Patent Application
20080069861
This invention pertains to a method of promoting gastrointestinal health by ingesting a composition comprising one or more probiotic microorganisms, one or more carbohydrate sources, and one or more resistant protein products. Large intestinal health is benefited by one or more of the following: increased fecal weight, lower fecal pH, increased carbohydrate fermentation, and amelioration of (decreased) protein fermentation.
Probiotic bacteria or microorganisms are intended to include one or more species which beneficially affects the host mammal after ingestion and may include any species known in the art including without limitation Bifiobacterium such as Bif. Pseudolongum, Bif. Lactis, Bif. Longum, Bif. Infantis, and Bif. bifidum; Bacteroids such as Bact. Vulgatus and Bact. Fragilis, Clostridium such as Cl. butyricum; Eubacteria; Fusobacterium; Propionibacterium; Streptococcus; Enterococcus; Lactococcus; Staphylococcus; Peptostreptococcus; and Lactobacillus such as Lactobacillus acidophilus. Probiotic microorganisms also include yeasts such as Saccharomyces. The invention is not, however, limited to these particular microorganisms. In one embodiment, the probiotic microorganism is Bifiobacterium.
The carbohydrate source used may be any carbohydrate source known in the art, including without limitation starches, flours, grains, legumes, polysaccharides and oligosaccharides. The carbohydrate source is selected from high amylose carbohydrates, high amylose resistant starch products, and fructo-oligosaccharides. In an embodiment, the carbohydrate source is one which at least partially resists digestion and is fermented, such that butyrate is produced. In one aspect of the invention, the carbohydrate source used is a resistant starch product, including RS1, RS2, RS3 and RS4 types. In another aspect of the invention, the carbohydrate source is a high amylose resistant starch product. In yet another aspect of the invention, the carbohydrate source is a fructo-oligosaccharide.
The resistant starch product may be one found in nature or one made using starches found in nature. A native starch as used herein, is one as it is found in nature. Also suitable are starches derived from a plant obtained by standard breeding techniques including crossbreeding, translocation, inversion, transformation or any other method of gene or chromosome engineering to include variations thereof. In addition, starch derived from a plant grown from induced mutations and variations of the above generic composition which may be produced by known standard methods of mutation breeding are also suitable herein.
Typical sources for the starches are cereals, tubers, roots, legumes and fruits. The native source can include corn (maize), pea, potato, sweet potato, banana, barley, wheat, rice, sago, amaranth, tapioca, arrowroot, canna, or sorghum, as well high amylopectin or high amylose varieties thereof. As used herein, the term “high amylopectin” is intended to include a starch containing at least about 90, particularly at least about 95, more particularly at least about 98% amylopectin by weight. As used herein, the term “high amylose” is intended to include a starch containing at least about 27% amylose for wheat or rice flour and at least about 50% amylose for other sources, particularly at least about 70, more particularly at least about 80% amylopectin by weight.
In one embodiment, the carbohydrate source used is extracted from a plant source having an amylose extender genotype, the starch of such material comprising less than about 10% by weight amylopectin. This material is derived from a plant breeding population, particularly corn, which is a genetic composite of germplasm selections and comprises at least 75% by weight normal amylose, and in one embodiment at least 85% normal amylose as measured by butanol fractionation/exclusion chromatography techniques. The starch further comprises less than about 10%, by weight, and in one embodiment less than 5%, amylopectin, and additionally from about 8 to 25% low molecular weight amylose. The starch is extracted from the grain of a starch bearing plant having a recessive amylose extender genotype coupled with numerous amylose extender modifier genes. This starch and the method of preparation are described in U.S. Pat. No. 5,300,145, which is incorporated herein by reference.
In one embodiment, the carbohydrate source used is a high amylose resistant starch product. In one embodiment, the resistant starch product is a chemically, physically and/or enzymatically treated or modified starch.
