Allulose-containing composition for promoting excretion of vegetable lipids from the body

Abstract

The present application relates to an allulose-containing composition for promoting excretion of vegetable lipids from the body and a food comprising the composition of the present application and vegetable lipids.

Claims

1. A margarine composition comprising allulose and vegetable oils in an emulsion form, the margarine composition made by a process comprising the steps of: providing an amount of the vegetable oils, wherein the vegetable oils comprise corn oil, palm olein oil, palm stearin oil and coconut oil, and combining the amount of the vegetable oils with an amount of the allulose that is 30 to 50 parts by weigh based on 100 parts by weight of the amount of vegetable oils, based on dry solids.

2. The margarine composition of claim 1, further comprising one or more ingredients selected from the group consisting of sodium chloride, milk, lecithin, and an organic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows changes in the body weight observed in C57BL/6J mice fed with a high-fat diet (HFD) containing vegetable lipids along with allulose for 8 weeks. In FIGS. 1a to 1d, abbreviations for a control group represent as follows: OL: olive oil provision, MG: margarine provision, PO: palm oil provision, SO: soybean oil provision. In FIGS. 1a to 1d, abbreviations for a test group are as follows: OLA: olive oil+5% allulose provision, w/w, MGA: margarine+5% allulose provision, w/w, POA: palm oil+5% allulose provision, w/w, SOA: soybean oil+5% allulose provision, w/w.

(2) Meanwhile, FIG. 1e shows changes in the body weight observed in C57BL/6J mice fed with a high-fat diet (HFD) containing creamer along with allulose for 8 weeks. Herein, PR for a control group represents the provision with a high-fat diet (HFD) along with creamer and PRA represents the provision with a high-fat diet (HFD)+creamer+5% allulose (w/w).

(3) FIG. 2a shows changes in the fecal lipid excretion after 8 weeks observed in C57BL/6J mice fed with a high-fat diet (HFD) containing vegetable lipids along with allulose. Herein, data were represented as means±SE. Significance between the group without allulose and the group with 5% allulose is as follows: *p<0.05, **p<0.01, ***p<0.001. In FIG. 2a, each abbreviation represents as follows: OLA, HFD+olive oil+5% allulose (w/w); OL, HFD+olive oil; MGA, HFD+margarine+5% allulose (w/w); MG, HFD+margarine; POA, HFD+palm oil+5% allulose (w/w); PO, HFD+palm oil; SOA, HFD+soybean oil+5% allulose (w/w); SO, HFD+soybean oil.

(4) FIG. 2b shows changes in the fecal lipid excretion after 8 weeks observed in C57BL/6J mice fed with a high-fat diet (HFD) containing creamer along with allulose. Herein, data were represented as means±SE. Significance between the group without allulose and the group with 5% allulose is as follows: *p<0.05, **p<0.01, ***p<0.001. In FIG. 2, PRA represents “HFD+creamer+5% allulose (w/w)” and PR represents “creamer+HFD.”

MODE FOR CARRYING OUT THE INVENTION

(5) Hereinafter, preferred examples are presented to help an understanding of the present application. However, the following examples are provided for easier understanding of the present application, and the contents of the present application are not limited by examples.

(6) Experimental Methods

(7) 1. Experimental Animal Breeding

(8) 1-1. Palm Oil, Olive Oil, Soybean Oil, and Margarine

(9) Twenty C57BL/6J mice (male, 4-week-old) were purchased from The Jackson Laboratory (USA) and used. After the animals were acclimated to the feeding environment with a lab-chow diet (Purina Co., USA) for 4 weeks, they were divided by a randomized block design into two groups: a negative control group without the ingestion of allulose (n=10/one kind of lipid); and a test group with the ingestion of allulose (n=10/one kind of lipid) and they were fed with the diets for 8 weeks.

(10) For the negative control group, the high-fat diet was applied to the AIN-76 diet, and the vegetable lipids [palm oil (Wilmar, Malaysia), olive oil (CJ CheilJedang Corp., South Korea), soybean oil (CJ CheilJedang Corp., South Korea), margarine (Ottogi Foods Co., Ltd., ‘Corn margarine’, raw materials: consisted of 80 wt % of vegetable lipids (palm olein oil, palm stearin oil, coconut oil, corn oil)] were included. For the test group, among the ingredients of the diet for the negative control group, 5 wt % of sucrose was replaced with allulose (crystalline allulose, 98 wt % of allulose based on dry solids, CJ CheilJedang Corporation) (Table 1). All the animal experiments were carried out under the approval of the Ethics Committee for Animal Studies at Kyungpook National University, South Korea (approval no. KNU-2013-18).

