IMPROVED SUGAR-DEPLETED FRUIT OR VEGETABLE JUICE AND JUICE-RETAINING FRUIT OR VEGETABLE DERIVED MATTER, METHODS OF PRODUCING THE SAME AND THE USE THEREOF TO MAINTAIN HEALTH AND TO TREAT AND PREVENT MEDICAL AILMENTS

Abstract

The present invention provides a sugar-depleted fruit or vegetable juice product, wherein said juice product is a fruit or vegetable juice or juice-retaining fruit or vegetable derived matter, wherein said juice product contains at least about 5 g/l gluconic acid and said juice product contains any two or three, of (i) at least about 0.5 g/l Ca.sup.2+, (iii) at least about 1 g/l K.sup.+, and (iii) at least about 0.1 g/l Mg2+. Also provided are methods of producing the same and the use thereof to assist in maintaining the health and well-being of a subject and in the treatment and prevention of medical ailments, specifically those associated with the over-consumption of glucose and/or sucrose or inappropriate metabolism of glucose, e.g. metabolic syndrome, diabetes mellitus type II, obesity, dyslipidemia, insulin resistance, hypertension and liver steatosis.

Claims

1. A sugar-depleted fruit or vegetable juice product, wherein said juice product is a fruit or vegetable juice or juice-retaining fruit or vegetable derived matter, wherein said juice product contains at least about 5 g/l gluconic acid and said juice product contains any two or three, of (i) at least about 0.5 g/l Ca.sup.2+, (ii) at least about 1 g/l K.sup.+, and (iii) at least about 0.1 g/lMg.sup.2+.

2. The sugar-depleted fruit or vegetable juice product of claim 1, wherein the combined mass concentration of free glucose and sucrose in the sugar-depleted juice product is no more than about 20 g/l when said juice product is adjusted in volume with water to give a gluconic acid concentration of about 5 g/l to about 100 g/l.

3. The sugar-depleted fruit or vegetable juice product of claim 2, wherein the combined mass concentration of free glucose and sucrose in the sugar-depleted juice product is no more than about 15 g/l.

4. The sugar-depleted fruit or vegetable juice product of claim 1, wherein the sugar-depleted juice product contains no more than about 5 g/l free glucose, preferably no more than about 1 g/l free glucose, and no more than about 5 g/l sucrose, preferably no more than about 1 g/l sucrose, when said juice product is adjusted in volume with water to give a gluconic acid concentration of about 5 g/l to about 100 g/l or the specific sub-ranges disclosed below.

5. The sugar-depleted fruit or vegetable juice product of claim 1, wherein the sugar-depleted juice product is essentially devoid of free glucose and sucrose.

6. The sugar-depleted fruit or vegetable juice product of claim 1, wherein the sugar-depleted juice product contains at least 6 g/l gluconic acid.

7. The sugar-depleted fruit or vegetable juice product of claim 1, wherein the sugar-depleted juice product contains at least about 0.5 g/l Ca.sup.2+, wherein the sugar-depleted juice product contains at least about 1 g/l K.sup.+, wherein the sugar-depleted juice product contains at least about 0.1 g/l Mg.sup.2+, or a combination thereof.

8. (canceled)

9. (canceled)

10. The sugar-depleted fruit or vegetable juice product of claim 1, wherein the sugar-depleted juice product contains 5 to about 100 g/l gluconic acid.

11. The sugar-depleted fruit or vegetable juice product of claim 10, wherein the sugar-depleted juice product contains about 0.5 to about 10 g/l Ca.sup.2+, wherein the sugar-depleted juice product contains about 1 to about 20 g/l K.sup.+, wherein the sugar-depleted juice product contains about 0.1 to about 2 g/l Mg.sup.2+, wherein the sugar-depleted juice product contains about 0.1 to about 2 g/l Mg.sup.2+, or a combination thereof.

12. (canceled)

13. (canceled)

14. The sugar-depleted fruit or vegetable juice product of claim 1, wherein said sugar-depleted juice product contains at least about 5 g/l gluconic acid and wherein said juice product contains any two or three, of Ca.sup.2+, K.sup.+, and Mg.sup.2+ at a mass concentration which, when said juice product is adjusted in volume with water to give a gluconic acid concentration of about 5 g/l, gives: (i) a mass concentration of Ca.sup.2+ of about 0.5 to about 10 g/l (ii) a mass concentration of K.sup.+ of about 1 to about 20 g/l, (iii) a mass concentration of Mg.sup.2+ of about 0.1 to about 2 g/l.

15. The sugar-depleted fruit or vegetable juice product of claim 1, wherein the sugar-depleted juice product has a pH of equal to or greater than about 3 and equal to or less than about 5.

16. The sugar-depleted fruit or vegetable juice product of claim 1, wherein the sugar-depleted juice contains Na.sup.+ at a mass concentration which, when said juice product is adjusted in volume with water to give a gluconic acid concentration of between about 5 g/l and about 100 g/l, gives a mass concentration of Na.sup.+ of no more than about 0.5 g/l.

