HK1128035A - Long-chain inulin - Google Patents

Description

Long-chain inulin

The application is a divisional application of Chinese patent application 200680011892.9 entitled "long-chain inulin" filed on 12.4.2006 by the inventor.

Technical Field

The invention relates to long-chain inulin and a method for the production thereof from artichoke roots, the use thereof in foodstuffs and cosmetic preparations, and foodstuffs and cosmetic preparations containing long-chain inulin.

Background

Inulin is a polysaccharide belonging to the family of fructans. Consists of beta-2-1 linked fructose molecular chains, and has alpha-D glucose units at the reducing end of the chains. In many plants, such as chicory roots and dahlia tubers, inulin is present in economically exploited amounts. In addition, the presence of inulin has also been found in e.g. Jerusalem artichoke and artichoke. The average chain length of different inulins and their physiochemical properties vary from plant species to plant species.

When inulin is extracted from plant tissues with hot water, the extract liquid contains monosaccharides such as glucose and fructose, disaccharides such as sucrose and fructooligosaccharides (DP 3-10) in addition to the polymerized crude inulin. These by-products, mono-, di-and fructo-oligosaccharides (DP 3-10), interfere with the further processing of the inulin. For example, monosaccharides and disaccharides are not required in the production of dietary food. The sweet taste of mono-, di-and fructo-oligosaccharides (DP 3-10) may interfere with some applications in the food industry. Due to its hygroscopicity and stickiness, fructooligosaccharides (DP 3-10) greatly interfere with the use of crude inulin in foodstuffs during handling and storage. In the further processing of the crude inulin, for example by chemical derivatization, mono-, di-and fructo-oligosaccharides (DP 3-10) lead to the formation of undefined product mixtures which cannot be purified or can only be purified in an expensive manner.

It is therefore desirable to provide inulin with a lower content of mono-, di-and fructo-oligosaccharides (DP 3-10) compared to crude inulin.

The invention is therefore based on the object of providing inulin with novel properties.

The embodiments identified in the claims provided achieve this object.

Disclosure of Invention

The invention relates to the average degree of polymerization DP_wInulin between 54 and 61, preferably between 55 and 60, particularly preferably between 56 and 57.

For this reason and in connection with the present invention, the term "between" is also intended to include the numerical boundaries indicated respectively.

The term "inulin" in connection with the present invention refers to polyfructose consisting of beta-2-1 linked fructose molecule chains. The chain preferably has a reduced alpha-D glucose unit at the end. The degree of branching of the fructose units linked by beta-2, 6-linkage is less than 5%, preferably less than 2%.

The term "average degree of polymerization DP" in connection with the present invention_w"(average DP weight) means the weight average molecular weight M_wWith monomer molecular weight M_oThe quotient thereof. Weight average molecular weight M_wIs obtained from

Wherein Ni is M having a molecular weight_iThe number of molecules of (c).

In connection with the present invention, the "average degree of polymerization DP" is measured_w"gel permeation chromatography with light scattering and refractive index detection (GPC-RI-MALLS system)" described hereinafter is preferably used.

The inulin of the invention also exhibits the unexpected advantage over inulin previously described in the art that it can be processed into emulsions, exhibits a very high stability under heat treatment or acid treatment, and is therefore more suitable for particular industrial applications or for applications in the cosmetics and/or food industry, for example. Furthermore, emulsions comprising the inulin of the invention show an unexpectedly high stability to shear forces. The inulin of the invention therefore exhibits further advantages over conventional inulin, allowing better processing in industrial processes with strong shearing forces.

Furthermore, the inulin of the invention also outperforms outstanding excellent viscosity properties and high gel strength.

The inulin of the invention also shows a slower fermentation compared to the products which are currently advantageous for the prevention of distal colon diseases.

Furthermore, the inulin of the invention shows a stronger prebiotic effect than the products currently used. In particular, the inulin of the invention stimulates the production of bifidobacteria in an advantageous manner while also reducing unwanted and/or pathogenic bacteria. The inulin of the invention is therefore suitable for use in food and/or pharmaceutical products for the prevention and treatment of intestinal dysfunction and diseases, in particular of the distal colon.

Finally, the inulin of the invention also imparts advantageous use properties to certain food products, such as, for example, viscosity building, emulsifying capacity, water holding capacity and crumb formation.

Drawings

FIG. 1 shows the molar mass distribution analysis of different inulins;

FIG. 2 shows the typical melting behavior of different inulins with excess water.

Detailed Description

In a further embodiment, the inulin of the invention has a fructooligosaccharide (oligofructan) content with a DP of between 3 and 10 of less than 3%, preferably less than 1.5%, particularly preferably less than 0.7%, very particularly preferably less than 0.3%.

In connection with the present invention, fructooligosaccharide contents with a DP of between 3 and 10 are measured by the following methods (see conventional methods: "analysis of inulin by ion exchange chromatography and pulse amperometric detection (HPAEC-PAD)", and "determination of the percentage content of inulin oligomers with a chain length of DP ═ 3 to DP ═ 10 in the total inulin" by HPAEC-PAD ").

In a further embodiment, the inulin of the invention has a glucose content of less than 2%, preferably less than 1%, particularly preferably less than 0.5%, very particularly preferably less than 0.2%.

