阅读说明：本技术 用于借助于转化酶（ec 3.2.1.26）减少食物产品中果聚糖的方法 (Method for reducing fructan in food products by means of invertase (EC 3.2.1.26) ) 是由 彼得鲁斯·雅各布斯·西奥多罗斯·德克 于 2020-02-18 设计创作，主要内容包括：本发明涉及一种用于减少含果聚糖的食物产品中果聚糖的方法,包括向所述食物产品添加属于酶分类EC 3.2.1.26的转化酶,以及将所述食物产品与所述转化酶一起温育,其中所述含果聚糖的食物产品中的果聚糖被水解。本发明还涉及属于酶分类EC 3.2.1.26的转化酶在用于制备治疗患有肠易激综合征的人的药物或饮食补充剂中的用途。转化酶(EC 3.2.1.26)可以任选地与菊粉酶(EC 3.2.1.7)和/或β-果糖苷酶(EC 3.2.1.80)进一步组合。(The present invention relates to a method for reducing fructan in a fructan-containing food product, comprising adding to said food product an invertase belonging to the enzyme classification EC 3.2.1.26, and incubating said food product with said invertase, wherein fructan in said fructan-containing food product is hydrolyzed. The invention also relates to the use of a invertase belonging to the enzyme class EC 3.2.1.26 for the preparation of a medicament or a dietary supplement for the treatment of a person suffering from irritable bowel syndrome. The invertase (EC 3.2.1.26) may optionally be further combined with inulinase (EC 3.2.1.7) and/or β -fructosidase (EC 3.2.1.80).)

1. A method for reducing the amount of fructan in a fructan-containing food product, comprising adding to the food product an invertase belonging to the enzyme classification EC 3.2.1.26, and incubating the food product with the invertase, wherein fructan in the fructan-containing food product is hydrolyzed.

2. The method of claim 1, wherein the invertase is added in an amount of 2 to 2000SU per gram of food product.

3. The method of claim 1 or 2, wherein the fructan comprises fructan oligosaccharide with a degree of polymerization of DP3, DP4 and/or DP 5.

4. The method of claim 3, wherein at least 50% of the amount of the fructan oligosaccharide with DP3, DP4 and/or DP5 originally present in the food product is hydrolyzed.

5. The method according to any one of claims 1 to 4, further comprising adding inulinase EC 3.2.1.7 and/or fructan β -fructosidase EC 3.2.1.80 to the food product.

6. The method according to any one of claims 1 to 5, wherein the fructan-containing food product is a non-fermented food product, wherein the non-fermented food product has not been prepared with yeast and/or bacteria.

7. The method of any one of claims 1 to 6, wherein the non-fermented food product comprises pasta, noodles, cakes, biscuits, biscuit, pancake, snack, pretzels, corn chips, corn tortillas, low or no alcohol beer, malt beverages, onions, garlic.

8. The method according to any one of claims 1 to 7, wherein the food product comprises wheat, rye, barley or spelt.

9. The method according to any one of claims 1 to 8, wherein the fructan-containing food product causes less discomfort to a person with irritable bowel syndrome.

10. A fructan-containing food product obtainable by the method according to any one of claims 1 to 9.

11. A fructan-containing food product or an intermediate form of a fructan-containing food product, comprising an invertase belonging to the enzyme classification EC 3.2.1.26.

12. Use of a invertase belonging to the enzyme classification EC 3.2.1.26 for the preparation of a medicament or dietary supplement for the treatment of a person suffering from irritable bowel syndrome.

13. The use of claim 12, wherein the treatment comprises applying the formulation prior to consumption of a food product or simultaneously with consumption of a food product.

14. Use according to claim 12 or 13, further comprising the use of an inulinase belonging to enzyme class EC 3.2.1.7 and/or a fructan β -fructosidase belonging to enzyme class EC 3.2.1.80.

Technical Field

The present invention relates to a method for reducing levan in a food product.

Background

Fructans, such as inulin and levan, are present in several plants such as cereals (e.g. wheat, rye and barley) and in onions, garlic or artichoke. Fructans are considered as beneficial for health as prebiotics, but at the same time fructans such as those with a Degree of Polymerization (DP) of DP3 to DP5 and which are part of fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPS) are considered to cause symptoms in patients with Irritable Bowel Syndrome (IBS).

Several studies have been performed on the hydrolysis of fructans.