17. (canceled)

18. A method for the preparation of a sugar-depleted fruit or vegetable juice product as defined in claim 1, said method comprising providing a fruit or vegetable juice product containing free glucose and/or sucrose and: (a) contacting said juice product with an enzyme which hydrolyses sucrose to glucose and fructose, (b) contacting the enzyme treated juice product of step (a) with an enzyme which converts glucose into gluconic acid, and (c) supplementing said juice product with one or more, preferably any two or three, of a source of Ca.sup.2+, a source of Mg.sup.2+ and a source of K.sup.+ in an amount sufficient to give said mass concentrations of Ca.sup.2+, Mg.sup.2+ and/or K.sup.+, respectively, wherein steps (a) and/or (b) may be performed simultaneously with step (c) or before or after step (c); or said method comprising providing a sucrose-depleted fruit or vegetable juice product containing free glucose and: (d) contacting said juice product with an enzyme which converts glucose into gluconic acid, and (e) supplementing said juice product with one or more, preferably any two or three, of a source of Ca.sup.2+, a source of Mg.sup.2+ and a source of K.sup.+ in an amount sufficient to give said mass concentrations of Ca.sup.2+, Mg.sup.2+ and/or K.sup.+, respectively, wherein steps (d) and (e) may be performed simultaneously or separately in any order; or said method comprising providing a sucrose-depleted fruit or vegetable juice product containing free glucose and said mass concentrations of any two or three of Ca.sup.2+, Mg.sup.2+ and/or K.sup.+, respectively, and: (f) contacting said juice with an enzyme which converts glucose into gluconic acid; or said method comprising providing a free glucose-depleted fruit or vegetable juice product, wherein said juice product contains sucrose and at least about 5 g/l gluconic acid and: (g) contacting said juice product with an enzyme which hydrolyses sucrose to glucose and fructose, and fh) supplementing said juice product with one or more, preferably any two or three, of a source of Ca.sup.2+, a source of Mg.sup.2+ and a source of K.sup.+ in an amount sufficient to give said mass concentrations of Ca.sup.2+, Mg.sup.2+ and/or K.sup.+, respectively, wherein steps (g) and (h) may be performed simultaneously or separately in any order; or said method comprising providing a free glucose-depleted fruit or vegetable juice product, wherein said juice product contains sucrose, at least about 5 g/l gluconic acid and said mass concentrations of any two or three of Ca.sup.2+, Mg.sup.2+ and/or K.sup.+, respectively and: (i) contacting said juice product with an enzyme which hydrolyses sucrose to glucose and fructose; or said method comprising providing a free glucose and optionally sucrose depleted fruit or vegetable juice product, wherein said juice product contains at least about 5 g/l gluconic acid and: (j) supplementing said juice product with one or more, preferably any two or three, of a source of Ca.sup.2+, a source of Mg.sup.2+ and a source of K.sup.+ in an amount sufficient to give said mass concentrations of Ca.sup.2+, Mg.sup.2+ and/or K.sup.+, respectively; or said method comprising providing a fruit or vegetable juice product containing free glucose and/or sucrose and said mass concentrations of any two or three of Ca.sup.2+, Mg.sup.2+ and/or K.sup.+, respectively, and: (k) contacting said juice product with an enzyme which hydrolyses sucrose to glucose and fructose, and (l) contacting the enzyme treated juice of step (k) with an enzyme which converts glucose into gluconic acid. wherein steps (k) and (l) may be performed simultaneously or separately; or said method comprising providing a fruit or vegetable juice product containing free glucose and optionally sucrose, and: (m) contacting said juice product with an enzyme which converts glucose into gluconic acid, and (n) supplementing said juice product with one or more, preferably any two or three, of a source of Ca.sup.2+, a source of Mg.sup.2+ and a source of K.sup.+ in an amount sufficient to give said mass concentrations of any two or three of Ca.sup.2+, Mg.sup.2+ and/or K.sup.+, respectively, wherein steps (m) and (n) may be performed simultaneously or separately in any order; said method comprising providing a fruit or vegetable juice product containing free glucose and optionally sucrose and said mass concentrations of any two or three of Ca.sup.2+, Mg.sup.2+ and/or K.sup.+, respectively, and: (o) contacting said juice product with an enzyme which converts glucose into gluconic acid.

19. (canceled)

20. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

26. (canceled)

27. The method of claim 18, wherein oxygen is supplied during one of more steps, preferably in the form of pure O.sub.2.

28. The method of claim 18, wherein said enzyme which converts glucose into gluconic acid is a glucose oxidase.

29. The method of claim 28, wherein the step of contacting the juice product with the glucose oxidase takes place in the presence of a hydrogen peroxide degrading enzyme, preferably catalase.

30. The method of claim 28 wherein the step of contacting the juice product with the glucose oxidase takes place at about pH 3-6, preferably about 3.4-5, about 3.6-4.8, about 3.6-4.6, or about 3.8-4.4.

31. The method of claim 18, wherein said source of Ca.sup.2+, Mg.sup.2+ and/or K.sup.+, are selected from salts, preferably halide salts, including fluoride, chloride, bromide, iodide salts; organic salts, including acetate, citrate, glutamate; oxides; hydroxides; sulphates; phosphates; nitrites; nitrates and carbonates, preferably an oxide and/or a hydroxide.

32. The method of claim 30, wherein said pH is controlled by one or more of the sources of Ca.sup.2+, Mg.sup.2+ and/or K.sup.+ defined in claim 31, preferably an oxide and/or a hydroxide.

33. (canceled)

34. A method for the preparation of food products, preferably purees, sauces and toppings, jams, jellies, fruit butters and spreads, dessert products, ice cream, juice based candies juice based gelatins, mixed juice products, smoothies, yoghurts, diabetic, low carbohydrate and low calorie products, dietary supplements containing juice, and wines and ciders, said method comprising the use of a sugar-depleted juice product of claim 1 as an ingredient in said food products.

35. A method of assisting in maintaining the health and well-being of a subject or for maintaining or promoting health and well-being in a subject, said method comprising the subject consuming a sugar-depleted juice product of claim 1.

36. A method for the treatment or prevention of a disease or condition associated with the over-consumption of glucose and/or sucrose and/or inappropriate metabolism of glucose, said method comprising administering a sugar-depleted juice product of claim 1 to a subject on a calorie-controlled diet.

37. The method of claim 36, wherein said disease or condition associated with the over-consumption of glucose and/or sucrose and/or inappropriate metabolism of glucose is selected from metabolic syndrome, diabetes mellitus type II, obesity, abdominal obesity, dyslipidaemia, insulin resistance, hyperinsulinemia, impaired glucose metabolism, hypertension, liver steatosis, steatohepatitis, hypertriglyceridemia, hypercholesterolemia, low HDL levels, high LDL levels, pancreatitis, neurodegenerative disease, retinopathy, nephropathy and neuropathy.