(11) [Table 1] Composition of test feed (% of diet, w/w)

(12) TABLE-US-00001 TABLE 1 Palm Olive Soybean oil oil oil Margarine Classification (PO) (OL) (SO) (MG) POA OLA SOA MGA Casein 20 20 20 20 20 20 20 20 DL- 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Methionine Corn 11.1 11.1 11.1 11.1 11.1 11.1 11.1 11.1 starch Sucrose 37 37 37 37 32 32 32 32 Cellulose 5 5 5 5 5 5 5 5 Palm oil 14.6 — — — 14.6 — — — Olive oil — 14.6 — — — 14.6 — — Soybean oil — — 14.6 — — — 14.6 — Margarine — — — 14.6 — — — 14.6 Lard 5.4 5.4 5.4 5.4 5.4 5.4 5.4 5.4 Mineral 4.2 4.2 4.2 4.2 4.2 4.2 4.2 4.2 mix.sup.1) Vitamin 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 mix.sup.2) Choline 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 bitartrate Cholesterol 1 1 1 1 1 1 1 1 tert- 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 Butylhy droquinone Allulose — — — — 5 5 5 5 Total (%) 100 100 100 100 100 100 100 100 kcal/g diet 4.584 4.584 4.584 4.34 4.384 4.384 4.384 4.14 Note 1) Mineral mix: AIN-76 mineral mixture (gram/kg): calcium phosphate, 500; sodium chloride, 74; potassium citrate, 2220; potassium sulfate, 52; magnesium oxide, 24; managanous carbonate, 3.5; ferric citrate, 6; zinc carbonate, 1.6; cupric carbonate, 0.3; potassium iodate, 0.01; sodium celenite, 0.01; chromium potassium sulfate, 0.55; sucrose 118.03 Note 2) Vitamin mix: AIN-76 vitamin mixture (gram/kg): thiamin HCL, 0.6; riboflavin, 0.6; pyridoxine HCL, 0.7; nicotinic acid, 0.003; D-calcium pantothenate, 0.0016; folate, 0.2; D-biotin, 0.02; cyanocobalamin (vitamin B12), 0.001; retinyl palmitate premix, 0.8; DL-alpha tocopheryl acetate, premix, 20; cholecalciferol (vitamin D3), 0.0025; menaquinone (vitamin K), 0.05; antioxidant, 0.01; sucrose, finely powdered, 972.8

(13) 1-2. Creamer

(14) Sixteen C57BL/6J mice (male, 4-week-old) were purchased from The Jackson Laboratory (USA) and used. After the animals were acclimated to the feeding environment with a lab-chow diet (Purina Co., USA) for 4 weeks, they were divided by a randomized block design into two groups: a negative control group without the ingestion of allulose (PR, n=8); and a test group with the ingestion of allulose (PRA, n=8) and they were fed with the diets for 8 weeks.

(15) For the negative control group, the high-fat diet was applied to the AIN-76 diet, and as the vegetable lipid, creamer [Dongsuh foods corporation ‘Prima’, South Korea, raw materials: vegetable hydrogenated oil 30-38 wt % (coconut hydrogenated oil, palm hydrogenated oil), starch syrup (including maltose), sodium casein, potassium phosphate, dibasic, calcium phosphate, tribasic] was used. For the test group, among the ingredients of the diet for the negative control group, 5 wt % of sucrose was replaced with allulose (crystalline allulose, 98 wt % of allulose based on dry solids, CJ CheilJedang Corporation) (Table 2). All the animal experiments were carried out under the approval of the Ethics Committee for Animal Studies at Kyungpook National University, South Korea (approval no. KNU-2013-18).

