Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
  • A23G1/30—Cocoa products, e.g. chocolate; Substitutes therefor
  • A23G1/32—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds
  • A23G1/48—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds containing plants or parts thereof, e.g. fruits, seeds, extracts
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
  • A23G1/04—Apparatus specially adapted for manufacture or treatment of cocoa or cocoa products
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
  • A23G1/30—Cocoa products, e.g. chocolate; Substitutes therefor
  • A23G1/32—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds
  • A23G1/42—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
  • A23G1/426—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing vitamins, antibiotics
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
  • A23G1/30—Cocoa products, e.g. chocolate; Substitutes therefor
  • A23G1/56—Cocoa products, e.g. chocolate; Substitutes therefor making liquid products, e.g. for making chocolate milk drinks and the products for their preparation, pastes for spreading, milk crumb
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
  • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
  • A23L33/22—Comminuted fibrous parts of plants, e.g. bagasse or pulp
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
  • A23G2200/00—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF containing organic compounds, e.g. synthetic flavouring agents
  • A23G2200/14—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF containing organic compounds, e.g. synthetic flavouring agents containing fruits, nuts, e.g. almonds, seeds, plants, plant extracts, essential oils

Definitions

• the present invention relates to cacao extract containing dietary fiber, more specifically cacao extract containing dietary fiber useful for the treatment of diabetes obtained as a residue of solvent extraction of the cacao bean husk
• CBH cacao bean
• a high content of dietary fiber is contained, and especially, insoluble dietary fiber (IDF) is in excess of soluble dietary fiber (SDF);
• Cacao (Theobroma cacao L.) is a Latin America originated perennial belonging to Byttneriaceae family, which grows as high as 6-8m. It produces an oval-type pod, in which about 30-40 seeds are embedded by pulp.
• Cacao comprises a shell (or testa), a nib (or cotyledon) and a germ, and the main ingredient of chocolate is a ground mass made by grinding the nib with high butter content. This mass is called cacao liquor or cacao mass (CM) because it is in a paste phase below the melting point of butter. While nibs are used as a main ingredient of chocolate, the husks winnowed in the CM production process are by-products of about 15 wt% (about 400,000 M/T worldwide) and wasted entirely.
• cacao bean husk This is called the cacao bean husk (CBH). While the current chocolate market in Korea is fairly large, the recognition about chocolate is changing recently with the opening of foreign chocolate import. Especially, together with the recent dieting boom or increased consumers' interest in health food related to various intestinal and metabolic diseases, such as diabetes, tooth decay and arteriosclerosis, chocolate is being recognized negatively due to its high sugar content and high-fat calories.
• dietary fiber which is abundantly contained in cacao. Researches on dietary fiber is presented in the reference ["Dietary Fiber", Food Technol, 43(10), 133-139, 1989; “Dietary Fiber and Disease", /. Am. Med. Assoc, 229, 1068-1074, 1974]. However, since dietary fiber is a structural residue of plant cells indigestible with human digestive enzymes, it has been recognized as having no nutritional value. Although dietary fiber is contained very abundantly in CB or CBH, researches on characteristics and physiological functions are not enough, compared with polyphenols.
• dietary fiber is contained in various food stuffs, and there are many kinds of dietary fiber.
• Dietary fiber is largely divided into soluble dietary fiber (SDF: indigestible dextrin, pullulan, polydextrose, glucomannan, pectin, enzymolyzed residue of guar gum, rice barn, hemicellulose, agar, etc.) and insoluble dietary fiber (IDF: wheat bran, spirulina, chitosan, corn, apple, soybean, soybean husk, cellulose, ginseng, etc.). Dietary fiber content of various plant food ranges 1-43 wt%.
• the physiological activity of dietary fiber is affected by its physical and chemical characteristics, such as water holding capacity (WHC), cation exchange capacity, bile acid binding capacity, fermentability, etc. and varies with composition, content, binding state, cooking condition, source, etc. of dietary fiber ["Physiological Activity and Use of Dietary Fiber", Food Science and Industry(Korean), 28(3), 2-23, 1995; "The Effect of the Polysaccharide Composition and Structure of Dietary Fiber on Cecal Fermentation and Fecal Extraction", Am. Soc. Clin. Nutr., 4A, 51-55, 1986].
• WPC water holding capacity
• water absorption capacity of dietary fiber is known to be related to the mechanism lowering digestive capacity , increasing volume and weight of feces, and lowering serum triglyceride.