38. A method for increasing the absorption and retention of dietary minerals or the mineralisation of bone, said method comprising administering a sugar-depleted juice of claim 1 to a subject.

39. The method of claim 38, wherein said method treats or prevents perturbations, caused by insufficient absorbance or retention of dietary minerals, in blood coagulation, energy-yielding metabolism, muscle function, neurotransmission, digestive enzyme function, cell division and differentiation, development and maintenance of bones and teeth, blood pressure, the reduction of tiredness and fatigue, electrolyte balance, protein synthesis, psychological function, spermatogenesis, maintenance of hair and nails, immune system function, thyroid function, protection of DNA, proteins and lipids from oxidative damage, DNA synthesis, carbohydrate and macronutrient metabolism, cognitive function, fertility and reproduction, maintenance of serum testosterone concentrations, vitamin A metabolism, formation of red blood cells and haemoglobin, oxygen transport, and cognitive development of children.

40. The method of claim 38, wherein said method treats or prevents bone loss disorders, preferably osteoporosis and arthritis.

Description

[0209] The invention will be further described with reference to the following non-limiting Examples in which:

[0210] FIG. 1 shows the measurements of pH and glucose, sucrose, fructose and gluconic acid (g/l) taken during the experiment of Example 1. Invertase is added at 0 hr [101], Glucose oxidase/catalase and 1.25 g/l MgO is added at 12 hrs [102], 1 g/l MgO was added at 12 hrs 30 min [103], 0.2 g/l MgO was added at 13 hrs 50 min [104] and reaction was stopped at 14 hrs 15 min (finished product prior to packaging) [105]. Open squares: fructose; open triangles: free glucose; closed circles: gluconic acid; closed triangles: sucrose; crosses: pH.

[0211] FIG. 2 shows (A) incremental capillary blood glucose and (B) incremental venous plasma insulin levels before and after oral ingestion of 500 mL control apple juice without (control) and a sugar depleted apple juice product of the invention(verum) in 30 men with impaired fasting glucose (IFG). Least square means (LSM)±SEM.

EXAMPLES

Example 1

Preparation of a Sugar Depleted Apple Juice Product

[0212] An example of a preferred process and sugar-depleted juice product according to the present invention is exemplified by the following preparation of an apple juice product. Standard large scale apple juice production techniques were first used to produce an apple juice, which was then pasteurised and used in a method according to the present invention to produce a sugar-depleted juice product according to the present invention. Although the preparation of an apple juice product is exemplified herein, this could of course be modified to produce other juice products in accordance with the present invention.

Materials and Methods

[0213] A pilot scale production run with three batches has been carried out. Results are shown in FIG. 1 and Table 1. The apples used in the production were 17.5% Red Gravenstein (1400 kg) and the remainder Aroma (6600 kg). All the apples were bought from Sognefrukt BA, Sognefjordvegen 130, 6863 Leikanger. The juice was pressed from the apples and pasteurized. Then invertase (Maxinvert L4000, DSM) was added to 2000 l pasteurized apple juice (0.5 g, 5000 U/l) to split the non-reducing disaccharide sucrose into fructose and glucose over a 12 hour/overnight incubation at ca. 20° C. At the same time a commercially available pectinase was added at 30 g/1000 l, this is the same as standard apple juice production process. To regulate pH prior to glucose oxidase/catalase treatment magnesium hydroxide slurry (Mg(OH).sub.2), made from adding magnesium oxide (MgO) to water, was added until the pH of the juice was about 3.8-4.4. The enzymes glucose oxidase/catalase (as Hyderase L, Amano/Mitsubishi) were added to the juice (0.2 g/l, 3000 U/l) followed by oxygen added and maintained at a concentration in the juice of 3-4 mg/l. pH and oxygen were monitored (data not shown) throughout the glucose oxidase incubation via an automated computer controlled system. pH was maintained at 3.8-4.4 by batch addition of Mg(OH).sub.2 when necessary. A typical incubation time of 3-4 hours at room temperature is sufficient to reduce almost all glucose to gluconic acid. Glucose was monitored over the incubation period by measuring the glucose concentration in juice samples using a rapid and simple reflectometric based kit (Reflectoquant from Merck). The sucrose content after invertase incubation can also be measured in this way. Fructose and gluconic acid were determined via enzymatic assay (r-biopharm). Once most glucose had disappeared all enzyme activity was terminated by pasteurisation. The final product was referred to as a new apple drink, and is a sugar-depleted juice product in accordance with the present invention. A wide range of nutritional and chemical parameters of the finished and packaged products were also determined within 2 months. A reference apple juice (conventional juice) was also analysed simultaneously as a control, and it comprised a blend of equal parts of the same apple juice (1:1:1 by weight) used as the starting juice in each pilot scale production batch.

Results

[0214] FIG. 1 discloses the experimental results, and is a typical example of pH, concentration of glucose, sucrose, fructose and gluconic acid monitored during the enzymatic conversion process to make a new apple juice product in accordance with the present invention. The following main findings were shown:

[0215] The sucrose content was reduced to levels below the limit of detection by the action of invertase over a period of 12 hours at room temperature. The Limit of Detection is 1 g/L.

[0216] The glucose content was slightly increased by the action of invertase on sucrose over a period of 12 hours at room temperature and then significantly reduced to remove almost all glucose by the action of glucose oxidase/over a further 2-3 hour period. Thus the glucose content is decreased significantly from approximately 14 g/L in the conventional control juice to less than 3 g/L in the packaged juice product (mean value of 1.8 g/L over 3 batches tested).