(16) [Table 2] Composition of test feed (% of diet, w/w)

(17) TABLE-US-00002 TABLE 2 Neg. Cont. Test Classification Group (PR) Group (PRA) Casein 20 20 DL-Methionine 0.3 0.3 Corn starch 11.1 11.1 Socrose 37 32 Cellulose 5 5 Creamer(Prima) 14.6 14.6 Lard 5.4 5.4 Mineral mix.sup.1) 4.2 4.2 Vitamin mix.sup.2) 1.2 1.2 Choline bitartrate 0.2 0.2 Cholesterol 1 1 tert-Butylhydroquinone 0.004 0.004 Allulose — 5 Total (%) 100 100 kcal/g diet 4.047 4.847 Note .sup.1)Mineral mix: it is the same as in Table 1 above and thus will be omitted here. Note .sup.2)Vitamin mix: it is the same as in Table 1 above and thus will be omitted here.

(18) All groups were allowed to ingest the iso-energetic diet of the same level by pair feeding based on the test group so as to exclude the calorie-reducing effect due to the allulose and the diets were kept refrigerated at 4° C. during the feeding period. The animals were housed in an individual cage under constant temperature (25±2° C.), constant humidity (50±5%), and a 12 h dark-light cycle.

(19) 2. Measurement of Dietary Intake and Body Weight

(20) Dietary intake was measured at a constant time every day, and the body weight was measured at a constant time every week.

(21) 3. Fecal Sample Collection and Analysis

(22) 3-1. Fecal Sample Collection

(23) The feces were collected for 84 hours (3.5 days) after termination of the feeding, dried and then stored in the freezer.

(24) 3-2. Fecal Lipid Extraction

(25) For the measurement, neutral lipid, cholesterol, and free fatty acids in the feces were extracted by modifying and supplementing the method of Folch et al. (1957). Specifically, the dried feces were ground in a mortar to take 0.5 g and then, 5 mL of a solution of chloroform:methanol (2:1, v/v) was added thereto, and the lipids were extracted at 4° C. for 24 hours. The extract was centrifuged at 3000×g (4° C.) for 10 minutes, and then 3 ml of the supernatant was taken out and dried under nitrogen gas at 37° C. and then dissolved again in 1 ml of the same extraction solvent. 200 μL of each sample for the measurement of neutral lipid, cholesterol, and free fatty acids was taken, dried again under nitrogen gas and the samples for the measurement of the neutral lipid and total cholesterol were dissolved in 500 μL of ethanol. The sample for the measurement of the free fatty acids was dissolved in 2.25 ml of NaOH and then 1 M HCl solution was added thereto to adjust the pH to the range of 2-3. To remove the turbidity occurring from the development with 3 mM cholic acid (sodium salt), 0.5% Triton X-100 as an emulsifier was mixed with an enzyme test solution during the quantification of total cholesterol and neutral lipids.

(26) 3-3. Quantification of Total Cholesterol in Feces

(27) For the measurement of total cholesterol, 10 μL of a solution dissolved in 500 μL of ethanol and 690 μL of the emulsifier were mixed and 800 μL of a test solution for the measurement (kit, Asan pharmaceutical Co., Ltd.) to which the enzyme method of Allain et al. (1974) was applied was mixed therewith. For the quantification of both free cholesterol (FC) and ester-type cholesterol (CE) in the feces, CE was converted by cholesterol esterase into FC and fatty acids. Among them, FC was subjected to the reaction with cholesterol oxidase to convert into Δ.sup.4-cholestenone. The resulting product and H2O2 which is the substrate were subjected to the reaction with peroxidase, phenol, and 4-amino-antipyrine to obtain a color developing material and the absorbance was measured at 500 nm. Quantification was carried out by comparing the measured value with the cholesterol standard curve.

(28) 3-4. Quantification of Fecal Neutral Lipids

(29) For the measurement of the neutral lipid, 10 μL of a solution dissolved in 500 μL of ethanol and 690 μL of the emulsifier were mixed and 800 μL of a test solution for the measurement (kit, Asan pharmaceutical Co., Ltd.) to which the enzyme method of McGowan et al. (1983) was applied was mixed therewith. The neutral lipid was degraded by lipoprotein lipase into glycerol and fatty acids. Of the degradation products, glycerol formed, by ATP and the action of glycerol kinase (GK), L-α-glycerol phosphate, which reacted with O2 and glycerophosphoxidase (GPO) to produce H2O2. Peroxidase and 4-amino-antipyrane were treated thereto to develop red color and then absorbance was measured at 550 nm and quantification was carried out by comparison with the glycerol standard curve.