In another embodiment, the resistant starch product used is one containing the RS2 type of resistant starch, including those modified by heat-moisture treatment to increase the percent resistant starch by weight of the product, those typically found in high amylose carbohydrate materials and/or those described in U.S. Pat. Nos. 5,593,503 and 5,902,410. In yet another embodiment, the carbohydrate source used is a resistant starch product containing the RS3 type of resistant starch, including those described in U.S. Pat. Nos. 5,281,276 and 5,409,542. In a further embodiment, the carbohydrate source used is a resistant starch product containing the RS4 type of resistant starch, including chemically modified starches selected from the group consisting of oxidation, crossbonding, etherification, esterification, acidification, dextrinisation, and mixtures thereof and those described in U.S. Pat. No. 5,855,946. In a still further embodiment, the degree of enzyme susceptibility of the resistant starch is modified by altering the conformation or structure of the starch, such as by acid or enzyme thinning and cross bonding using difunctional reagents. In yet another embodiment, the amylolysis of the high amylose resistant starch is modified to give starch granules characterised by pits or erosions which can extend from the surface to the interior of the granules. These pits allow the entry of enzymes to the more enzyme susceptible core of the starch granule which is solubilized. Low or normal amylose containing starches can also provide a source of RS, particularly if they are consumed in their native form or if they have been modified in some manner (such as by physical, hydrothermal, enzymatic, acidic, chemical, etc.).
In a still further embodiment, the carbohydrate source used is an oligosaccharide and in a yet further embodiment, the carbohydrate source is a fructo-oligosaccharide. Fructo-oligosaccharides suitable for the present invention may include any fructo-oligosaccharides available for consumption. Commercial fructo-oligosaccharides presently available include without limitation fructo-, galacto, malto-, isomalto-, gentio-, xylo-, palatinose-, soybean- (includes raffinose and stachyose), chito-, agaro-, neoagaro-, .alpha.-gluco-, .beta.-gluco-, cyclo-inulo-, glycosylsucrose, lactulose, lactosucrose and xylsucrose. It will be appreciated by one skilled in the art, however, that other oligosaccharides would also be suitable for inclusion in the composition of the present invention.
The protein source may be any resistant protein product, including those of single or multiple cell animal or vegetable origin. Resistant protein products include without limitation proteins sourced from meat, fish, maize, rice, wheat, oats, millet, peas, peanuts, soy, sago, cassaya, arrowroot, taro, and beans. In one embodiment, the resistant protein product is of vegetable origin. In another embodiment, the protein source is synthetic, including oligomethionine. In yet another embodiment, the resistant starch product is derived from a cereal source and in yet another from potato. The source of protein may also be in the form of flour, extracts or isolates.
In one aspect of the invention, the probiotic microorganism is a Bifidobacteria, the carbohydrate source is a high amylose corn (maize) resistant starch, and the resistant protein product is potato protein in the form of an isolate.
Each component of the composition may be present in any ratio. In one embodiment, the probiotic is present in an amount of from about 104-1012 CFU of probiotic microorganisms per 10 g of composition, and in one embodiment from about 106-1011 CFU of probiotic microorganisms per 10 g of probiotic microorganism/carbohydrate source/resistant protein composition and in yet another embodiment from about 108-1010 CFU of probiotic microorganisms per 10 g of probiotic microorganism/carbohydrate source/resistant protein composition. In one embodiment, the carbohydrate source is present in an amount of from about 0.5 g to about 9.5 g per 10 g of probiotic microorganism/carbohydrate source/resistant protein composition, and in one embodiment is present in an amount of from about 1 g to about 8 g per 10 g of probiotic microorganism/carbohydrate source/resistant protein composition, and in yet another embodiment from about 5 g to about 7 g per 10 g of probiotic microorganism/carbohydrate source/resistant protein composition. In one embodiment, the resistant protein is present in an amount of from about 0.5 g to about 9.5 g per 10 g of probiotic microorganism/carbohydrate source/resistant protein composition, and in one embodiment is present in an amount of from about 1 g to about 6 g per 10 g of probiotic microorganism/carbohydrate source/resistant protein composition, and in yet another embodiment from about 2 g to about 4 g per 10 g of probiotic microorganism/carbohydrate source/resistant protein corn position.