• water absorption capacity of grain fiber is an important decisive factor in increasing the volume of feces. This water absorption capacity is greatly affected by the type, content and particle size of the dietary fiber, and depends mainly on the component, fineness, pH and ionic strength of the dietary fiber.
• an object of this invention is to provide a cacao extract containing dietary fiber with excellent effect of diabetes treatment from formerly wasted CBH.
• the present invention is characterized by cacao extract obtained from extraction residue of CBH wasted during cacao processing and containing dietary fiber of 43-60 wt%.
• the present invention relates to cacao extract including dietary fiber solvent-extracted from the formerly wasted CBH, more specifically cacao extract including excess dietary fiber with excellent diabetes treatment compared to cacao bean or other plant sources due to its good physiological activity on the depression of blood sugar and the improvement of blood cholesterol level.
• Cacao bean is preheated, ground, selected and winnowed to obtain CBH, and then ethanol is added to CBH. Thereby the cacao extract of the present invention is obtained as residue of extraction.
• Extraction residue of CBH is prepared using the L-BTC (Better Taste and Color) method as shown in Fig. 1.
• L-BTC etter Taste and Color
• Cacao bean is preheated at the temperature of 160-180 ° C and under the pressure of 0.5-1 bar, crushed, selected, and winnowed to give CBH. If the pressure is below 0.5 bar, CBH becomes excess in the nib, and if the pressure exceeds 1 bar, the nib yield becomes poor.
• the purpose of the thermal pretreatment is to make bean soft and easily breakable before grinding it. If the temperature of the thermal pretreatment is below 160 ° C, CBH is not separated well from the nib, and if the temperature exceeds 180 ° C, the nib yield becomes poor.
• Extraction residue of CBH is obtained by adding ethanol to the said preheated CBH and centrifuging the same.
• the polyphenol extract is put aside and used for beverage.
• Solvents such as ethanol, methanol, butanol, etc. are used for the extraction, and ethanol is the most desirable among them.
• the dietary fiber composition is separated using the Prosky method as shown in Fig. 2 in order to identify the dietary fiber content of the residue.
• the results of adding enzymes such as temamyl, protease and amyloglucosidase show that the cacao extract of the present invention consists of the mixture of SDF and IDF.
• CM Cacao mass
• Cacao powder, cacao nibs, cacao mass or mixture of these can be mixed additionally to the said extract in the range of 5-15 wt%.
• the total dietary fiber content of the defatted CBH, powder, nibs and CM of the present invention is 30-60 wt%, and desirably 43-60 wt%.
• the dietary fiber content is larger than the conventional content of 1-43 wt% in plant materials, and physiological activities such as absorption retardation of glucose and bile acid are superior.
• IDF content of the CBH residue with polyphenol composition removed by the ethanol extraction is higher than that of CBH as shown in Fig. 3.
• IDF and SDF contents of the supernatant are very low as 0.2-0.4 wt% and 2-3 wt%, respectively.
• IDF content of the residue is high because SDF is lost or transferred to the supernatant during the polyphenol extraction process.
• the CBH extraction residue is high in the carbohydrate content (50-60 wt%) and low in the reduced sugar content.
• dietary fiber exists mostly in the residue.
• the protein content (33.43 ⁇ 0.09 wt%) and crude ash content (18.03 ⁇ 0.67 wt%) are higher in the supernatant. It is because soluble materials such as protein and salts were transferred to the supernatant. Accordingly, this residue is used as high dietary fiber source.
• IDF content of CBH is 25-45 wt% and higher than its SDF content of 5-15 wt%.
• IDF content of cacao bean extract is 29-33 wt% and its SDF content is 3-5 wt%.
• the higher IDF content compared with the SDF content enables cacao bean extract to be used as high dietary fiber source.
• cacao bean is not as economical as CBH.
• IDF content of CM extract is 25-35 wt% and higher than its SDF content of 1.5-3 wt%.
• CM alone is applicable for the source of chocolate, but limited and economically unfavorable for high dietary fiber source.
• the dietary fiber contained in the cacao extract of the present invention shows high water holding capacity (WHC), oil binding capacity (OBC) and viscosity and excellent retardation effect of glucose and bile acid absorption.
• WHC of dietary fiber extracted from CBH, cacao bean (CB) and CM is very powerful as 9 times, compared with 8 times of pectin on the market.
• WHC of cacao husk and cacao bean themselves are only about a half of the corresponding extracts.
• Oil binding capacity (OBC) of the dietary fiber contained in the cacao extract of the present invention is also excellent.