[0217] The fructose content was slightly increased by the action of invertase on sucrose over a period of 12 hours at room temperature, and then remained constant during the rest of the production process. Thus, the fructose content in the packaged juice increased by approximately 20% (from approximately 58.2 g/L in the conventional juice) to a mean of 68.7 g/L over 3 batches tested. The extra fructose in the finished and packaged apple juice product may be desirable because it contributes to maintain sweetness.

[0218] The sum of sugars in the finished and packaged apple juice product (mean 70.6 g/L over 3 batches) is less than that in the conventional apple juice (96.6 g/L), equating to a reduction of 27%.

[0219] The D-gluconic acid content increased to a maximum in the finished product following the action of glucose oxidase/catalase on glucose. In this way nearly all the starting apple juice glucose was converted into gluconic acid in the apple juice product according to the present invention. In the 3 batch experiments, it was shown that the gluconic acid in the packaged juice product is present at a mean of 21.1 g/l compared to below detection limits in the conventional juice. This term “gluconic acid” is generic and represents all the equilibrium species of gluconic acid in an aqueous medium—e.g. lactones, gluconate salts forms, acid.

[0220] The magnesium content in the finished packaged juice product was increased to 1200 mg/kg (approximately equivalent to 1.2 g/l) compared to less than the detection limit (<50 mg/kg) in the conventional/reference apple juice.

[0221] The Vitamin C content in the finished packaged juice product was decreased from 0.625 mg/100 g in the reference apple juice to an undetectable level (>0.5 mg/100 g).

[0222] There was no significant difference between the reference apple juice and the finished packaged apple drink for a range of other chemical parameters e.g. amino acids, vitamins, essential and trace elements, nutrients etc.

[0223] It should be noted that the classical industry standard of Brix measurement of free sugar content (sucrose, fructose, glucose) in fruit juice is not appropriate for monitoring the production process according to the present invention because gluconic acid makes an almost identical contribution to the measurement as glucose. Further, the addition of magnesium salt also makes a small contribution to the Brix measurement.

[0224] Table 1 below shows analysis of sugar in the 3 production batches of packaged apple juice product according to the present invention, and compares this with conventional apple juice made from the same raw materials. The conventional apple juice meets the requirements for fruit juice laid down by Council Directive 2001/112/EC (Council of the European Union, 2001), and is a representative example of the “starting” material to the juice product according to the present invention.

TABLE-US-00001 TABLE 1 Sugar analysis of apple juice product Sugar-depleted juice product Untreated Parameter Units Batch 1 Batch 2 Batch 3 Juice Calculation on Sugars Sugar free extract g/l 55.0 47.1 48.3 15.1 (enzymatic) % sucrose % 0 0 0 26 Sum of sugars g/l 71.2 73.0 67.7 96.6 (enzymatic) Individual Sugars Glucose g/l 1.7 2.1 1.7 13.5 Fructose g/l 69.4 70.8 66.0 58.2 Sucrose g/l <6 <6 <6 24.9 Sugar Alcohol Sorbitol g/100 g 0.22 0.23 0.22 0.22

Example 2

Glycemic and Insulin Responses to Sugar Depleted Apple Juice Product I

[0225] Evidence for a beneficial effect of conversion of glucose to gluconate with the aid of glucose oxidase on postprandial glycemic response to juice is provided by comparing the reference food item of 400 ml apple juice (control) with the test product (400 ml apple juice treated in accordance with the invention) in overweight (BMI 25-30) adult human males. Testing follows the FAO/World Health Organization (WHO) guidelines.

[0226] Test products are administered in randomized order. The drinks are ingested within 5 min. Between the tests at least 2 days are interposed. At each visit the capillary blood glucose is measured 9 times over 2 hours by a Haemacue meter. Capillary blood collections and glucose measurements are performed during the two hour interval as the recommended technique to reduce the measurement errors. Blood is drawn twice at baseline and then at times 0 (start of drink), 15 min, 30 min, 45 min, 60 min, 90 min and 120 min.

[0227] Venous blood is drawn at baseline, times 0, 15, 30, 45, 60, 90 and 120 min for plasma insulin determination. The incremental AUCs obtained for each test meal are used for comparing the two meals. The 15 incremental AUC obtained for each test meal are used for comparing the two meals. AUC of glycaemic response to treated juice is expressed as percentage of AUC of the response to untreated juice.

Example 3

Glycemic and Insulin Responses to Sugar Depleted Apple Juice Product II

Introduction

[0228] This study was designed to assess postprandial glycemic and insulin responses to oral ingestion of a sugar depleted apple juice product of the invention and to directly compare those responses to those of the untreated apple juice. According to the European Food Safety Authority (EFSA) the reduction of post-prandial blood glucose responses may be considered a beneficial physiological effect (e.g. for subjects with impaired glucose tolerance) as long as insulin responses are not disproportionally increased and low postprandial glycemia is given priority in food choice according to the Food and Agriculture Organization of the United Nations and the World Health Organization.

Subjects and Methods

Design

[0229] This mono-center, double-blind, randomized, placebo-controlled, cross-over study was conducted in 30 male volunteers aged 18 years presenting with a diagnosed impaired fasting plasma glucose (IFG) of 5.6-6.9 mmol/L.

[0230] The study received approval from an independent ethics committee (The Ethical Committee of the Medical Council of Schleswig-Holstein, Bad Segeberg, Germany) and was conducted in line with the principles of the current version (2013) of the Declaration of Helsinki (WMA Declaration of Helsinki—Ethical Principles for Medical Research Involving Human Subjects adopted by the 18th WMA (World Medical Association) General Assembly, Helsinki, Finland, June 1964, and amended for the last time by the 64th WMA General Assembly, Fortaleza, Brazil, October 2013, the recommendations for Good Clinical Practice (ICH E6), and in accordance with European and National regulatory requirements.