The composition may be ingested by mammals, including humans, to promote gastrointestinal health, in one embodiment large intestinal health and in another distal intestinal health. Large intestinal health is benefited by one or more of the following: increased fecal weight, lower fecal pH, increased carbohydrate fermentation, and reduced protein fermentation. Lower fecal pH is due to increased short chain fatty acid content, particularly acetate and butyrate as well as decreased ammonia. Carbohydrate fermentation is preferred over protein fermentation as the by-products of carbohydrate fermentation are primarily beneficial while the by-products of protein fermentation are not. Ingestion of the composition also resulted in highest butyrate to ammonia ratio and highest fecal weight to cresol ratio. The composition may be used to prevent and/or treat gastrointestinal cancer, hepatic encephalopathy, prostate cancer, constipation, diarrhea, inflammatory bowel disease, irritable bowel disease, irritable bowel syndrome, diverticular disease, hemhorroids, osteoporosis, bone fractures, insulin resistance and insulin sensitivity. The composition may also be used to increase bone mineral density, and control insulin, glucose and mineral bioavailability/balances.
The composition of this invention may be used in any food or beverage product (hereinafter collectively referred to as foods). In one embodiment, the food is a cultured dairy product such as yoghurts, cheeses, and sour creams. The composition may be added at any amount which is acceptable to the consumer from both an organoleptic and a physiological standpoint and in one embodiment is used in an amount of from about 0.1 to 50%, and in another embodiment in an amount of from about 1 to 25%, by weight of the food.
The composition may also be used in a pharmaceutical or nutritional product, including but not limited to synbiotic compositions, diabetic foods and supplements, dietetic foods, foods to control glycemic response and glycemic index, and tablets and other pharmaceutical dosage forms.
The composition may also be used in a feed for livestock or companion animals.
The following examples are presented to further illustrate and explain the present invention and should not be taken as limiting in any regard. All parts and percentages are given by weight and all temperatures in degrees Celsius (° C.) unless otherwise noted.
The following ingredients were used throughout the examples.
1) resistant starch product from high amylose corn, where the resistant starch content was 65%, as is basis (commercially available from National Starch and Chemical Company, USA).
2) resistant protein product isolated from potato, where the resistant protein content was 16.0% (commercially available from Hokuren Co. Japan); prepared from potato juice by steam coagulation after adjustment of pH to 5.0-5.5
3) Bifidobacterium lactis (LAFTI B94), 1010 CFU/g, (commercially available from DSM Specialties, Australia)
The following test procedures were used throughout the examples.
A. Simulated Digestion—(Modified from Englyst et al., European Journal of Clinical Nutrition, 1992, 46, S33-S50)—Food samples are ground/minced as if masticated. Powder starch samples are screened to a particle size of 250 microns or less. A 500-600 mg±0.1 mg of sample is weighed and added to the sample tube. 10 ml of a pepsin (0.5%), guar gum (0.5%), and HCl (0.05 M) solution is added to each tube.
Blank and glucose standard tubes are prepared. The blank is 20 ml of a buffer containing 0.25 M sodium acetate and 0.02% calcium chloride. Glucose standards are prepared by mixing 10 ml sodium acetate buffer (described above) and 10 ml of 50 mg/ml glucose solution. Standards are prepared in duplicate.
The enzyme mix is prepared by adding 18 g of porcine pancreatin (Sigma P-7545) to 120 ml of deionized water, mixing well, then centrifuging at 3000 g for 10 minutes. The supernatant is collected and 48 mg of dry invertase (Sigma 1-4504) and 0.5 ml AMG E (Novo Nordisk) are added.
The sample tubes are pre-incubated at 37° C. for 30 min, then removed from the bath and 10 ml of sodium acetate buffer is added along with glass balls/marbles (to aid in physical breakdown of the sample during shaking).
5 ml of the enzyme mixture is added to the samples, blank, and standards. The tubes are shaken horizontally in a 37° C. waterbath at approximately 180 strokes/min. Time “zero” represents the first addition of the enzyme mixture to the first tube.