• OBC of the dietary fiber is 35-70% of WHC, and for CM, OBC is 60-70% of WHC. So, efficient adsorption can be attained if used for the processed food containing both water and oil.
• the dietary fiber of the cacao extract of the present invention since the dietary fiber of the cacao extract of the present invention has viscosity and gel formation ability, it increases the viscosity of the digested material. Hence, it affects the mobility and absorption of the digested material in the digestive duct.
• the dietary fiber of the extract residue obtained from the CBH of the present invention shows somewhat higher viscosity than the dietary fiber of CB, and the same has higher viscosity in IDF than in SDF.
• the in vitro measurement results of the cacao extract containing dietary fiber of the present invention show very excellent retardation of glucose and bile acid permeation.
• the retardation rate of glucose absorption of IDF and SDF of the cacao extract residue of the present invention are 17-20% and 25-30% respectively. It is because SDF's viscosity is higher than that of IDF, since WHC of dietary fiber increases generally with time and gel structure is formed so that physical property is changed and glucose is entrapped. Accordingly, the cacao extract of the present invention is very useful for the high dietary fiber product, because the dietary fiber content is higher than 9-11% which is extracted from ⁇ -cellulose currently on the market.
• glucose retardation rate of CBH itself is higher for IDF as 15-30% than that of SDF, being 10-25%. This is because the glucose absorption retardation effect is changed due to the solvent treatment of ethanol extraction, whereas CBH does not go through such treatment.
• ethanol extraction residue with polyphenol removed is superior to CBH itself.
• sampling time and sampling place of cacao bean affect the results.
• the bile acid absorption retardation of IDF and SDF of the cacao extract residue of the present invention are 30-35% and 60-65%, respectively. It is because SDF's viscosity is higher than that of IDF as explained above. Accordingly, bile acid retardation rate of the cacao extract of the present invention is superior to 5-10% of the conventional SDFs such as citrous pectin, guar gum, etc.. Also, the bile acid retardation rates of IDF and SDF of CBH are higher than the conventional, being 35-40% and 55-63% respectively.
• the bile acid retardation rates of CB and CM are 30-35%. Therefore, the bile acid retardation effect is very excellent, with SDF's bile acid retardation rates of all extracts except CB extract being almost double that of IDF. It is because SDF's viscosity is higher than that of IDF as explained above.
• Physiological activity measurement through in vitro experiment of cacao extract of the present invention shows the reduction of blood insulin level, because sugar absorption at the intestine is retarded due to the reduced mobility of carbohydrate from the stomach to the intestine and inhibited digestion of starch and other saccharides, and blood insulin level is reduced due to the inhibited entry of digested material into the surface of epithelial cells and the consequent inhibited sugar absorption at the intestine.
• the cacao extract containing dietary fiber of the present invention has powerful effect on prevention and treatment of diabetes and adult disease, because it reduces the neutral lipid level, phospholipid level and cholesterol level of the serum, and regulates the lipid metabolism imbalance.
• Cacao Theobroma cacao L.
• BTC Bit Taste and Color
• Fig. 1 the latest CM processing method of nib roasting process wherein the nib is homogeneously crushed and then roasted, was used.
• Cacao bean was thermally pretreated at 170 ° C and under the pressure of 0.8 bar. After grinding and selecting, the cacao bean was winnowed to obtain CBH. After adding ethanol, the same was extracted, centrifuged at 1,000-3,000 rpm and then fractionated into the supernatant and residue. Winnowed nib obtained during the said winnowing process was treated with nib reaction for 10 min under the pressure of 1.2 bar to give reacted nib. The same was dried, roasted and ground under the condition of 132 °C and D.R. (discharge rate) 1,000kg/ hr to obtain CM, the major source of chocolate.
• Example 5 Dietary fiber was extracted and fractionated from the extract residue prepared from the above Example 1 using Prosky method as shown in Fig. 2. The residue was passed through a 30-mesh sieve and skimmed with diethylether for 12 hours using Soxhlet method. After drying at 70 °C in the drying oven, the same was deodored and preserved in the refrigerator after sealing for the use of dietary extract sample. Phosphate buffer solution (0.08M, pH 6, 25ml) was added to 0.5g of the prepared residue sample, and the same was treated with temamyl for at 95-10 ° C for 30 min and pH was adjusted to 7.5 using NaOH (0.275N, 5m4), and then treated with protease at 60 °C for 30 min.