Subjects

[0231] For enrollment the following inclusion criteria had to be fulfilled: Men aged 18 years diagnosis of IFG, signed Informed Consent Form. Exclusion criteria were: Current enrollment in another clinical study, enrollment in another clinical study within the last 4 weeks before inclusion, hypersensitivity, allergy or idiosyncratic reaction to apple, apple juice or other apple containing food, acute or chronic infections, renal insufficiency, gastrointestinal illness, history of gastrointestinal surgery, known fructose intolerance, overt diabetes mellitus, endocrine disorders, any disease or condition which might compromise significantly the hematopoietic, renal, endocrine, pulmonary, hepatic, cardiovascular, immunological, central nervous, dermatological or any other body system with the exception of the conditions defined by the inclusion criteria, history of hepatitis B and C, history of HIV infection, history of coagulation disorders or pharmaceutical anti-coagulation (with the exception of acetylsalicylic acid), regular medical treatment including OTC, which may have impact on the study aims (e.g. antidiabetic drugs, laxatives etc.), major cognitive or psychiatric disorders, subjects who are scheduled to undergo hospitalization during the study period, eating disorders (e.g. anorexia, bulimia) or special diets (e.g. vegan, vegetarian), present drug or alcohol abuse, legal incapacity.

[0232] The volunteers were free to withdraw from the study at any time without prejudice to their continued care. Specific reasons for discontinuing the study were defined as: Safety reasons as judged by the Investigator, development of specific exclusion criteria during the study, which have impact on subject's safety, incorrect enrollment or randomization of the subject, subject's wish to withdraw prematurely from the study, severe non-compliance to protocol as judged by the Investigator. Individuals withdrawing or discontinuing prematurely before finishing all study visits were supposed to be replaced, in order to have a complete set of 30 subjects having completed all study visits with exploitable results of the primary and secondary parameters.

Random Sequence Generation and Allocation Concealment

[0233] Volunteers were randomly assigned to either Verum (sugar-depleted apple juice product of the invention) or Control product (untreated apple juice). In order to avoid selection bias, the randomization scheme was generated by data managers in line with Cochrane guidelines. The randomization list was kept confidential at the premises of Nofima AS and remained confidential with the exception of those involved in product production and statistical managers (after the first part of data locking was performed).

Test Products and Blinding of Participants and Personnel (Table 1)

[0234] Control juice. Non-pasteurized conventional apple juice was purchased from Askim Frukt—og Brpresseri AS, 1815 Askim, Norway. The general apple juice production process includes: dumping, washing and grinding the apples, then pectinase treatment, cold pressing and separation. This control product contained 17 g/L free glucose and ca. 13.5 g/L glucose bound to sucrose (Table 2).

[0235] Verum. Sugar depleted apple juice was manufactured as follows. 95 litres of control apple juice was transferred to a kettle with, mixing, heating and cooling options (Proline Touch—Mix Kipgryde, Denmark). With lid the juice was warmed to 85° C. and held at this temperature for 5 min. The juice was then cooled down (running cold water in outer jacket of the kettle) until it was 24° C. Then invertase (Maxinvert L4000, DSM) was added (5000 U/l) to split the non-reducing disaccharide sucrose into fructose and glucose in a overnight incubation at room temperature (ca. 18-21° C.). At the end of the reaction (next morning) the content of sucrose was determined to be <0.1 g/l.

[0236] To regulate pH prior to glucose oxidase/catalase treatment calcium hydroxide and potassium hydroxide powder was added. The enzymes glucose oxidase/catalase (as Hyderase L, Amano/Mitsubishi) were added to the juice (3000 U/l) followed by pure oxygen to maintain a constant supply into the reaction tank of 3 mg/l. pH was maintained at 3.6-4.6 by batch addition of calcium hydroxide and potassium hydroxide powders when necessary. An incubation time of 12 hours at room temperature was sufficient to reduce almost all glucose to gluconic acid (remaining glucose<0.1 g/l; Table 2). Glucose was monitored over the incubation period by reflectometric based kit (Reflectoquant from Merck). The sucrose content after invertase incubation was also be measured in this way (0.0 g/l; Table 2). Gluconic acid was determined via enzymatic assay (R-Biopharm). All enzyme activity was terminated by shutting off the oxygen supply. The organoleptic properties were optimized by further addition of calcium and/or potassium hydroxide powder. The final pH of the sugar depleted drink was approximately 4. The final product was pasteurised in a KTM-Troxler (Germany) pasteur and bottled hot, corked, cooled and eventually stored in a fridge.

TABLE-US-00002 TABLE 2 Composition of control apple juice and sugar depleted apple juice Ingredient Apple Juice g/L Untreated (Control) Sugar depleted (Verum) Glucose 17 0.1 Fructose 65.2 86.3 Sucrose 26.7 Below detection limit Sugar* 109 86.4 Gluconic Acid Below detection limit 36.4 Calcium 0.032 1.5 Potassium 0.960 3.100

[0237] Verum and control were similar in flavour, color, texture, and appearance and identical in packaging throughout the study and coded by consecutive numbers in order to avoid performance bias by blinding both study participants and key study personnel including outcome assessors. Code-breaking systems were available in case an adverse event occurred and medical personnel needed to be aware of what the participant received: (verum or control product). Raw data were also blinded during the blind review. The code was broken after the database was locked.

[0238] Each study participant consumed 500 mL test product at the morning of the interventional day (Visit 1 and 2). The 500 mL bottle content was shaken well before opening and had to be ingested within 5 minutes.

[0239] The test products were provided by Nofima AS, Norway in a brown glass bottles and delivered to Kiel by courier service.The investigating site ensured that the study products were stored safely and properly according to the instructions given by Nofima AS and kept in a secured location to which only the investigator and designated study staff had access. The test products could be stored at room temperature with a shelf-life of two years after production. The shipment and dispensing of study products was recorded in a product accountability log. Monitoring of product accountability was performed by the quality manager after the visits and at the end of the trial.

