anesthetized rats (sodium pentobarbital, 40 mg/kg) to measure the arteriovenous difference along the intestine. In order to measure the blood flow rate, a saline solution of bromosulphthalein (4.7 mmol/L) was infused in one of the small veins at the surface of the cecum at a rate of 100 IM/min. The dilution of this marker in the vein draining the cecum enabled calculation of the blood flow rate through the cecum. This intestinal blood flow is approximately proportional to the mass of the intestinal wall (0.9-1.3 mL - min~ - g intestinal wall~'). Blood was collected from the intestinal vein and the abdominal aorta and then put in tubes containing heparin and centrifuged at 10,000 g for 2 minutes. The plasma samples were stored at 4"C for analysis of the lipids and lipoproteins. Part of the livers was frozen and stored at -80*C for measurement of the lipids. The hepatic microsomes were prepared according the procedure described by Younes et al. (Lipids 30, pp. 847-853, 1995) and stored at -80*C until measurement of the 3-hydroxy-3-methylglutaryl-CoA (HMGR) activity. The protein content of the microsomes was determined using the Pierce NCA reagent kit. The cecum was also collected and weighed. Duplicate samples were WO 99161036 PCTIFR99/01146 11 collected in 2-ml microtubes, frozen immediately and stored at -20*C. The supernatants were obtained by centrifugation at 20,000 g for 10 minutes at 4*C on one of the two tubes, prior to analysis of the short-chain fatty acids (SCFAs). 2) Effects on the digestion. The SCFAs were measured by liquid-gas chromatography according to Rim6sy and Demign6 (Biochem. J. 141, pp. 85-91, 1974). The incorporation of 8% corn arabinoxylanes in the diet did not modify the growth of the rat (daily feed consumption and body weight). As shown in figure 2a, a rapid increase in the intestinal mass (100%) was seen starting on D+3 in the rats receiving an arabinoxylane diet compared to the control animals; likewise, there was a progressive increase up to D+10 in the mass of the intestinal wall after which these values stabilized. This hypertrophy of the intestinal wall was accompanied by a stimulation of cell division. As shown in figure 2b, the intestinal pH became acid (less than 6) after D+3 of the arabinoxylane diet compared to the controls (7.2). This acidification did not appear to be due to a slight accumulation of lactic acids but rather to an increase in the production of SCFAs (+280%) as shown in figure 3. In fact, the presence of the arabinoxylanes entailed a progressive increase in the concentrations of acetate (95 mmol/L compared to 66 mmol/L) and propionate (80 mmol/L compared to 22 mmol/L) and a decrease in the concentration of butyrate (5 mmol/L compared to 12 mmol/L). It is known that propionate is the sole neoglucogenic short-chain fatty acid and that propionate or its acylCoA derivatives have effects on various metabolic pathways such as neoglucogenesis, WO 99161036 PCT/FR99/01146 12 ureagenesis and ketogenesis (R6m6sy and Demigne, Abbott International Ltd., III, 14-21, 1989). 3) Effects on the lipid metabolism. The bile acids and neutral sterols were extracted from the feces by dispersion in 10 volumes of ethanol KOH (0.5 M) using a Polytron homogenizer. After a second extract step, the samples were heated at 80*C for 1 hour and then centrifuged at 8000 g for 5 minutes. The bile acid concentration was measured on this extract by the reaction catalyzed by 3-hydroxysteroid dehydrogenase (Turley and Dietschy, J. Lip. Res. 19, 924-928, 1978). The cholesterol concentration was determined with a kit from Societ6 BioMerieux (France). The liver lipids were extracted with chloroform/methanol (2:1, vol./vol.) according to the method described by Folch et al. (J. Biol. Chem. 226, 497-506, 1957). The plasma lipoproteins were separated by density gradient ultracentrifuga tion at 100,000 g for 24 h at 18*C on pooled samples. Twenty-four 500-pl fractions were stored at 4*C for analysis of the lipids. The liver microsomal HMGR activity was measured by the method described by Wilce and Kroone (Lipoprotein Analysis, Oxford University Press 1992). The activity was expressed in pmot of [14C]HMG-CoA transformed to [14C]mevalolactone/minute - mg of microsomal protein. The diet supplemented with arabinoxylanes enabled greater elimination of cholesterol in the feces (-26.4 ± 2.7 compared to the control value -18.8 1 1.9 WO 99/61036 PCT/FR99/01146 13 pmol/day) and a greater excretion of neutral steroids (cholesterol + coprosterol) (116 pmol/day versus a control value of 58,9 pmol/day), which corresponds to 87% of the daily consumption of cholesterol. On the other hand, it is difficult to see the effect of arabinoxylanes on the excretion of bile acids because the decrease in the absorption of cholesterol (13% versus 50% in the control rats) by arabinoxylanes led to a reduced transformation of cholesterol into bile acids in the liver. A decrease in the plasma concentration of cholesterol was also seen in the rats receiving an arabinoxylane diet whereas the concentration of triglycerides was not affected. A comparable effect was seen for the hepatic cholesterol (decrease of 40% in the rats receiving the cholesterol + arabinoxylanes diet compared to the rats receiving only a cholesterol diet). The HMGR microsomal activity, practically nonexistent in the presence of cholesterol, increased in a remarkable manner (elevation of 70% of the losses in total steroids). 4) Effects on the mineral balance. The nitrogen present in the intestine and feces was determined by the method of Kjeldahl. In accordance with this method, the homogenized samples were treated with 36 M sulfuric acid in the presence of catalysts (K 2 S0 4 , Se). The ammonia extracted and trapped in a boric acid solution was determined by direct titration with phosphoric acid. The Ca and Mg were determined on the soluble (supernatant) and total fractions of the intestine as well as on the fecal material after treatment at 500 0