• Phosphate buffer solution (0.08M, pH 6, 25ml
• Example 1 The yield of Example 1 was 12.63 ⁇ 1.38 wt % for CBH, 86.77 ⁇ 1.52 wt% for winnowed nib, 95.95 ⁇ 0.49 wt% for reacted nib and 84.18 ⁇ 0.23 wt% for CM. for CM. Extract was prepared by adding solvent and mixing each, the solvent was removed and the same was dried. The dietary fiber was fractionated and extracted as in the Example 2 for the identification of the dietary fiber content.
• IDF content of the prepared cacao extract was determined using the Prosky method. Two 0.5g portions of the each extract were treated with enzymatic hydrolysis with total dietary analysis method of AOAC (Association of Official Analytical Chemists) method. The solution (enzyme digest) was suction-filtered to B2 crucible and 500 ⁇ f. suction flask. The same was washed with acetone (200 ⁇ n , 2 times) and suction filtered again. One of the two B2 crucibles containing dietary fiber was incinerated to obtain the ash content (A), and the other was used to obtain the protein content (P).
• AOAC Association of Official Analytical Chemists
• Equation 1 The ash content was measured with direct incineration at 550 ° C and the protein content was measured with Kjeldahl (Kjeltec Auto 1035 Sampler System, Tecator, Sweden) method. The same procedure was performed for the blank. The measurement obtained from the blank (B) was subtracted from the dry weight, and IDF content was calculated using the following Equation 1. The results are shown in Table 1. Equation 1
• Blank(mg) ' + Rj - P - A
• Ri, R 2 Weight of residues
• Si, S 2 Weight of samples
• P Protein content of IDF
• A Ash content of IDF
• SDF content of the prepared cacao extract was determined using the Prosky method as in Experiment 1. Distilled water was added to the filtrate of the 500-- ⁇ # suction flask saved during the IDF analysis to lOOm After pouring the same to a 600 ⁇ £ beaker, ethanol (95 wt%, 400 ⁇ 0.) preheated to 60 ° C was added. The same was left stationary for 60 min at room temperature, and SDF was aggregated and precipitated. This solution was suction filtered to 2 glass crucibles (1G3 crucible), and was washed two times with 20ml. of ethanol (95 wt%) and two times with 20m£ of acetone.
• Equation 2 ash content (A) was determined from one of the two 1G3 crucibles and protein content (P) was determined from the other. The same procedure was performed for blank and SDF content was calculated using the following Equation 2. The results are shown in Fig. 1. Equation 2
• Blank(mg) R ⁇ +R2 _ p _ A
• NDF neutral detergent fiber
• ADF acid detergent fiber
• Hemicellulose (wt%) NDF - ADF
• NDF content was determined by the following procedure. Partially modified van Soest and Wine's method was used for quantification. 1.7g of ⁇ -amylase (Type VI-B, A-3176, from porcine pancreas) and lOOi-il of phosphate buffer solution (0.1M, pH 7.0) were taken to a 200m£ flask, and dissolved under agitation for 15 min. The same was centrifuged at 4 ° C under 1,500* g for 10 min. The supernatant was filtrated to 1G3 crucible at
• neutral detergent solution a solution prepared by dissolving 30g of sodium laurylsulphate, 18.61g of disodium EDTA, 6.81g of sodium borate decahydrate and 4.56g of hydrogen phosphate (anhydride) sequentially in HI of distilled water, adding lOinl of 2-ethoxyethanlol and adjusting pH to 6.9-7.1 with H3PO4 solution at room temparature) and 2ml of decahydronaphthalene were added sequentially.
• neutral detergent solution a solution prepared by dissolving 30g of sodium laurylsulphate, 18.61g of disodium EDTA, 6.81g of sodium borate decahydrate and 4.56g of hydrogen phosphate (anhydride) sequentially in HI of distilled water, adding lOinl of 2-ethoxyethanlol and adjusting pH to 6.9-7.1 with H3PO4 solution at room temparature) and 2ml of decahydronaphthalene were added sequentially.