(30) 3-5. Quantification of Fecal Free Fatty Acids

(31) The concentration of free fatty acids was measured using a test solution for the measurement of free fatty acids (Non-esterified fatty acid; NEFA kit, Wako, Japan) according to the color development principle using an enzyme method. First, acyl-coenzyme A synthase was applied to plasma free fatty acid to produce acyl-CoA, AMP, and pyrophosphoric acid. Then, acyl-coenzyme A oxidase was added thereto to generate 2,3-trans-enoyl-CoA and hydrogen peroxide. Peroxidase, 4-aminoantipyrine, and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine were treated thereto to develop red color and then absorbance was measured at 546 nm and quantification was carried out by comparison with the free fatty acid standard curve.

(32) Experimental Results

(33) 1. Confirmation of the Effect of Inhibiting Body Weight Gain Due to Allulose

(34) 1-1. Palm Oil, Olive Oil, Soybean Oil, and Margarine

(35) At the time of the 0th week of the diet, the body weights of the negative control group and the test group were similar (Table 3), but after 8 weeks of the diet, the effect of inhibiting the body weight gain in the test group was confirmed compared to the negative control group (Table 4 and FIGS. 1a to 1d). Specifically, in the cases of the MGA group and the OLA group, from the 5th week of the diet, and in the case of the POA group, from the 6th week of the diet, the significant effect of inhibiting the body weight gain was confirmed compared to the negative control group (FIGS. 1a to 1c). Particularly, in the case of the SOA group, the effect of inhibiting the body weight gain was exhibited from the 1st week of the diet and thus the excellent effect of inhibiting the body weight gain was confirmed compared to other vegetable lipids (FIG. 1d).

(36) TABLE-US-00003 TABLE 3 Olive Palm Soybean p-value* oil Magarine oil oil (ANOVA) Initial With- 21.81± 21.71± 21.56 ± 21.22± 0.899 body out 0.41 0.60 0.81 0.43 weight allulose With 21.28± 21.57± 21.62± 21.23± 0.933 allulose 0.43 0.53 0.68 0.42 (5%) p- 0.386 0.873 0.957 0.985 value** (t-test) Data were represented as means ± SE. *ANOVA is a comparative analysis among four groups. **t-test is the comparison value between the HFD without allulose and the HFD containing 5 wt % allulose in each group.

(37) TABLE-US-00004 TABLE 4 p- value* Olive Palm Soybean (A- oil Magarine oil oil NOVA) Body With- 32.86± 36.47± 34.12± 34.39± 0.101 weight out 0.66 0.94 1.44 0.69 on allulose 8th With 28.87± 32.30± 29.57± 28.84± 0.115 week allulose 1.23.sup.# 1.16.sup.# 1.05.sup.# 0.95.sup.### (5%) p- 0.014 0.016 0.025 0.001 value** (t-test) Data were represented as means ± SE. *ANOVA is a comparative analysis among four groups. **t-test is the comparison value between the HFD without allulose and the HFD containing 5 wt % allulose in each group (.sup.#p < 0.05, .sup.###p < 0.001).

(38) 1-2. Creamer

(39) At the time of the 0th week of the diet, the body weights of the negative control group (PR) and the test group (PRA) were similar (Table 5). However, the body weight of the negative control group increased significantly from 1 week, whereas the body weight of the test group was remarkably inhibited from the 1st week of the diet and thus after 8 weeks of the diet, the significant effect of inhibiting the body weight gain in the test group was confirmed (Table 5 and FIG. 1e).

(40) TABLE-US-00005 TABLE 5 Without With allulose allulose p-value** (PR) (PRA) (t-test) Body 0 week 21.93 ± 0.46 21.47 ± 0.50 0.517 weight 8th week 33.50 ± 0.69 27.41 ± 0.48 0.000 Data were represented as means ± SE. **t-test is the comparison value between PR without allulose and PRA containing 5 wt % allulose in each group.

(41) 2. Confirmation of the Effect of Excreting Vegetable Lipids Due to Allulose

(42) 2-1. Palm Oil, Olive Oil, Soybean Oil, and Margarine

(43) The effect of excreting vegetable lipids due to allulose was confirmed by the lipid excretion in the feces.