Procedure/Conduct

[0240] Screening visit (V0). Prior to the inclusion procedure (for the assessment of eligibility of the subject), the subject was informed in detail by written information as well as verbally by the investigator about the study and was given the opportunity to ask the Investigator any questions. After signing and dating the Informed Consent Form by both the subject and the investigator subject's identity was verified, the subject's demographics and ethnics were documented, medical history was assessed, concomitant medication and alimentary supplements, smoking and alcohol use were documented, fasting for at least 12 hours was ascertained, vital signs (blood pressure, pulse) and anthropometric data (body weight, body height) were assessed, inclusion and exclusion criteria were assessed and blood samples for confirmation of the inclusion parameter IFG (impaired fasting glucose) were taken. Blood was drawn from the median cubital vein using a 21 G butterfly needle. Subjects were requested to appear the next visit after an overnight fasting of at least 12 hours and provided with a subject diary for daily reply regarding adverse event and medication and a questionnaire (EPIC FFQ) for recalling the food frequency with regard to the last 12 month, which had to be completed until the randomization visit.

[0241] Impaired fasting glucose (5.6-6.9 mmol/L resp. 100-125 mg/dL) was confirmed by two timely independent measures subjects are eligible for inclusion (one from the data base and one from V0).

[0242] Randomization and interventional visit 1 (V1). At V1 the study subjects were randomized and the first intervention was performed. This visit followed V0 within four weeks. Adverse events happening since V0 were documented. If the eligibility of the subject was confirmed by the investigator the subject was randomized. Fasting for 12 hour prior to this visit was checked and an intravenous catheter (Vasofix® Braunüle®, Braun Melsungen, Germany) was inserted into a forearm vein for blood withdrawal at baseline, directly before (time point 0) and 15, 30, 45, 60, 90 and 120 minutes after starting the ingestion of the test product. From all samples plasma insulin was measured. From the blood samples taken at baseline and 120 minutes after consumption of the test product safety parameters were determined (Na, K, Ca, Mg, AST, ALT, γGT, CHE, AP, LDH, CK, bilirubin, creatinine, urea-N, uric acid, complete blood count, cholesterol, HDL-C, LDL-C, triglycerides, CRP). Capillary blood was taken from the finger pad using a HemoCue® Safety Lancet at baseline (twice) and once directly before (time point 0) and 15, 30, 45, 60, 90 and 120 minutes after ingestion of the test product.

[0243] Arterial blood pressure, pulse and waist was assessed before and 120 minutes after ingestion. Subjects completed questionnaires on gastrointestinal symptoms (Gastrointestinal Symptom Rating Scale (GSRS)) directly before ingestion (time point 0) with regard to the last three days before the visit day (V1) and with regard to the last hour before starting ingestion. The GSRS was also assessed 60 and 120 minutes after ingestion with respect to the last hours, each. Stool frequency and stool form was assessed directly before ingestion (time point 0) with regard to the last three days before the visit day (V1) and with regard to the last two hours before starting ingestion and also 120 minutes after ingestion with respect to the last two hours. Satiety, hunger, fullness and prospective food consumption were monitored before (time point 0) and 30, 60, 90 and 120 minutes after ingestion using questionnaires.

[0244] Subjects were allowed to walk around, sit or lay down, but asked to abstain from eating or drinking or exercising during the test phase. The subjects were surveyed during the whole observation period at the test day and adverse events were monitored.

[0245] They were provided with a diary for daily assessment of adverse events and medication. GSRS, stool frequency and stool form were assessed during the three day lasting observation period starting with ingestion of the test drink at visit day V1 and two subsequent days.

[0246] Interventional visit 2 (V2): This visit was scheduled on the seventh day after V1 at the earliest. Subjects were requested to return their diaries and questionnaires. Adverse events happening since V1 were documented. Fasting for 12 hour prior to this visit was checked and the test was conducted as described for V1. Again they were provided with a diary for daily assessment of adverse events and medication. GSRS, stool frequency and stool form were assessed during the three day lasting observation period starting with ingestion of the test drink at visit day V1 and two subsequent days. Subjects received a stamped envelope and were requested to send back their diaries and filled questionnaires.

Assessments/Parameters

[0247] Primary target parameter. The incremental area under the curve (iAUC.sub.120) of the capillary blood glucose levels from baseline to 120 min after ingestion of the test drinks (according to FAO/WHO) was defined as primary parameter. Although capillary and venous blood glucose values have been shown to be highly correlated, capillary blood samples is regarded preferable to venous blood samples for reliable GI testing. Glucose was determined using a Hemocue 201 analyzer, Hemocue AB, Ängelholm, Sweden, which had been tested for glycemic index.

[0248] Secondary target parameter. The incremental AUC (iAUC.sub.120) of the plasma insulin levels from baseline to 120 min after ingestion of the test drinks was defined as secondary parameter. Insulin was determined using ELISA (LIASION® Insulin, Diasorin S.p.A, Saluggia, Italy).

[0249] Exploratory parameters: The incremental AUC (iAUC.sub.60) of glucose and insulin levels from baseline to 120 min after ingestion of the test drinks, the postprandial glucose peak G.sub.max, the amplitude between baseline and G.sub.max (G.sub.max−G.sub.baseline) and the maximal amplitude of glucose excursions (G.sub.max−G.sub.min) were calculated for further characterization of postprandial glucose response according to Brand-Miller (Brand-Miller et al, 2009, Am J Clin Nutr, 89: 97-105). Proportional reduction in glycaemic load (Liu, 2000) was calculated by 100-100 (iAUC.sub.120verum×CH.sub.verum)/(iAUC.sub.120control×CH.sub.control), whereby CH was carbohydrate (sugar) content of verum and control, respectively. Postprandial insulin sensitivity was expressed by ISI=2/[AUC insulin×AUC glucose+1] according to Belfiore (Belfiore F, et al., 1998, Mol Genet Metab; 63: 134-141; and Belfiore F., 2000, Diab Care; 23:1595). Satiety, hunger, fullness and prospective food uptake were assessed before and 30, 60, 90 and 120 min after ingestion of test drinks according to established questionnaires.