• Equation 4 After dissolving 0.5g of sodium sulphite into the solution, the same was heated for 60 min in a boiling constant-temperature water bath with a countercurrent cooler, and the 1G3 crucible was incinerated for 1 hr in a Maffle's furnace at 525 ° C, was dried for 3 hr in a drying oven and weighed. Heated solution was suction filtered to this 1G3 crucible, and washed two times with hot distilled water. 1G3 crucible containing NDF was dried for 12 hr in a drying oven, and left to be cooled. After incarnating for 3 hr at 525 ° C , the same was weighed. The same procedure was performed for the blank, and NDF content was determined by the following Equation 4. Equation 4
• ADF content was measured using AOAC method. After weighing lg of the sample to a 500ml reflux flask, 100ml of acid detergent solution (20g of acetyltrimethyl ammonium bromide dissolved in II of IN sulfuric acid) was added and the same was heated for 60 min in a boiling constant-temperature water-bath with a countercurrent cooler. After the 1G3 crucible was incinerated for 1 hr at 525 ° C, the same was dried in a drying oven for 3 hr at 105 ° C and weighed after cooling. The heated solution was suction-filtered to this 1G3 crucible and washed thoroughly with about 100ml of hot distilled water and detergent, and then rinsed two times with 20ml of acetone. 1G3 crucible containing ADF was dried in a drying oven for 12 hr at 105 ° C , and weighed after cooling. ADF content was calculated using the following Equation 5. Equation 5
• Lignin content was calculated using AOAC method.
• the 1G3 crucible containing ADF was put into a 50ml beaker, and 72 wt% of sulfuric acid (15 ° C) was added to the crucible to cover the surface of the residue.
• the solution was homogenized by stirring with a glass rod and 72 wt% of sulfuric acid was poured to about 2/3 of the crucible. The same was kept stationary for 3 hr while agitating at 20-23 ° C every 1 hr.
• the resultant solution was suction-filtered, and acid was thoroughly removed by washing with about 100ml of hot distilled water.
• the crucible containing lignin was dried in a drying oven for 12 hr at 105 ° C and weighed after cooling. The same was weighed again after incinerating for 3 hr at 500 ° C .
• the same procedure was performed for the blank, and the lignin content was calculated by subtracting this weight from the weight after drying. Equation
• CBH the content of cellulose, hemicellulose, lignin and pectin was 18.55 wt%, 5.35 wt%, 17.75 wt% and 12.45 wt%, respectively.
• cellulose content, hemicellulose content, lignin content and pectin content was 20.05 wt%, 4.57 wt%, 19.33 wt% and 9.66 wt%, respectively. So, all the contents increased in a degree compared with CBH due to the increased IDF content.
• CB cellulose content, hemicellulose content, lignin content and pectin content was 9.26 wt%, 2 wt%, 19.25 wt% and 2.84 wt% respectively.
• cellulose content, hemicellulose content, lignin content and pectin content was 7.27 wt%, 3.3 wt%, 20.33 wt% and 2.72 wt% respectively.
• cellulose content, hemicellulose content, lignin content and pectin content was 6.9 wt%, 2.25 wt%, 21.8 wt% and 2.02 wt% respectively.
• CM cellulose content, hemicellulose content, lignin content and pectin content was 6.05 wt%, 3.75 wt%, 20.45 wt% and 0.53 wt%, respectively.
• Each dietary fiber of CBH, CB and CM extracts prepared from the Examples 1-3 was suspended, either alone or by mixing 0.1-0.5 wt% of stabilizer (pectin) and suspending in distilled water, to 1-3 wt% suspension. After heating at 60 ° C and cooling to 30 ° C, the viscosity was measured with a rotating viscometer (Brookfield DN- II +, Brookfield Eng. Labs Inc.) or a capillary viscometer (Cannon-Fenske, Cannon Instrument Co.) The viscosity of SDF of CBH and CB (1% (w/v)) was 1.363 and 1.339mPa-s respectively.
• the viscosity of the suspension was 1.5 times (1.902-2.907mPa-s) and two times (3.803-3.858mPa-s), respectively.
• the viscosity of the suspension was 1.57 and 3.16mPa-s for 0.1% of pectin and guar gum, respectively.
• the viscosity of dietary fiber of CBH and CB (1% (w/v)) in 0.1% of pectin solution was 4.47 and 3.97mPa-s, respectively, and the viscosity of dietary fiber of CBH and CB (1% (w/v)) in 0.1% of guar gum solution was 8.22 and 7.48mPa-s respectively.
• the apparent viscosity of 2% CBH and CB in 0.1% guar gum solution at 50 rpm was 21.76 and 17.01mPa-s, respectively.
• Dialyzing membrane (Sigma D7884: M.W. cut-off ⁇ 1200) with 3.2 ⁇ of area and 10cm of length was submerged in 0.1 wt% of sodium azide solution overnight and one end of the dialyzing membrane was tied tightly with cotton thread.