After 120 minutes, a 0.5-ml aliquot is removed from the incubating samples and placed into a separate tube of 20 ml 66% ethanol (to stop the reaction). After 1 hour, an aliquot is centrifuged at 3000 g for 10 minutes.
The glucose concentration in the tube is measured using the glucose oxidase/peroxidase method (Megazyme Glucose Assay Procedure GLC9/96). This is a calorimetric procedure.
The degree of starch digestion is determined by calculating the glucose concentration against the glucose standards, using a conversion factor of 0.9. The amount of resistant starch is determined by subtracting the % starch digested (dry weight basis) at 120 minutes from 100%.
Every sample analysis batch includes a reference sample of uncooked cornstarch. The accepted range of % digestion values for cornstarch are:
1Melogel ® starch, cornstarch commercially available from National Starch and Chemical Company, Bridgewater, NJ, USA. Thus, cornstarch would contain about 20% resistant starch.
0.5 g of a starch (1.0 g of a ground grain) sample was heated in 10 mls of concentrated calcium chloride (about 30% by weight) to 95° C. for 30 minutes. The sample was cooled to room temperature, diluted with 5 mls of a 2.5% uranyl acetate solution, mixed well, and centrifuged for 5 minutes at 2000 rpm. The sample was then filtered to give a clear solution.
The starch concentration was determined polarimetrically using a 1 cm polarimetric cell. An aliquot of the sample (normally 5 mls) was then directly titrated with a standardized 0.01 N iodine solution while recording the potential using a platinum electrode with a KCl reference electrode. The amount of iodine needed to reach the inflection point was measured directly as bound iodine. The amount of amylose was calculated by assuming 1.0 gram of amylose will bind with 200 miligrams of iodine.
Protein digestibility was determined in vivo with Wistar rats, using casein as a reference protein. Rats were housed individually in metal screened cages, and fed one of three diets for 10 days—protein-free diet, 10% protein isolate diet, and 10% casein diet. Food intake and body weight was recorded daily, and feces were collected for the last 3 days of the experimental period. Dietary nitrogen intake and fecal nitrogen were measured.
Resistant protein (%)=100−true protein digestibility (%)
where
True protein digestibility (%)=[nitrogen intake−(fecal nitrogen excreted−fecal nitrogen excreted on the protein-free diet)]/nitrogen intake×100
Composition of the Experimental Diet.
D. Rat Study. Effect of a Probiotic/Non-Probiotic Combination.
Male Sprague-Dawley rats were housed in plastic cages with wire bottom screens. 12 rats per group were fed one of 8 different diets for a period of 4 weeks. The diets were a modified AIN-76 diet (see the table below), consisting of a no-fibre diet, or the same diet to which the three components were added either individually, as a two-part combination, or as a three-part combination. The protein isolate replaced casein (at 19% of diet), the high amylose resistant starch product replaced regular cornstarch (at 10% of the diet), and the probiotic was added at 1% of the diet.
At the end of the experimental period, fresh fecal samples were collected from each rat for 3 days. On the final day each rat was sacrificed, and the large intestine removed, sectioned, and cleaned for subsequent analysis. Large intestinal contents were also removed for subsequent analysis.
Measured parameters included: (1) Feces—weight, pH, short chain fatty acids (SCFA) including total, acetate, propionate and butyrate, and ammonia; and (2) urine—cresol (see table below).
Rats eating the diet with the three-part combination (Diet 8) displayed higher fecal weight and lower fecal pH than rats eating no, one or two parts of the combination. In addition ratios of fecal butyrate:ammonia, fecal weight:ammonia and fecal weight:urine cresol were highest for the three-part combination. Concentrations of total short chain fatty acids, as well as acetate and butyrate were high for the three-part combination. Overall, diet 8 (three-part combination) showed greater impact on the parameters measured than for any part of the combination.
| Number | Date | Country | |
|---|---|---|---|
| 60845652 | Sep 2006 | US |