In a further embodiment, the inulin of the invention has a fructose content of less than 2.5%, preferably less than 1.5%, particularly preferably less than 1.0%, very particularly preferably less than 0.3%.

In a further embodiment, the inulin of the invention has a sucrose content of less than 2%, preferably less than 1%, particularly preferably less than 0.5%, very particularly preferably less than 0.3%.

In an embodiment of the inulin of the invention which is particularly advantageous for food applications, the composition of mono-and disaccharides is below 0.5%.

All percentages are percentages based on the total weight of the dry mixture of inulin and other substances, unless otherwise stated.

In connection with the present invention, the fructose, glucose and sucrose contents were measured by the following optically enzymatic method (conventional method: "determination of sugar").

In a further embodiment, the inulin of the invention has a weight-average molecular weight M_wBetween 8740g/mol and 9890g/mol, preferably between 8910g/mol and 9720g/mol, particularly preferably between 8910g/mol and 9250 g/mol.

In connection with the present invention, the weight average molecular weight M is preferably measured by the method "gel permeation chromatography with light scattering and refractive index detection (GPC-RI-MALLS system)" described hereinafter_w。

In a further embodiment, the inulin of the invention has an average degree of polymerization DP, as measured using Gel Permeation Chromatography (GPC)_n(GPC)Between 44 and 48, preferably between 45 and 48, particularly preferably between 46 and 48.

In connection with the present invention, it is preferred to measure the "average degree of polymerization DP" by the "gel permeation chromatography with light scattering and refractive index detection (GPC-RI-MALLS System)" method described hereinafter_n”。

In connection with the present invention, the term "average degree of polymerization DP_n"(average DP number) number average molecular weight M_nWith bound monomers M_oQuotient of molecular weight (anhydrofructose 162 g/mol). Number average molecular weight derived from

Wherein Ni is M having a molecular weight_iNumber of molecules (2)Amount of the compound (A).

In a further embodiment, the inulin of the invention has a molecular weight distribution of between 1620g/mol and 40000 g/mol, preferably between 2268g/mol and 32000 g/mol, particularly preferably between 2592g/mol and 29160 g/mol.

In connection with the present invention, it is preferable to measure the distribution of molecular weight by the method of "gel permeation chromatography with light scattering and refractive index detection (GPC-RI-MALLS system)" described hereinafter.

The average degree of polymerization DP can also be determined by acid hydrolysis with perchloroacetic acid (PCA, equivalent to trichloroacetic acid-TCA)_n. When this method is used, the inulin of the present invention has an average polymerization degree DP_n(PCA)Between 48 and 56, preferably between 48 and 52. The hydrolysis method using PCA was carried out as described in the following examples.

Furthermore, the object of the present invention is to produce aqueous pastes with the inulin of the invention, which can be obtained by the following procedure: dispersing inulin in water, shearing the obtained dispersion to homogeneity, storing the obtained product at 4-15 deg.C for 12-24h, adjusting to room temperature, and stirring to obtain uniform paste. Preferred pastes contain 5-40 wt.%, more preferably 5-30 wt.%, and especially preferably 10-20 wt.% inulin, based on the total weight of the paste.

The inulin of the present invention exhibits unexpectedly high acid stability against acids. In particular, the inulin-formed aqueous paste of the invention exhibits high stability against acids. The viscosity of the aqueous inulin paste of the invention after storage for 2 weeks at pH4 was increased by less than 10% compared to the initial value of the viscosity. Preferably, the increase in viscosity is less than 7%, most preferably less than 5%, respectively, compared to the initial value of viscosity.

The initial value of viscosity is the value of viscosity exhibited by the paste after dispersion of the inulin in water, storage at 4-15 ℃ for 12-24 hours, conditioning to room temperature and stirring to a homogeneous smooth paste. Details regarding the production of the paste are described in the working examples.

The values of acid stability are measured under the following conditions: room temperature, rotational viscometry (Rotovisco VT 550), diagonal blade stirrer, 128 revolutions per minute, measurement time 15 minutes, inulin concentration in water preferably 10-30% w/v (1% w/v. 10 g/l), particularly preferably 15% w/v. Detailed descriptions of the methods are given in the examples. The above-mentioned low increase in viscosity (after thickening) is advantageous for the use of inulin in foodstuffs at acidic pH.

The aqueous inulin paste of the invention also outperforms shear stability compared to commercially available products. After 15 minutes of shearing, the viscosity value of the aqueous inulin paste is still higher than 85%, preferably higher than 90%, most preferably higher than 95% of the initial value. The paste is obtained by the method described above. The values for shear stability were determined under the following conditions: room temperature, rotational viscometry (RotoviscoVT 550), diagonal blade stirrer, 128 revolutions per minute, measurement time 15 minutes, inulin concentration in water preferably 15% w/v (1% w/v ═ 10 g/liter). Detailed descriptions of the methods are given in the examples. In the manufacture of food and cosmetic products, high shear stability is advantageous, especially in processes with stirring.

The inulin of the invention shows unexpectedly high gel strength compared to other commercially available inulins. The inulin has a gel strength of 30-100N, preferably 40-100N, more preferably 50-100N, after dissolving inulin in water at 90 deg.C and cooling to room temperature for more than 20 hours. The gel thus obtained has particle-like characteristics (particulate gel). The method of determining gel strength is described in detail in the working examples.