EP2271220B1 discloses a method for reducing fructans in dough or baked products using inulinase EC 3.2.1.7 and/or fructan β -fructosidase EC 3.2.1.80 in order to increase the softness of the baked products. Inulinases and fructan β -fructosidases disclosed in EP2271220B1 degrade fructans (inulin and levan) into short chain fructooligosaccharides (scFOS) and fructose.

Nilsson et al (1987), J.of Cereal Science 6,53-60 disclose invertase from Saccharomyces cerevisiae (Saccharomyces cerevisiae) hydrolyzing short chain fructans with DP3 to DP 5. Nilsson et al (1987), J.of Cereal Science 6,53-60 and Verspret et al (2013), J.Agric.food chem.61, 1397-1404 disclose that fructans are degraded by Saccharomyces cerevisiae, which shows invertase activity during dough fermentation. The amount of fructan that is hydrolysed depends on the type of yeast used.

EP 3266318 discloses the preparation of sugar-free bread from dough supplemented with inulin (fructan), inulinase (EC 3.2.1.7 and EC 3.2.1.80) and invertase (EC 3.2.1.26). The inulin (fructan) added to the dough is degraded into sweet sugars after the addition of all three enzymes and no additional sugars need to be added.

WO2017/220864 discloses an enzyme isolated from Lactobacillus crispatus capable of degrading fructan (SEQ ID NO: 1 in WO 2017/220864). The enzyme can be used in food products such as bakery products or fructans that include fructan-low vegetables that are suitable for low FODMAP diets.

There is a need for an improved method for reducing fructan in food products, such as non-fermented food products.

Disclosure of Invention

The present invention relates to a method for reducing the amount of fructan in a fructan-containing food product, comprising adding an invertase (β -fructofuranosidase) belonging to the enzyme classification EC 3.2.1.26 to the food product, and incubating the food product with the invertase, wherein the fructan in the fructan-containing food product is hydrolyzed.

Further disclosed herein is the use of an invertase in the manufacture of a medicament or dietary supplement for the treatment of a human with irritable bowel syndrome.

Detailed Description

The present invention relates to a method for reducing the amount of fructan in a fructan-containing food product, comprising adding an invertase (β -fructofuranosidase) belonging to the enzyme classification EC 3.2.1.26 to the food product, and incubating the food product with the invertase, wherein the fructan in the fructan-containing food product is hydrolyzed.

Surprisingly, it was found in the method as disclosed herein that by adding an invertase belonging to the enzyme class EC 3.2.1.26 and incubating the food product with the invertase in the method as disclosed herein, at least 50% of the amount of fructan with a Degree of Polymerization (DP) of DP3, DP4 and/or DP5 originally present in the food product and/or the intermediate form of the food product is hydrolyzed, preferably at least 60%, 70%, 80%, 90% or 95% of the amount of fructan with DP3, DP4 and/or DP5 originally present in the food product or the intermediate form of the food product is hydrolyzed, or all fructans with DP3, DP4 and/or DP5 originally present in the food product or the intermediate form of the food product is hydrolyzed. It was found that the fructan-containing food product produced in the process as disclosed herein surprisingly causes less discomfort to a person suffering from irritable bowel syndrome compared to a fructan-containing food product without the addition of the invertase EC 3.2.1.26.

The Degree of Polymerization (DP) is defined herein as the number of monomeric units. For example, a fructan with DP3 contains 3 units of monofructose.

Thus, the method as disclosed herein comprises reducing the amount of fructan with DP3, DP4 and/or DP5 originally present in the food product by at least 50%, preferably reducing the amount of fructan with DP3, DP4 and/or DP5 originally present in the food product and/or the intermediate form of the food product by at least 60%, 70%, 80%, 90% or 95%. The method as disclosed herein may comprise reducing all or 100% of fructan with DP3, DP4 and/or DP5 originally present in the food product and/or the intermediate form of the food product.

Examples of fructooligosaccharides with DP3 (fructan fraction) are 1-kestose, 6-kestose, and examples of fructooligosaccharides with DP4 are nystose, neokestose and/or secale bifida oligosaccharides, and examples of fructooligosaccharides with DP5 are different forms of nystose, such as fructosyl nystose.

Invertase may be added to a food product in the following amounts: 0.2 to 2500 SU/gram food product, e.g. 2 to 2000 SU/gram food product, e.g. 5 to 1000 SU/gram food product, such as 10 to 500 SU/gram food product, such as 20 to 400 SU/gram food product, such as 50 to 300 SU/gram food product, such as 100 to 200 SU/gram food product. The invertase activity was measured according to the invertase Semner Unit (SU) activity method described in the food products chemical code (FCC 6, American pharmacopoeia convention, Rockville MD., pp 1123-1124).