[0250] Plasma sodium, potassium, calcium, magnesium, AST, ALT, γGT, cholinesterase, alkaline phosphatase, LDH, CK, bilirubin, creatinine, urea-N, uric acid, cholesterol, LDL-C, HDL-C, triglycerides, CRP, complete blood count, blood pressure and pulse were measured before and 120 min after ingestion of test drinks.

[0251] Three days before and 3 days beginning with ingestion of the test drinks as well as 1 h before and the first and second hour after its ingestion the gastrointestinal symptoms using the GSRS, stool frequency and stool form using Bristol Stool Form Scale were assessed. Bristol Stool Form Scale was transformed by from 1, 2, 3, 4, 5, 6 and 7 to +3, +2, +1, 0, −1, −2 and −3 for expressing deviation from normal.

[0252] Adverse events were monitored throughout the total study period and their severity grade (mild, moderate, severe), relationship to the study products (suspected/not suspected), duration (start and end dates or if continuing at final examination), the action taken and its potential categorisation as serious adverse event (SAE) were documented.

Statistical Analysis

[0253] Estimation of sample size: Since iAUC data were not available from testing apple juice, sample size estimation was based on data reported by Johnston et al., 2003, Am J Clin Nutr; 78: 728-33. After a 25 g glucose load they found an iAUC=55.6±20.4 (mean±SD) of glycemic response. The glucose content of 500 mL apple juice was expected only about 7 (8.5) g, the content of fructose (29.1 (33) g, GI=19%) and sucrose (12.5 (13.5) g, GI=68%) summing up to an expected glycemic response (7+0.19×29.1+0.68×12.5=21.03)(8.5+0.19×33+0.68×13.5=24.0) of similar magnitude. We further assumed a reduction in glycemic response by at least 30% by enzymatic treatment of the juice. Assuming a reduction of iAUC by 16.7 and a SD=23.0 of the change and a power of 0.95, a sample size=27 was calculated for paired t-test. Taking the weaknesses of assumptions into account a sample size of n=30 was defined for the trial.

[0254] Preventing bias: In order to meet the recommendations of the Cochrane Collaboration for preventing detection bias blinding of outcome assessment was ensured by a blind review of raw data and by un-blinding only after data base was locked, and by conducting statistical analysis in compliance with the statistical analysis plan. In order to avoid attrition bias, distribution of the missing data across intervention groups and the magnitude compared to the effect size were assessed, and missing data were replaced by the Last Observation Carried Forward (LOCF) method using the last post-baseline value for one subject at the previous time. Reporting bias by selective outcome reporting was prevented by the availability of the study protocol and pre-specification of (primary and secondary) outcomes and by adhering to these specifications.

[0255] Definition of sets to be analysed: The Intention-To-Treat (ITT) collective was defined to comprise all subjects randomized and having taken at least one dose of the test products (intervention 1 at V1). The Per-protocol (PP) Set comprised all subjects randomized, who have no major protocol deviation.

[0256] Tests: The baseline and demographic characteristics of the two groups with different order of intervention (verum-control versus control-verum) were compared using Student t or Mann-Whitney test as appropriate depending on distribution of data. Verum and control were compared by repeated measures ANOVA, in order to take cross-over design and potential effects by the order of intervention into account. The significance level of the primary and secondary parameters was adjusted to multiple testing according to Bonferroni-Holm.

Results

Subject Characteristics

[0257] Distribution of volunteers through the study: N=51 subjects having had IFG in previous studies at the study site were screened for meeting inclusion criteria and for exclusion criteria. In N=19 IFG was not verified and in N=1 individual an allergy was reported which was not reported before. Thus N=20 subjects were excluded at screening and N=31 were enrolled. Between screening visit (V0) and randomization (V1) an erysipela occurred in N=1 individual. There were no drop-outs and no major deviations from study protocol. Thus the per protocol population (PP) was identical with intent-to-treat population (ITT) and N=30 individuals, who were supposed to complete all study visits according to the study protocol with exploitable results of the primary and secondary parameters (PP), actually completed the study.

[0258] Population characteristics at baseline (Table 3): The total population (ITT and PP) showed features of the metabolic syndrome. The baseline characteristics in the group with the order verum-control (VC) did not differ from those in the group with the order control-verum (CV).

TABLE-US-00003 TABLE 3 Population characteristics at baseline (Mean ± SEM) t-Test/ Mann- Total Group Group Order Group Order Whitney (n = 30) VC (n = 15) CV (n = 15) Test* Age [years] 68.0 ± 2.4 68.7 ± 1.4 67.4 ± 1.9 p = 0.602 Body Height 178.0 ± 1.8  177.9 ± 2.0  178.1 ± 1.5  p = 0.911 [m] Body Weight 100.2 ± 4.2  98.3 ± 3.3 102.0 ± 5.1  p = 0.542 [kg] BMI [kg/m.sup.2] 31.6 ± 1.2 31.1 ± 1.0 32.1 ± 1.5 p = 0.581 Waist [cm] 110.9 ± 2.3  110.3 ± 2.7  111.8 ± 3.7  p = 0..699 Syst. Blood 131.7 ± 3.9  129.3 ± 3.9  134.0 ± 3.8  p = 0.403 Pressure [mmHg] Diastol. Blood 81.8 ± 1.8 81.0 ± 2.0 82.7 ± 1.6 p = 0.524 Pressure [mmHg] Fasting Plasma 6.04 ± 0.1  6.0 ± 0.1 6.08 ± 0.1 * p = 0.467 Glucose [mmol/L] Fasting Plasma 166.8 ± 17.9 163.6 ± 22.0 169.9 ± 28.9 p = 0.864 Triglycerides [mg/dL] Fasting Plasma 49.3 ± 1.8 49.5 ± 2.4 49.2 ± 2.7 p = 0.927 HDL-C [mg/dL]

[0259] Characteristics of groups whereby those of the group with the order verum-control (VC) were compared with those of the group with the order control-verum (CV) by Student t test in case of normal distribution of data and by *Mann-Whitney test, if data were not normally distributed.