Furthermore, the inulin of the invention exhibits unexpected viscosity properties in the dissolved state in water. Inulin in water at a concentration of 30% w/v at a measurement temperature of 90 ℃ and for 20s^-1At a shear rate (CVO120HR Bohlin/Malvern rheometer, cone plate geometry), a viscosity of 300-. The details of the measurement method are described with reference to the method in the additional embodiment.These values are higher than what is expected to be achieved with inulin known today. This high viscosity value at relatively low concentrations is particularly advantageous in food applications.

In addition to the inulin of the invention described above, the invention also relates to compositions comprising the inulin of the invention described above and one or more edible or pharmaceutical ingredients. Representative compositions include human and animal foods, beverages, functional foods, pharmaceuticals and pharmaceutical compositions (including prophylactic and therapeutic compositions), and intermediates thereof.

According to the present invention, functional foods refer to foods containing ingredients that can provide health-beneficial effects in addition to conventional nutrients (according to the definition of national academy of sciences medical research institute (USA; 1994)).

The edible or pharmaceutical ingredients mentioned are preferably selected from the group consisting of sugars (e.g.glucose, fructose, sucrose, lactose, galactose, maltose, isomaltulose), polyols (e.g.sorbitol, lactitol, maltitol, isomalt, mannitol, xylitol), maltodextrin, sweeteners, hydrogenated glucose syrups, food and feed additives, food and feed intermediates, food and feed products, edible liquids, beverages, bioavailable mineral sources, pharmaceutically acceptable excipients, substances with pharmaceutical and therapeutic activity, pharmaceutical compositions and medicaments.

Particularly preferred compositions of the invention comprise the co-presence of the inulin of the invention with an edible or pharmaceutically acceptable, bioavailable mineral source, in particular a calcium and/or magnesium and/or iron source, such as dairy products, salts and complexes of calcium, magnesium and iron.

As mentioned before, it is an object of the present invention to provide inulin having outstanding beneficial properties when applied in foodstuffs, wherein the terms foodstuff and foodstuff are equivalent according to the present invention. Furthermore, the present invention therefore also relates to foods and dietary supplements containing the aforementioned inulin. According to the invention, the term food comprises food for humans and food and feed for animals, respectively. Dietary supplements include those for humans and animals.

Particularly preferred, the food is selected from dairy, yogurt, ice cream, dairy-based smoothies, whipped cream (dairy toppings), puddings, shakes, custards, cheese, nutritional bars, energy bars, breakfast bars, preserves, baked bread, crackers, cookies, biscuits, cereal chips (grain chips), assorted dried fruits (trail mix), iced tea granules (ice tea mix), fruit juice smoothies, weight control beverages (weight management drink), ready-to-drink beverages, sports drinks, endurance drinks, powdered mix drinks (supple powder prepared drink mix), infant formula, high calcium orange juice, bread, croissants, cereal products, pasta products, bread spreads, sugar-free and chocolate, lime chews, meat products, sauces, salad dressings, nut butter, frozen entrees, mayonnaise, soup and ready-to-eat meals.

In one embodiment of the invention, the food is a food product made by an extrusion process, such as cereal.

In a further aspect the invention relates to a cosmetic preparation comprising the aforementioned inulin. Particularly preferred cosmetic preparations are emulsions, in particular for application to the skin and the face.

In another aspect the invention relates to the use of the aforementioned inulin as a supplement in foods, functional foods and cosmetic preparations. In particular, the use also relates to all the specific food and cosmetic preparations mentioned above.

In another aspect, the invention relates to the use of the inulin of the invention for the preparation of a pharmaceutical composition or a medicament.

The inulin of the invention may be advantageously used in foodstuffs, functional foodstuffs, pharmaceutical compositions or medicaments for modifying or modulating the bacterial flora composition in the colon, in particular in the distal part of the colon, of a human, mammal or other vertebrate.

The inulin of the invention may also be used in foodstuffs, functional foodstuffs, pharmaceutical compositions or pharmaceuticals for modifying or regulating the fermentation pattern of inulin in the colon, in particular in the distal part of the colon, of humans, mammals or other vertebrates.

Finally, the inulin of the invention may be used in foods, functional foods, pharmaceutical compositions or pharmaceuticals, giving the following beneficial effects: dietary fiber effects, modulation of intestinal function, prebiotic and/or bifidogenic effects, increased absorption of minerals such as calcium, magnesium and iron, increased bone mineral density, increased bone mineral content, increased peak bone mass, improved bone structure, reduced loss of bone mineral density, reduced loss of bone structure, modulation of lipid metabolism, stimulation of the immune system, prevention and reduction of cancer risk, prevention and reduction of colon cancer, reduction of risk of colon cancer and prevention of breast cancer.

The invention will be illustrated by the following examples, but is not intended to limit the general inventive concept.