The invertase added to the food product as disclosed herein belongs to the enzyme classification EC 3.2.1.26 and is also referred to as β -fructofuranosidase or β -D-fructofuranosidase. The invertase may be from any suitable microorganism, such as a fungus, yeast or bacterium, e.g. from the genus aspergillus, e.g. aspergillus fumigatus, aspergillus niger, fusarium oxysporum or saccharomyces cerevisiae. The invertase as disclosed herein may be from saccharomyces cerevisiae. Commercial products containing the invertase EC 3.2.1.26 are available from DSM Food-Specialties, the Netherlands

The invertase may be added to the food product during preparation of the food product in any suitable manner, for example by mixing the invertase with suitable ingredients for preparing the food product. Adding the invertase to the food product may include adding the invertase to an intermediate form of the food product. The intermediate form of the food product may be a dough, batter or paste. For example, the invertase may be added to a premix or dough for preparing a baked product, or a batter for preparing a beer, such as a non-alcoholic or low-alcoholic beer.

A method for reducing the amount of fructan in a fructan-containing food product comprises preparing a food product as disclosed herein. For example, the method as disclosed herein comprises preparing a dough or batter and baking or drying the dough or batter at a suitable temperature depending on the food product to be prepared.

The dough is typically a mixture of (cereal) flour, water and optionally salt. Typically, the dough is firm enough to knead or roll. The dough may be fresh, frozen, prepared or baked. For fermentation products, baker's yeast is mainly used and optionally chemical fermentation compounds can be used, such as a combination of an acid (generating compound) and a bicarbonate. Within the scope of the present disclosure, fructan-containing grains that can be made into flour include wheat, rye, barley, or spelt. The term dough as used herein also includes batter. Batter is a semi-liquid mixture of one or more flours in combination with a liquid such as water, milk or eggs for preparing various foods including cakes, sufficiently thin to drip or pour from a spoon.

A premix as defined herein is a mixture of baking agents, typically comprising flour, starch, maltodextrin and/or salt, which can be used not only in industrial bread baking plants/facilities, but also in retail bakeries. The premix as disclosed herein may comprise an invertase belonging to the enzyme class EC 3.2.1.26. The premix as disclosed herein may comprise further enzymes such as lipase and amylase, and may also comprise inulinase belonging to the enzyme classification EC 3.2.1.7 and/or levan β -fructosidase belonging to the enzyme classification EC 3.2.1.80.

The invertase may be added at any suitable step during the preparation of a beer, such as a low or alcohol-free beer, which can be prepared by methods known in the art. Generally, processes for preparing beer (including low-alcohol and alcohol-free beer) include preparing a mash, separating wort from the mash, and boiling the wort. Preparing a mash in a method as disclosed herein may comprise preparing a mash from a flour of non-malted grain, from malted grain, or from a mixture of malted and non-malted grain. The grain as used herein may also be malt. Examples of cereals from which mash and subsequent beer can be prepared are barley and wheat.

Mashing, i.e. the process for preparing a paste, typically involves pausing (resting) at certain temperatures, e.g. at 43 ℃ to 51 ℃, 62 ℃ to 65 ℃, 72 ℃ to 74 ℃ and/or 77 ℃ to 78 ℃. After boiling the wort, the wort is usually filtered and yeast can be added in order to ferment the wort. For low alcohol beers, the wort may be fermented very quickly or in limited fermentations known in the art. The invertase may be added to the mash before or after the wort is boiled or during fermentation. The wort can be concentrated to a malt extract or dried. Dried malt extract can be used, for example, in malt beverages.

Incubating the food product with the invertase may comprise incubating the intermediate form of the food product with the invertase. The incubation may be performed in any suitable manner. Incubating the food product or the intermediate form of the food product with the invertase may comprise incubating the (intermediate form) of the food product with the invertase at: from 15 ℃ to 80 ℃, e.g. from 20 ℃ to 70 ℃, e.g. from 25 ℃ to 65 ℃, e.g. from 30 ℃ to 60 ℃, e.g. from 35 ℃ to 55 ℃, e.g. from 40 ℃ to 50 ℃. Incubation in the methods as disclosed herein includes incubation during 5 minutes to 24 hours, e.g., 10 minutes to 12 hours, e.g., 20 minutes to 8 hours, e.g., 1 hour to 6 hours. If invertase is added during mashing during the process for the preparation of beer, the incubation may comprise mashing.