Composition of Test Products (Table 2)

[0260] By enzymatic treatment glucose and sucrose were mostly removed from apple juice by enzymatic treatment, whereas fructose increased after cleavage of sucrose by invertase. The sugar content in g/L was reduced by 21%. The pH-value was similar between verum and control after addition of potassium and calcium hydroxides to the enzymatically treated juice. Potassium and calcium content accordingly differed between verum and control.

Postprandial Glycemia (FIG. 2, Table 4)

[0261] The curves of capillary blood glucose levels after ingestion of the test drinks differed considerably between verum and control (FIG. 2B). The iAUC.sub.120 of glucose (primary parameter) differed significantly between verum and control. Similar differences were seen for iAUC.sub.60, glucose maxima, the postprandial increase from baseline and the maximal glucose excursion (Table 4). The order of intervention had no impact indicating that there were no significant carry-over effects (Table 4). By enzymatic treatment of apple juice glycemic response to its oral ingestion was significantly reduced by 68% resulting in a reduction of glycemic load by 74.9%.

TABLE-US-00004 TABLE 4 Glycemic and insulin response to apple juice without (control) and with enzymatic treatment (verum) Carry- verum Control Over Parameter (N = 30) (N = 30) V versus C p Effect p iAUC.sub.120 63.6 ± 12.3 198.0 ± 12.3  <0.001 0.806 Glucose [min × mmol/L] iAUC.sub.60 29.7 ± 5.3  108.0 ± 5.3  <0.001 0.945 Glucose [min × mmol/L] Gmax 6.97 ± 0.21 8.77 ± 0.21 <0.001 0.876 [mmol/L] Gmax-G 0.984 ± 0.141 2.796 ± 0.141 <0.001 0.701 base [mmol/L] Gmax-Gmin 1.157 ± 0.141 3.026 ± 0.141 <0.001 0.579 [mmol/L] iAUC.sub.120 2045 ± 285  3864 ± 285  <0.001 0.608 Insulin [min × mU/L] iAUC.sub.60 Insulin 739 ± 125 1603 ± 125  <0.001 0.401* [min × mU/L] ISI × 10.sup.6 32.2 ± 3.83 4.36 ± 3.83 <0.001 0.471 iAUC.sub.120 Glucose and iAUC.sub.60 Glucose express the incremental area under the curve of capillary blood glucose levels from ingestion to 120 and 60 min, respectively, after that. Gmax is the postprandial peak glucose level. Gmax-Gbase express the increase of glucose level from baseline to Gmax. Gmax-Gmin express the maximal glucose excursion. iAUC.sub.120 Insulin and iAUC.sub.60 Insulin express the incremental area under the curve of venous plasma insulin levels from ingestion to 120 and 60 min, respectively, after that. ISI = 2/[AUC insulin × AUC glucose + 1] after Belfiore. P was assessed by ANOVA RM; *Normality failed

Postprandial Insulin (FIG. 2, Table 4)

[0262] The curves of venous plasma insulin levels after ingestion of the test drinks differed considerably between verum and control (FIG. 2B). The iAUC.sub.120 (secondary parameter) differed significantly. By enzymatic treatment of apple juice insulin response to its oral ingestion was reduced by 47%. Similar differences between verum and control were seen for iAUC.sub.60 (Table 4). Postprandial insulin sensitivity as assessed by ISI differed, too (Table 4). The order of intervention had no impact indicating that there were no significant carry-over effects (Table 4).

Postprandial Safety Parameters

[0263] Plasma sodium, potassium, calcium, magnesium, AST, ALT, γGT, cholinesterase, alkaline phosphatase, LDH, CK, bilirubin, creatinine, urea-N, uric acid, cholesterol, LDL-C, HDL-C, CRP, complete blood count, blood pressure and pulse did not show clinically relevant changes and remained within the normal range 120 min after ingestion of test drinks.

Postprandial Satiety, Hunger, Fullness and Prospective Food Uptake

[0264] Satiety, hunger and prospective food uptake did not differ, neither in the fasting state nor postprandially. Fullness differed in the fasting state between verum (12.9±4.7) and control (24.7±4.7) (p=0.04; ANOVA RM), but no longer in the following, postprandial assessments.

Gastrointestinal Symptoms

[0265] GSRS: Gastrointestinal symptoms, as assessed by the Gastrointestinal Symptom Rating Scale (GSRS), did not differ 1 hour before ingestion of the drinks, neither the total score, nor any of the dimensions pain, reflux, indigestion, diarrhoea or constipation. Within the first hour after ingestion the total score was higher (p=0.028) in case of verum (1.140±0.038) compared to control (1.053±0.038) and the indigestion score was also higher (1.275±0.083) versus 1.117±0.083; p=0.008). During the second hour after ingestion no differences between verum and control were seen. This held true within the 3 days period beginning with ingestion of the test drinks.

[0266] Stool frequency did not differ between verum and control within the two hours before ingestion, but was higher (p=0.009) 2 hours after verum (0.567±0.123) compared to control (0.467±0.123). Accordingly stool form, as assessed by a transformed Bristol Stool Form Scale, was looser (p=0.002) after verum (−1.20±0.23) than after control (−0.67±0.23). Within the 3 days period beginning with ingestion of the test drinks no differences were reported, neither in stool frequency nor in stool form. In none of the volunteers diarrhoea as defined by WHO (three or more loose stools per day) occurred.

Discussion

[0267] By enzymatic treatment of apple juice its sugar content in g/L could be reduced by 21% and glycemic and insulin response to oral ingestion was significantly reduced by 68% and 47%, respectively resulting in a reduction of glycemic load by 74.9%.