Examples

Conventional methods

1. Inulin purification and fractionation

1kg of one-age artichoke roots with a diameter of 0.5-5.0cm was used for the preparation. The starting material was homogenized in 200g portions with 500ml of water at 85 ℃ in a homogenizer (Waring Blender, VWR International GmbH, Darmstadt, Germany). The resulting homogenate was collected in a metal container, water-washed at 85 ℃ with the extraction volume increased to 5l of water, and inulin extracted from the material was stirred at 80-85 ℃ for 45 minutes. The hot extract was filtered through a 125 μm sieve. The filter cake was transferred to cotton and drained. The squeezed solution was mixed with the filtrate (about 5 liters in total).

Removing non-saccharides in the crude inulin extract by lime clarification and carbon dioxide saturation purification. In the pre-lime clarification, by reaction with Ca (OH)₂Stirring, and adjusting pH of the raw material extractive solution to 11.2. The extract was kept at this pH for 30 minutes, the temperature of the extract being 65 ℃. For the main lime clarification process, Ca (OH) is added₂Raising the pH to>12, stirring the extract at 85 ℃ for 30-45 minutes. To make it possible toThe precipitated haze is more easily filtered by rapid CO injection during the first carbon dioxide saturation step₂The pH of the extract was lowered to 10.8. The temperature in this step was about 65 ℃. The precipitate formed was removed by centrifugation (2800g, 10 min). In the second carbon dioxide saturation step, by introducing CO₂The pH of the supernatant was lowered to pH 8.9. The precipitate formed was removed from the extract by centrifugation (2800g, 10 min) and filtered through filter paper using a vacuum filter funnel (Schleicher)&Schuell, order number 10311614).

In a batch process, the extract was decolorized by gradually adding 10-15% (w/v) ion exchanger (mixed bed resin TMD8, Sigma, order number M8157) with stirring. After removing the ion exchanger by filtration through a 30 μm sieve, the solution was adjusted to pH 7.0 with KOH. For selective concentration of longer chain inulin polymers (DP >10), absolute ethanol was added to the solution to a final concentration of 40% (v/v), mixed well and incubated overnight at 8 ℃. The inulin precipitate was sedimented by centrifugation (2800g, 10 min). The inulin precipitate was washed twice with 1.6 l of 80% (v/v) ethanol. The washed inulin was dissolved in 1 liter of water in a water bath at 95 ℃ and the suspended matter was removed by filtration through a 30 μm sieve, the inulin solution was frozen at-80 ℃. The moisture was then removed by freeze drying (Alpha 1-4, by Christ, Germany) and the dried inulin powder was isolated.

2. Measurement of fructan

2.1 Fructosan assay protocol

The fructan content of a sample can be determined using the "fructan assay protocol" kit (by Megazyme International Ireland Ltd, Wicklow, Ireland). The principle of this analysis is based on the hydrolysis of fructan into its reducing monomers glucose and fructose and the photometric determination of the reducing sugar (glucose, fructose) content (wavelength 410nm) after development by the so-called "PAHBAH method" (the details of the method are described later).

In the first step, the sucrose present in the extract is hydrolyzed into glucose and fructose using a specific enzyme sucrase. The starch and maltodextrin in the extract is then degraded to glucose using a mixture of highly purified enzymes beta-amylase, pullulanase and maltase. The resulting reducing sugars are then removed from the solution after reduction to sugar alcohols by treatment with an alkaline borohydride solution. Dilute acetic acid was added to neutralize the solution and remove excess borohydride. The fructan is then hydrolysed to fructose and glucose using purified levanase (exoinulinase) and the monosaccharide content produced is determined by the PAHBAH method.

Chemistry and solutions in the "fructan assay protocol" kit:

1. a lyophilized powder containing 50U of sucrase (yeast), 500U of β -amylase (Bacillus cereus), 100U of pullulanase (K.pneumoniae) and 1000U of maltase (yeast) was dissolved in 22ml of 0.1M sodium maleate buffer solution (hereinafter referred to as "enzyme 1") having a pH of 6.5 for measurement.

2. A freeze-dried powder containing 8000U of levanase (exoinulinase) was dissolved in 22ml of 0.1M sodium acetate buffer (hereinafter referred to as "enzyme 2") at pH4.5 for measurement.

3. A standard fructose solution (1.5mg fructose/ml) dissolved in 0.2% benzoic acid.

4. Fructosan control powder

Dahlia fructan with known fructan content, was freeze-dried together with alpha-cellulose.

Solution not in kit:

PAHBAH reagent

Solution A:10 g of PAHBAH (. rho. -hydroxybenzoic acid hydrazide, Sigma order number H-9882) were placed in a 250ml beaker, 60ml of distilled water were added, and 10ml of concentrated hydrochloric acid was added to the suspension with stirring. The solution was made up to 200ml and stored at room temperature.

Solution B: 24.9g of trisodium citrate, then 2.20g of calcium chloride and finally 40g of sodium hydroxide are addedGradually dissolved into 500ml of distilled water with stirring. After addition of sodium hydroxide, the solution may be milky, but becomes clear after 2l of water. The solution was stored at room temperature.

Before use, 20ml of solution a was added to 180ml of solution B and mixed well (═ PAHBAH reagent). The solution must be stored on ice and used within 4 hours.