Incubating the fructan-containing food product or the intermediate form of the fructan-containing food product in the method as disclosed herein can be performed at any suitable pH, e.g., a pH of 3 to 9, such as a pH of 4 to 8, such as a pH value of 5 to 7.

In one embodiment, the method as disclosed herein further comprises adding inulinase belonging to enzyme class EC 3.2.1.7 and/or levan β -fructosidase belonging to enzyme class EC 3.2.1.80 to the food product or to an intermediate form of the food product. These enzymes can be added and incubated in a similar manner as disclosed above for the invertase EC 3.2.1.26. Inulinase EC 3.2.1.7 and levan β -fructosidase EC 3.2.1.80 can be produced by fermentation of aspergillus niger. A commercial product comprising both enzymes is Fructozyme L available from Novozymes A/S.

In one embodiment, the fructan-containing food product as disclosed herein is a food product comprising a fructan-containing cereal material, e.g. a cereal material comprising rye, wheat, barley or spelt wheat, or other material comprising fructan, such as onion or garlic. The fructan-containing food product does not contain isolated inulin or fructan added separately.

The fructan-containing food product in the process as disclosed herein may be a non-fermented fructan-containing food product. In the non-fermented fructan-containing food product, no yeast and/or bacteria such as lactic acid bacteria are added to the fructan-containing food product or to an intermediate form of the fructan-containing food product, such as a dough or a batter. Thus, a non-fermented food product is a food product wherein no yeast and/or bacteria have been added during the preparation of the food product, or a food product comprising no added yeast and/or no added bacteria, such as lactic acid bacteria. Examples of non-fermented fructan-containing food products include pasta, noodles, cakes, biscuits, pancakes, snacks, pretzels, corn chips, corn tortillas, non-or low-alcohol beers, malt beverages, onions or garlic. The food product as disclosed herein may comprise rye, wheat, barley or spelt wheat.

Food products such as pasta, noodles, cakes, biscuits, biscuit, pancakes, snacks, pretzels, corn chips or tortillas are prepared according to methods known in the art and typically include the preparation of a dough or batter as disclosed hereinabove.

Surprisingly, it was found that the fructan-containing food product prepared in the process as disclosed herein causes less discomfort to a person suffering from irritable bowel syndrome than a food product prepared without the addition of and incubation with an invertase.

In one aspect, the present disclosure relates to a fructan-containing food product obtainable by a method as disclosed herein. Further disclosed herein is a fructan-containing food product or an intermediate form of a fructan-containing food product comprising an invertase belonging to the enzyme classification EC 3.2.1.26. The fructan-containing food product or an intermediate form of the fructan-containing food product may further comprise inulinase belonging to enzyme class EC 3.2.1.7 and/or fructan β -fructosidase belonging to enzyme class EC 3.2.1.80. The fructan-containing food product may be a non-fermented food product as disclosed above. All embodiments disclosed above for the method for reducing fructan in a fructan-containing food product apply to the fructan-containing food product as disclosed herein.

In one embodiment of the method as disclosed herein, the fructan-containing food product causes less discomfort to a person with irritable bowel syndrome.

In one aspect, the disclosure relates to the use of an invertase in the manufacture of a medicament or dietary supplement for the treatment of a human with irritable bowel syndrome. Invertase in the use as disclosed herein belongs to the enzyme class EC 3.2.1.26. In one embodiment, the use of an invertase as disclosed herein further comprises the use of an inulinase belonging to enzyme class EC 3.2.1.7 and/or a fructan β -fructosidase belonging to enzyme class EC 3.2.1.80. The pharmaceutical and/or dietary supplement prepared herein may comprise invertase EC 3.2.1.26 and inulinase EC 3.2.1.7 and/or levan β -fructosidase EC 3.2.1.80.

Treatment of a person with irritable bowel syndrome may be performed in any suitable manner, for example treatment including application of the agent prior to or simultaneously with consumption of the food product.

The medicaments and dietary supplements as disclosed herein may be prepared in any suitable form known to the person skilled in the art, for example in solid form such as tablets or pills or in liquid form.

Examples

Enzyme

200000MG, an invertase from Saccharomyces cerevisiae is obtained from DSM Food Specialties, the Netherlands.

Enzyme Activity assay

The invertase activity was measured according to the invertase Semner Unit (SU) activity method described in the food products chemical code (FCC 6, American pharmacopoeia convention, Rockville MD., pp 1123-1124).