• the glucose content was measured using ABTS method. 5ml of ABTS indicator (a solution prepared by dissolving 60mg of glucose oxidase and 6mg of peroxidase in 250ml of 0.12M phosphate buffer solution) was added to dietary fiber sample of cacao extract dissolved in 0.1 wt% of sodium azide solution and lm of standard glucose solution. Absorbance was measured at 450nm after putting the same for 30-40 min at room temperature. Glucose change with time and retardation index were calculated, and the results are shown in Figs. 4a & 4b, and the following Table 3. Table 3
• Equation 8 0.05M phosphate buffer solution prepared from 0.1 wt% sodium azide solution was added to the container and maintained at 37 ° C .
• Permeation experiment was performed for 72 hr at 100 rpm in a constant-temperature water-bath, lin! of dialyzed solution was taken for constant intervals for measurement of bile acid content.
• the bile acid retardation effect was calculated by the following equation. Equation 8
• the bile acid content was measured using method of Boyd, et al.. 5ml of sulfuric acid solution was added to 1ml of the bile acid (Sigma B-8756) solution, and lull of 0.25 wt% furfural solution was added after 5 min. The same was put for 60 min, and the absorbance was measured at 510nm when the pink color was developed at its best. Change in the amount of taurocholic acid across the dialyzing membrane was measured, and the results are shown in Figs. 5a & 5b. The bile acid retardation rate (%) was calculated and the result is shown in the following Table 4.
• Experimental example 5 Diabetes treatment effect of cacao extract containing dietary fiber a) Diet & water intake and change of weight & fecal weight using experimental animal Animal experiment was performed using rats which were induced to diabetes with Streptozotocin (STZ, Sigma S-0130) in order to investigate the physiological effects such as blood glucose level and improvement of lipid metabolism. Experimental animals used were 5-week-old male S.D. (Sprague Dawley), bought from the Korea Animal Experiment Center Ltd.. The standard dietary composition (wt%) for the animal experiment is shown in Table 5, and water intake, weight and fecal weight are shown in Table 6. Table 5
• mice were raised under the condition of temperature of 20-22 °C, humidity of 50 + 10% and 12 hr of bright and dim period. They were adapted to solid feed for 1 week in the stainless steel cage, and were induced to diabetes. Diabetes induction was performed by intraperitoneally injecting Streptozotocin, which was dissolved in citrate buffer solution (pH 4.5) with the concentration of 50 mg/kg, and fasting the same for 12 hr. After taking out blood from the tails, only rats with blood glucose level higher than 180mg/ dl were used as diabetic rats. For control group, 0.01M of citrate buffer solution was injected by the same method as the diabetic group.
• mice After 2 weeks of adaptation, the experimental animals were classified as normal standard diet group, diabetic standard diet group, CBH-R group, CBH-S group, CM group and CM-NDF group. The animals were fed for additional 2 weeks with experimental diet, and 8 rats with similar weight were classified in the same group. Distilled water was provided and the diet was given freely. Diet intake, fecal weight and water intake were measured every day, and the weight was measured every two days.
• the blood sugar was measured at 500nm by GOD (glucose-oxidase) method using blood sugar measurement kit (AM 201 -K) of Asan Pharmaceuticals after isolating the blood serum.
• GOD glucose-oxidase
• the total cholesterol and HDL (high-density lipoprotein)-cholesterol level were measured at 500nm by enzymatic colorimetric method using the kit of Asan Pharmaceuticals (Cholesterol-enzyme and AM 203-K) after isolating the blood serum.
• LDL (low-density lipoprotein)-cholesterol was calculated with the following Friedewald's equation.
• Al atherogenic index
• A1 -- Tot ⁇ l cholesterol - HDL cholesterol HDL cholesterol The effect of dietary fiber of cacao extract on the total cholestrol, HDL-cholesterol, LDL-cholesterol and atherogenic index (Al) in the blood into the experimental animals were measured two weeks after the injection of STZ. The result is shown in Fig. 9.
• SAS Statistical Analysis System
• the cacao extract of the present invention uses CBH which has been wasted in the conventional CM processing, it provides the advantage of cost reduction and is environmental-friendly.
• the extract residue of CBH of the present invention contains a lot of dietary fiber, and the dietary fiber is rich in lignin. Therefore, it provides a probability of cacao extract product useful for the treatment of diabetes due to the excellent glucose and retardation effect of bile acid absorption, blood sugar depression and improvement of lipid metabolism.

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