II.50mM sodium hydroxide solution

Basic sodium borohydride solution

10mg/ml sodium borohydride in 50mM sodium hydroxide (Sigma, order number S-9125)

IV.100mM acetic acid

The detection method comprises the following steps:

1.(A) Fructosan control

20mg of fructan control powder was extracted in a 95 ℃ heating block with 1ml of double distilled water for 30 minutes. After centrifugation (13,000 Xg, 5 min), the supernatant was transferred to a new reaction vessel, and the precipitate was added again to 1ml of distilled water and extracted in a 95 ℃ heating block for 30 min. After centrifugation again (see above), the supernatant was removed and mixed with the first supernatant.

(B) Purified fructan/inulin

20mg of 1h were extracted in a 95 ℃ heating block with 2ml of double distilled water. After centrifugation (13,000 Xg, 5 min), the supernatant was transferred to a new reaction vessel for measurement.

Mu.l of the sample was mixed with 200. mu.l of enzyme 1 and incubated at 40 ℃ for 60 minutes (incubation time 30 minutes longer than the megazyme protocol).

3. 200 μ l of alkaline sodium borohydride solution was added, the solution was mixed well and incubated at 40 ℃ for 30 minutes to achieve complete conversion of the reducing sugar to sugar alcohol.

4. Excess borohydride was removed, 500 μ l of 100mM acetic acid was added and the solution was adjusted to pH4.5 with thorough mixing.

5. The extract from purified fructan/inulin (non-fructan control) was diluted 1:5 with double distilled water. Then 100. mu.l of each solution from the mixture and the fructan control was mixed with 100. mu.l of 100mM sodium acetate buffer, pH 4.5.

6.200 μ l of the solution was mixed with 100 μ l of enzyme 2 and incubated at 40 ℃ for 60 minutes (incubation time was 40 minutes longer than the megazyme kit to achieve complete hydrolysis of fructan).

7. A fructose standard was used as another sample. 200 μ l of standard fructose solution in the kit was treated with 900 μ l of 100mM sodium acetate buffer at pH4.5 and mixed. Then, 4X 200. mu.l of this mixture was mixed with 100. mu.l of 100mM sodium acetate buffer solution having pH 4.5.

8. All samples and additional blank samples (300 μ l pH4.5, 100mM sodium acetate buffer) were mixed with 5ml PAHBAH reagent and incubated precisely for 6 minutes in a boiling water bath.

9. The sample was immediately cooled in cold water (10-15 ℃) for about 5 minutes.

10. The absorption of the blank sample by all solutions was measured with a spectrophotometer at a wavelength of 410 nm.

The calculation is made according to the following formula:

fructan (% w/w) (. DELTA.E.times.Fx.5. times.V)_Ex×1.1/0.2×100/W×1/1000×162/180

PAHBAH uptake of samples measured for blank samples

Factor converting fructose absorption to fructose per μ g fructose (54.5 μ g fructose/absorption)

Factor for changing 200. mu.l to 1ml incubation volume

V_ExPrecise volume

1.1/0.2 ═ 0.2ml from 1.1ml of enzymatic digest

W-exact sample weight in mg

Coefficient of 100/W-expressed fructan as% of initial weight (W)

1/1000 μ g to mg

162/180 ═ coefficient for conversion of measured free fructose to anhydrofructose bound in fructans

2.2 measurement of fructan by exoinulase hydrolysis

1% (w/v) of the material was extracted with double distilled water at 95 ℃ for 30 minutes and then diluted with water (see above) 1: 25. For the exoinulase digestion (100. mu.l), 50. mu.l of the extract were incubated for 3 hours at 40 ℃ in 25U of sodium acetate pH 5.60.1M containing exoinulase (Megazyme International Ireland Ltd, Wicklow, Ireland, trade name E-EXO 1). The reaction was terminated by incubation at 95 ℃ for 10 minutes. The released glucose and fructose were determined photometrically as described in the "sugar determination" method. The fructan content was determined by adding the glucose and fructose content, and calculating the factor 162/180 (converting the measured free hexose sugars to the hexose sugars bound in the fructan).

3. Sugar determination (glucose, fructose and sucrose)

The glucose, fructose and sucrose content was determined by enzymatic analysis of the conversion of NAD + (nicotinamide adenine dinucleotide) to NADH (reduced nicotinamide adenine dinucleotide). The nicotinamide ring loses its aromatic character during the reduction reaction and the absorption spectrum changes. Changes in the absorption spectrum can be detected photometrically.

Glucose and fructose are converted to glucose-6-phosphate and fructose-6-phosphate by hexokinase and Adenosine Triphosphate (ATP). Then 6-phosphoglucose is oxidized into 6-phosphogluconate by 6-phosphoglucose dehydrogenase. NAD + is reduced to NADH in this reaction, and the total amount of NADH formed is measured photometrically. NADH is formed in a ratio of 1:1 to glucose present in the extract, so that NAD can be usedMolar extinction coefficient of H (6.3l mmol)^-1cm^-1) The glucose content was calculated from the NADH content according to Lambert-Beer's law.

After the oxidation of glucose-6-phosphate is completed, fructose-6-phosphate produced in the same manner in the solution is converted into glucose-6-phosphate by glucose-phosphate isomerase and then oxidized into gluconic-6-phosphate. The ratio of fructose to the amount of NADH formed was also 1: 1. The fructose content was calculated from the total amount of NADH formed as described for glucose.