C

WO 99161036 PCTIFR99/01146 14 for 10 hours. The ashes obtained were taken up in an acid solution of lanthanum. The mineral concentration was measured by atomic absorption spectrophotometry (Perkin-Elmer 420, Norwalk, CT) at the following wavelengths: 422 for the Ca and 285 for the Mg. No change in the pool of fecal Ca was seen during the first phase of adaptation to the arabinoxylane diet. However, after 10 days of adaptation, an accumulation of Ca in the cecum was observed. In addition, on D+3 we observed an increase (17 times) in the pool of cecal soluble Ca. The augmentation of the cecal volume and the pool of soluble Ca was manifested by the clear augmentation in the cecal absorption (3 times) of this cation. Thus, these fermentable carbohydrates promoted the absorption of Ca in the distal part of the digestive tract by induction of the following phenomena: - hypertrophy of the intestinal wall and larger exchange surface, - augmentation of the soluble Ca correlated with an acidification (production of SCFAs) and - acceleration of the blood flow. The SCFAs could also have a direct influence on the absorption of Ca by modifying the electrolyte exchanges. This Ca effect could have as a consequence a decrease in the solubility of the bile acids, which would promote their fecal elimination. Other benefits of Ca have been reported: - protection of the intestinal epithelium and WO 99161036 PCT/FR99/01146 15 - inhibition of the cytotoxicity of potential carcinogens such as the bile acids or fatty acids. The intestinal Mg did not follow the same evolution. In fact, its total concentration was not modified, but the soluble concentration reached a quintuple value (70 pmol) on D+3 in relation to the control value. The intestinal absorption of Mg increased in the presence of arabinoxylanes 0.23 ± 0.02 pmol/min compared to 0.05 E 0.0 1 pmolmin (control). These fermentable carbohydrates play an important role in increasing the mineral absorption in the large intestine and this effect is of great interest when the digestive mechanisms of absorption are ineffective, such as for example in the elderly. The arabinoxylanes of molecular weight of circa 240 kD exhibit good fermentability with a good distribution of the fermentations (moderately acid pH) along the colon, in contrast to inulin which is practically entirely fermented in the proximal part of the colon. The arabinoxylanes that are fermented by the bacteria of the colon, which are therefore referred to as slow soluble fibers, are thus likely to lead to less discomfort than inulin after ingestion of products containing them. Example 4: Measurement of the bifidogenic effects of the arabinoxylanes. As fermentable soluble fibers, the arabinoxylanes are fermented by the colonic flora which is a complex mixture of different bacteria whose activity plays an important role in health. In vitro tests were performed according to the protocol described below to demonstrate the stimulating effect of the arabinoxylanes on the colonic flora and especially on Bifidobacterium spp.

WO 99/61036 PCTIFR99/01 146 16 The arabinoxylanes, carbon source, are introduced into the culture media on a comparative basis versus another carbon source (glucose). The growth of the different known bacterial species of the colonic flora (Brfidobacterium, Lactobacillus, Eubacterium, Propionibacterium, Fusobacterium, etc.) is followed according to the method of K. Wada (In vitro fermentability of oligofructose and inulin by some species of human intestinal flora, internal report for the Tiense Suikerraffinaderij, 1990). In parallel, the growth of these same bacteria was followed in the presence of oligofructose, a substrate of known bifidogenic effect. These tests can be supplemented by in vivo tests in order to validate their results. This effect of the growth of the bacteria of the colonic flora will be correlated with the previously described fermentability characteristics of the arabinoxylanes.