Analysis and determination of fructans

Fructans with different Degrees of Polymerization (DP) in the supernatant of the extract were analyzed and quantified by pulsed amperometric detection (HPAEC-PAD) high performance anion exchange chromatography as described by Vergauwen et al (2000) J. Experimental Biology 51, 1261-1266. The column used was a Dionex CarboPac PA 1004X 250mm with CarboPac guard PA 1004X 50mm as protective column. The mobile phase was a (100% milliQ water); b (500mM NaOH); d (100mM NaOH +1M sodium acetate). The gradient applied is according to the following graph:

time (minutes)	Flow (ml/min)	％A	％B	％D
					-15	1	80	20	0
0	1	80	20	0
					15	1	64	16	20
20	1	0	0	100
					25	1	0	0	100
26	1	80	20	0

Fructan clearly separated from glucose, sucrose and maltose under these conditions. The fructan fraction is eluted from the column after:

1-kestose: 8.70 minutes

Neokestose: 10.19 minutes

Cane sugar tetrasaccharide: 10.39 minutes

Rye bifida oligosaccharide: 10.78 minutes

Other fructans DP 4: 10.8-11.5 minutes

Fructan DP 5: 11.5-12.1 min

Fructan DP 6: 12.1-13.0 minutes

Fructan DP 7: 13.0-14.5 minutes

Fructan DP 8: 14.5-16.0 min

Fructan DP 9: 16.0-17.2 minutes

Fructan DP 10: 17.2-17.9 min

Fructan > DP 10: 17.9-19.0

All fructans DP3 to DP10 and > DP10 were quantified relative to the area under the peaks for a known amount of 1-kestose.

Example 1: hydrolyzing fructan from wheat flour into glucose and fructose

Dough samples were prepared by mixing 1kg of common wheat flour with 500g of water and 10g of salt. The mixture was divided into 10 equal weight portions and 0, 2, 10, 20, 40, 100, 200, 400, 1000 or 2000SU was added per gram of dough200000MG (DSM Food specialties). The dough was kneaded into smooth balls. It was allowed to stand at 30 ℃ for 2 hours and dough samples were taken immediately after kneading and after 15, 30, 60, 90 and 120 minutes.

Soluble sugars were extracted from the dough samples using the method described in Verspret et al (J.Agric.food chem.2013,61, 1397-1404). Dough samples were immediately frozen using liquid nitrogen, freeze-dried and ground to a powder. The freeze-dried dough powder (50mg) was heated in ethanol (1.0mL, 90 ℃) until all the added ethanol was evaporated to inactivate the yeast invertase. The sample was extracted with hot water for 60 minutes (15mL, 80 ℃). The supernatant was collected for fructan analysis.

Example 2: hydrolysis of fructan in pasta dough

Dough samples were prepared by mixing 300 grams of Tipo '00' wheat flour, 300 grams of Farina di Semola and 6 large eggs, 6 grams of salt and 3 tablespoons of olive oil, and adding 2-2000SU/gOr no enzyme is added. Some water is added to the mixture if necessary. Knead into smooth dough in a kitchen machine for 5 minutes, cover the plastic foil and let it stand at room temperature for at least 30 minutes. Pasta is formed from the dough using conventional pasta machines. Fresh pasta is boiled in saline prior to consumption.

Example 3 hydrolysis of fructan in saccharification

Laboratory scale saccharification tests were performed in a saccharification bath (Lochner laboratory technik, Germany). In 200 ml of water80 grams of milled standard EBC malt was used. The paste was heated stepwise as shown in table 1. The paste was continuously stirred at 100rpm during the mashing process. At the end of saccharification, on a filter paper (Macherey-Nagel, MN 614)¹/₄320mm diameter) was filtered. The filtrate is called wort.

200000MG (2-2000SU/g) is added to the mash at the beginning of the preparation of the mash or to the wort before the wort is boiled.

Samples were taken from the wort and analyzed for the presence of fructan as disclosed above.

TABLE 1 saccharification protocol

Time from the beginning (minutes)	Temperature (. degree.C.)
		0	50
15	50
		28	63
43	63
		55	75
70	75
		73	78
78	78

The resulting wort can be used to make low or no alcohol beer in a limited time, e.g. 24 hours, e.g. at low temperature, e.g. below 10 ℃, preferably below 4 ℃, more preferably at about 0 ℃, e.g. in a fermentation limited process.

Example 4: effect of fructan hydrolysis on intestinal discomfort

A test panel of 20 volunteers each suffering from symptoms of IBS was tested. All members of the panel followed the low FODMAP diet prior to and during the test period. During the test period, the volunteers received a minimum of 2g fructan per serving of food or beverage with or without the dietary supplement comprising invertase.