Then, sucrose present in the extract was decomposed into glucose and fructose by sucrase (manufactured by Megazyme). The enzyme converts the released glucose and fructose molecules into 6-phosphogluconate by an NAD + dependent reaction. One molecule of sucrose is converted to 6-phosphogluconate to produce 2 molecules of NADH. The amount of NADH formed was likewise measured photometrically and the sucrose content was calculated using the molar extinction coefficient of NADH.

4. Molecular weight distribution analysis

4.1 gel permeation chromatography with light scattering and refractive index detection (GPC-RI-MALLS System)

Inulin/fructan was dissolved in water at a concentration of 0.5% (w/v). The solution was heated at 95 ℃ for 10 minutes. The polymer was analyzed with the following instrument: PL120 gel chromatography (Polymer Laboratories, Germany), Midas autosampler (Spark, Holland), equipped with lambda₀A 16-channel detector and K5 flow cell 690nm and an angle ranging from 14.9 ° to 162.9 ° and combined with a viscosity-refractive index detector η -1002(WGE dr&Co KG, Germany) of the DAWN-EOS light Scattering Detector (Wyatt Technology Santa Barbara, USA). The polymer was fractionated on the following columns: TSK pre-column, TSK6000PW, TSK3000PW (TosohBioscience GmbH Stuttgart, Germany). 100 μ l of the solution was injected. Fractionating at 30 deg.C with a flow rate of 0.8 ml/min and an eluent of 0.3M NaNO₃. The molecular weight distribution of the samples in GPC-RI-MALLS-MALLS was analyzed using the program Astra4.90.08 (Wyatt Technology Santa Barbara, USA).

4.2 determination of DP of inulin by hydrolysis with PCA_n

Inulin was completely hydrolyzed with perchloroacetic acid (PCA). The average chain length number (DP) of the inulin samples was determined from the ratio of fructose produced and glucose produced_n)。

Sample preparation:

50.0+/-5.0mg of inulin were accurately weighed into a 1ml flask. 700. mu.l of H was added₂O_bidestAnd (4) dissolving. The sample was then agitated to keep the sample material as far as possible off the bottom of the vessel and then placed in a boiling water bath (-99 ℃). During incubation, the flask was agitated every 30 seconds. After incubation, the sample was cooled to room temperature and then H was added₂O_bidestTo 1ml scale mark. The inulin concentration of the sample solution was 5.0 +/-0.5%. 200 μ l of the remaining ice was frozen to-20 ℃ for determination of sugars before hydrolysis. Before sugar measurement, the sample was thawed at room temperature, mixed, dissolved in a 95 ℃ heating block with 1400 rpm stirring for 5 minutes, and centrifuged at 4000 rpm for 2 minutes.

Hydrolysis and sample recovery:

250 μ l of a 5% inulin solution have been prepared in 250 μ l of 18% PCA (final inulin concentration 2.5%, PCA 9%). Alternatively, 900. mu.l of a 5% inulin solution have been prepared in 100. mu.l of 5% PCA (final inulin concentration 4.5%, PCA 0.5%). Mix at 4000 rpm and centrifuge the hydrolysis mixture for 1 minute. The hydrolysis mixture was then placed on a heater (heating block) at 37 ℃ or 56 ℃. At different time points, the hydrolysate was mixed again and after 1 minute at 4000 rpm separation, 100. mu.l of sample was recovered and immediately neutralized by adding the neutralization mixture of aqueous NaOH. The pH of the neutralized sample was checked with a pH indicator (pH paper). The time points for recovering the samples were 50 minutes, 2 hours, 3 hours, 4 hours and 24 hours. All neutralized samples were frozen at-20 ℃. Before measuring the sugar, the samples were thawed at room temperature, mixed and centrifuged at 4000 rpm for 2 minutes. Add 90. mu. l H to 10. mu.l of neutralized hydrolyzate₂O_bidestA1: 10 dilution was made for fructose measurements.

Measurement of sugars

To determine the fructose and glucose released in the hydrolysis in all samples, photometric glucose and fructose measurements were used. In the samples before hydrolysis, sucrose was measured in addition to glucose and fructose. Measurements were repeated in microtiter plates using a SPECTRAmax luminometer (Molecular Devices). All enzyme solutions used were prepared in a measurement buffer containing 50mM imidazole-HCl pH 6.9, 2.5mM MgCl₂1mM ATP and 0.4mM NADP. The conversion of NADP to NADPH was observed at 340 nm.

To measure the sugars before hydrolysis, undiluted 5% inulin solution was used. Mu.l of this solution was added to 200. mu.l of measurement buffer. Glucose was measured by adding a mixture of 2. mu.l hexokinase (from yeast, 0.3U/. mu.l) and glucose-6-phosphate dehydrogenase (from yeast, 0.14U/. mu.l). After complete conversion of glucose, fructose was measured by adding 2. mu.l of phosphoglucose isomerase (from yeast, 0.14U/. mu.l). Once the conversion of fructose is complete, an additional 2. mu.l of beta-fructosidase (3U/. mu.l) is added to cleave the existing sucrose.

Glucose and fructose were measured using the method described in item 3 (determination of sugars) above.