(44) As a result, it was confirmed that the contents of triglycerides and free fatty acids in the feces were significantly increased in the test groups of all kinds of oils compared to the negative control group. Particularly, it was confirmed that the contents of free fatty acids in the test groups were significantly higher than that in the negative control group (Tables 6 to 9 and FIGS. 2a (A) and (C)). In addition, it was confirmed that the SOA group also showed a significant increase in the cholesterol content in the feces compared to the SO group (FIG. 2a (B)).

(45) TABLE-US-00006 TABLE 6 OLA OL Triglycerides 0.26 ± 0.022*  0.19 ± 0.022 (mmol/day) Cholesterol 16.56 ± 0.61   15.19 ± 0.54  (mmol/day) Free Fatty Acids 5.67 ± 0.93** 1.50 ± 0.13 (mmol/day) Data were represented as means ± SE. There was a significant difference between OLA and OL: *p < 0.05, **p < 0.01. OLA, HFD + olive oil + 5% allulose; OL, HFD + olive oil.

(46) TABLE-US-00007 TABLE 7 MGA MG Triglycerides  0.44 ± 0.038***  0.23 ± 0.031 (mmol/day) Cholesterol 18.20 ± 1.16   16.00 ± 0.35  (mmol/day) Free Fatty Acids 5.83 ± 0.57*** 2.11 ± 0.10 (mmol/day) Data were represented as means ± SE. There was a significant difference between MGA and MG: ***p < 0.001. MGA, HFD + margarine + 5% allulose; MG, HFD + margarine.

(47) TABLE-US-00008 TABLE 8 POA PO Triglycerides  0.38 ± 0.026***  0.18 ± 0.013 (mmol/day) Cholesterol 17.07 ± 0.42   15.68 ± 0.54  (mmol/day) Free Fatty Acids 5.22 ± 0.45** 3.50 ± 0.32 (mmol/day) Data were represented as means ± SE. There was a significant difference between POA and PO: **p < 0.01, ***p < 0.001. POA, HFD + palm oil + 5% allulose; PO, HFD + palm oil.

(48) TABLE-US-00009 TABLE 9 SOA SO Triglycerides  0.29 ± 0.028* 0.18 ± 0.027 (mmol/day) Cholesterol 15.92 ± 0.57*  14.19 ± 0.37  (mmol/day) Free Fatty Acids 4.76 ± 0.95* 1.29 ± 0.054 (mmol/day) Data were represented as means ± SE. There was a significant difference between SOA and SO: *p < 0.05. SOA, HFD + soybean oil + 5% allulose; SO, HFD + soybean oil.

(49) 2-2. Creamer

(50) The effect of excreting lipids in the creamer due to allulose was confirmed by the lipid excretion in the feces.

(51) As a result, it was confirmed that the contents of triglycerides and free fatty acids in the feces were significantly increased in the test group compared to the negative control group. Particularly, it was confirmed that the content of free fatty acids in the test group was significantly higher than that in the negative control group (Tables 10 and FIG. 2b).

(52) TABLE-US-00010 TABLE 10 PRA PR Triglycerides 0.44 ± 0.053*  0.26 ± 0.032 (mmol/day) Cholesterol 18.28 ± 0.97   16.81 ± 0.52  (mmol/day) Free Fatty Acids  8.39 ± 0.75*** 2.78 ± 0.30 (mmol/day) Data were represented as means ± SE. There was a significant difference between PR and PRA: *p < 0.05, ***p < 0.001. PRA, HFD + creamer + 5% allulose; PR, HFD + creamer

(53) 2-3. Consideration

(54) It was confirmed that when allulose was consumed along with vegetable lipids in the creamer, the fecal excretion of lipids was promoted. In addition, it was confirmed that when allulose was consumed along with each of palm oil, olive oil, soybean oil, and margarine, the fecal excretion of lipids was also promoted. Especially, when allulose was taken together with soybean oil, the effect of excreting lipids was excellent compared to the ingestion of allulose along with other vegetable lipids and the excretion of cholesterol was also increased.

(55) The above-mentioned description of the present application is intended to be merely exemplary, and it will be understood by those skilled in the art to which the present application belongs that the present application can be easily modified into other specific forms without changing the technical concepts or essential features thereof. It is, therefore, to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.