In separate experiments, volunteers received food products or beverages containing a minimum of 2 grams of fructan per serving, or similar foods or beverages treated with invertase according to the preparation method as disclosed in examples 1 to 3.

The amount of hydrogen (ppm) in the exhaled breath was measured every hour up to 8 hours after a meal. Various gastrointestinal symptoms (pain, abdominal distension, flatulence, diarrhea, nausea, etc.) were recorded during this period.

During the first period, volunteers received untreated or enzyme treated food daily. After the washing period, they receive food that has not been received in the first period. Volunteers will complain less about enzyme treated foods than untreated foods.

Example 5: hydrolysis of fructans in dough

Wheat flour (Ibis: Meneba, cartes, netherlands) was used to make the dough. First, 100% flour, 2% salt and 62% water, plus 0, 2, 20, 200 or 2000SU/g flour of Maxinvert 200000MG were kneaded by hand and then for 6 minutes using a Hobart mixer at speed 2. The kneaded dough was divided into pieces of-10 grams each and stored in centrifuge tubes. All tubes were incubated at 30 ℃ for 15, 30, 60 or 120 minutes and the dough was then frozen by the addition of liquid nitrogen. Each frozen dough was freeze-dried and ground to a powder. To extract the soluble sugars in the dough powder, 200 mg of the powder was incubated in 10 ml of water at 90 ℃ for 60 minutes. 5ml samples were centrifuged at 14000rpm for 5 minutes, then the supernatant was filtered through a 0.2 μm filter and the filtrate was used for fructan detection with HPAEC-PAD as described above.

The total fructan content of wheat flour measured using the HPAEC-PAD method in this experiment was-0.70% (w/w). The results in table 2 show that incubation of flour with invertase (Maxinvert) reduced all fructan content by 90%. No additional fructan peaks were evident during the incubation period, indicating complete conversion of fructan to monosaccharide. The residual material after incubation at the area of the peak of neokestose can represent non-fructan oligosaccharides that resist the effect of Maxinvert.

Example 6 hydrolysis of fructan in pasta dough

Durum wheat semolina produced by De Cecco (De Cecco Semola di grano duro rimarina) is used for producing pasta dough. 100% flour, 40% whole egg, 8.3% olive oil, 16.7% water, plus 0, 2, 20, 200 or 2000SU/g flour in Maxinvert 200000MG were first kneaded by hand and then kneaded using a Hobart mixer at speed 2 for 4 minutes. The kneaded dough was divided into dough pieces of-10 grams each and stored in a centrifuge tube. All tubes were incubated at 22 ℃ for 30 minutes and the dough was then frozen by the addition of liquid nitrogen. Each frozen dough was freeze-dried and ground to a powder. To extract the soluble sugars in the dough powder, 200 mg of the powder was incubated in 10 ml of water at 90 ℃ for 60 minutes. 5ml samples were centrifuged at 14000rpm for 5 minutes, then the supernatant was filtered through a 0.2 μm filter and the filtrate was used for fructan detection with HPAEC-PAD as described above.

The total fructan content of wheat flour measured using the HPAEC-PAD method in this experiment was-0.85% (w/w). The amount of fructan (DP3-6) was clearly higher in the pasta flour and the high DP fructan was less abundant compared to the wheat flour used in the experiment of example 5.

The results in table 3 show that incubation of flour with invertase (Maxinvert) reduced the content of all fructans by as much as 94%. No additional fructan peaks were evident during the incubation period, indicating complete conversion of fructan to monosaccharide. Minute (min)

Example 7: hydrolysis of fructan in malt beverages

Commercial malt beverages (Supermalt-Royal Unibrew A/S) were incubated with 0, 0.4, 4, 40, 400 SU/ml Maxinvert 200000MG at 22 ℃ for 30 minutes. After incubation, the enzyme was inactivated by heating 2 ml of the solution at 90 ℃ for 30 minutes. The liquid was filtered through a 0.2 μm filter and used directly for fructan detection via HPAEC-PAD as described above. The results in Table 4 show that when malt beverages are incubated with 400 SU/ml invertase (Maxinvert), a 98-99% reduction in fructan 1-kestose and nystose is obtained. The elution profile of the larger fructan in malt beverages differs from that of examples 5 and 6 in wheat flour. This may be because the primary grain used to produce malt beverages is barley.

Table 4: fructan content of supernatant incubated with varying amounts of Maxinvert for 30 minutes