And (3) calculating:

in the calculation, the molar extinction coefficient of 6.23 l.m.mmol-1 cm-1 was used as the basis for the calculation of the conversion of NADP into NADPH. The concentration of glucose and fructose in the hydrolysate minus the concentration of glucose and fructose already present before hydrolysis. Likewise, glucose and fructose released from the hydrolysis of sucrose present in the sample prior to hydrolysis are also subtracted therefrom. The concentrations of fructose and glucose produced during hydrolysis of inulin are thus obtained. Thus, the number average chain length (DP) can be calculated according to the following formula_n)：

DP_n＝(c_Fructose/c_Glucose)+1

Here, it is assumed that each inulin molecule has only one terminal glucose.

As a control of the completeness of the hydrolysis, the recovery was determined indirectly by the concentration of glucose and fructose produced and the mass of inulin applied.

5. Analysis of inulin by ion exchange chromatography and with pulsed amperometric detection (HPAEC-PAD)

The mixture of inulin multimers was fractionated by anion exchange chromatography and pulsed amperometric detection was carried out using a DIONEX system (GP50 gradient pump, AS50 autosampler, model 585 column oven, ED50 detector, CarboPac PA-100 pre-column, CarboPac PA-100 separation column, manufactured by DIONEXcorporation, Germany). The temperature of the column furnace was 30 ℃. Eluent A was 150mM NaOH and eluent B was 1M sodium acetate in 150mM NaOH. The flow rate was 1 ml/min.

A0.5-2% (w/v) inulin solution was prepared with double distilled water and incubated at 95 ℃ until the inulin was completely dissolved.

The ED50 detector waveform has the following composition:

time (second) potential (V)

0.00 0.05

0.280.05 Start of integration

0.480.05 end of integration

0.49 0.65

0.60 0.65

0.61 -0.96

0.72 -0.96

0.73 0.05

The following gradient was used to maximize the fractional separation of inulin multimers:

gradient 1:time (min) eluent A (%)Eluent B (%)

(90 min) 01000

5 100 0

40 72 28

70 55 45

75 0 100

80 0 100

85 100 0

90 100 0

The chromatograms were analyzed using the DIONEX Chromeleon (version 6.6) software.

6. The percentage content of inulin oligomers having chain lengths of DP ═ 3 to DP ═ 10 in the total inulin was determined by HPAEC-PAD

The proportion of inulin multimers having chain lengths between DP3 and DP10 was determined by anion exchange chromatography and amperometric detection (see method 6) with varying detector settings and different salt gradients.

A2% (w/v) aqueous solution was prepared with purified artichoke inulin. Each anion exchange chromatography separated 100. mu.l of this solution with gradient 2, without activation of the ED50 detector, and the isolated oligofructans were collected after running the column. Four separations were carried out with a 2% strength inulin solution.

Gradient 2:time (min) eluent A (%) eluent B (%)

(50 min) 01000

5 100 0

35 76 24

37 0 100

42 0 100

45 100 0

50 100 0

The collected fractions were neutralized with acetic acid (pH 7), incubated with anion exchanger (TMD8 mixed bed resin, Sigma, order number M8157) for 5 minutes in batch processing at room temperature and desalted with shaking. The anion exchanger in the solution was removed by centrifugation and filtration through a 0.2 μm sieve (ultrafree-MC, amicon, order No. UFC 30 LG 25).

The fractions were frozen and concentrated in a vacuum concentrator to lyophilization. The corresponding fractions of the four runs were mixed in a total volume of 250. mu.l of deionized water.

To determine the ratio of the different oligomers, the individual fractions were hydrolyzed with exoinulinase into glucose and fructose. The fractions were diluted as needed. Mu.l of the fraction solution were digested with 0.25U of exoinulinase (Megazyme, order number E-EXO1) for 3 hours at 37 ℃. The reaction was terminated by incubation at 95 ℃ for 10 minutes. After cooling, the solution was filtered (ultrafree-MC, amicon, order No. UFC 30 LG 25). Mu.l of the filtrate was fractionated by ion exchange chromatography using a gradient 2 detector with the following waveform:

time (second) potential (V)

0.00 0.05

0.200.05 Start of integration

0.400.05 end of integration

0.41 0.75

0.60 0.75

0.61 -0.15

1.00 -0.15

The HPAEC-PAD system was calibrated with glucose and standard fructose solutions in the concentration range of 6. mu.M to 30. mu.M. The concentrations of glucose and fructose released in the fractions were determined with the aid of this calibration (in. mu. mol/l).

To calculate the percentage content of oligomers DP3-DP10 in total inulin, the total glucose and fructose released by the corresponding oligomers were divided by the weight of purified artichoke inulin (2% strength solution).

7. Determination of Water content

The water content was determined using AQUA 40.00 Karl-Fischer titrator (produced by Analytikjena AG). Using Hydranal-Coulomat AG (Order No. 34836) as an anolyte. Disodium tartrate dihydrate with water content of 15.61-15.71%, (Order number 32323) as a reference. 10-20mg of the sample was weighed into a 5ml sample bottle (N20-5DIN, Macherey-Nagel, order No. 70204.36), which was closed with a crimped cap (N20 TS/oA, Macherey-Nagel, order No. 702815), and the water content of the sample was determined with a Karl-Fischer titrator.

8. Preparation of inulin paste

10.5g of inulin (dry matter) were dispersed into 59.5ml of water in a 150ml glass beaker or citrate buffer at pH4.0 with an IKA RW16 basic stirrer (IKA AWerke GmbH und Co. KG., 79219 Staufen, Germany) driving a small stirrer (three rings, 2cm wide, 8.6cm long) at 6-7 steps. The suspension was then transferred into 250ml measuring cylinders (diameter 35mm, height 160mm) and sheared at 24000 rpm for 3 minutes using an Ultra-Turrax T25(T25 basic disperser) (IKA Werke GmbH und Co. KG., 79219 Staufen, Germany). The contents of the container are not cooled. The inulin paste was then transferred to a 100ml glass beaker, covered with a glass lid and stored in a refrigerator at 13 ℃ overnight. The inulin paste was left at room temperature for 1 hour before each subsequent measurement step. Then, the mixture was stirred with a long blade stirrer (2-blade stirrer on a shaft 1cm thick; blade width 1.4cm, blade length 5.9cm) for 5 minutes until smooth under a drive of 100 rpm with an IKA RW16 basic stirrer (IKA Werke GmbH und Co. KG., 79219 Staufen, Germany). Immediately after this, the pastes were transferred to the respective measuring containers.

9. Determination of shear stability of inulin paste

The shear stability of the aqueous inulin paste was measured with a Rotovisko VT550 viscometer (formerly Thermo Haake GmbH, now Thermo Electron GmbH, manufactured by 63303 Dreieich, Germany) at a measurement temperature of 20-22 ℃ in a measuring cup (diameter 42 mm; height 93mm) with a pitched blade stirrer rotating at 128 rpm. For this purpose, the inulin paste was stirred in a Rotovisko for 15 minutes and the paste viscosity was measured at the beginning (curve top) and at the end of the stirring period under each condition. The viscosity at the end of the stirring treatment (Visc) was then calculated₂) Viscosity at the beginning (Visc)₁) To determine the shear stability:

shear stability [% ]]＝Visc₂/Visc₁＊100

10. Determining the acid stability of inulin pastes

Inulin paste in citrate buffer at pH4 was prepared for determination of acid stability. Measurement ofThe stability in the acid matrix is similar to the determination of the shear stability, i.e.using a Rotovisko VT550 viscometer at a measurement temperature of 20-22 ℃ and a 128 rpm pitched-blade stirrer in a measuring cup (diameter 42 mm; height 93 mm). The inulin paste was stirred in a Rotovisko for 15 minutes, and the viscosity of the paste was measured at the beginning and end of the stirring period under each condition. The viscosity at the end of the stirring treatment (Visc) was then calculated₂) Viscosity at the beginning (Visc)₁) To determine the acid stability:

acid stability [% ]]＝Visc₂/Visc₁＊100

11. Determining the thermal stability of an inulin paste

The thermal stability of the inulin paste was determined with a DSR rheometer (formerly Bohlin Instruments GmbH, 75181 Pforzheim, Germany; from 10/2004 thereafter: Malvern Instruments GmbH, 71083Herrenberg, Germany) with the following settings:

the measurement system comprises: awl (4 degree/plate

Rate: 0.1Hz

Strain: 0.001

Initial pressure: 0.6Pa

Temperature range: 30 ℃ -90 ℃ (1 ℃/min)

The temperature at which the gel viscosity is less than 2Pas is defined as the dissolution temperature of the inulin paste.

12. Differential scanning calorimetry of inulin

Each 3g portion of inulin (dry matter) was weighed into 50ml graduated polypropylene bottles (30.0X 115mm, manufactured by Greiner, order number 227261). 18ml of double distilled water was added to each powder and shaken. All suspensions were dissolved by shaking several times in a water bath (95 ℃). After 20 minutes it was determined by eye that all suspensions had dissolved completely. Then, the solution was made up to 20ml according to the scale of the polypropylene tube to form a 15% strength solution (w/v).

Then, the hot solution was immediately put in its entirety into a Petri dish (100X 20mm, manufactured by Greiner, order No. 664102) and lid-opened and dried at 37 ℃ for 2 days. The dried material obtained was then transferred to a mortar and ground for 2-3 minutes. The powder was then granulated in a hammer mill (MM 300 from Retsch) with an additional apparatus, consisting of steel balls with a diameter of 20MM, at a frequency of 30 Hz for 30 seconds. The powder was then transferred to a closable container for DSC measurement.

The water content of the samples was determined using an automated Karl-Fischer titrator (see conventional method 7).

For DSC measurement, about 10mg inulin dry matter is weighed into a stainless steel pan (volume 50. mu.l), the exact weight is determined, and 30. mu.l distilled water is added. The disk was then sealed. An empty stainless steel pan was used as a reference. The sample was heated from 10-160 ℃ in a DSC apparatus (Perkin Elmer; Diamond) with an autosampler at a heating rate of 10 ℃/min. Data analysis was performed using the PYRIS7.0 software program (Perkin Elmer, 63110 Rodgau-Jugesheim, Germany). Under the present conditions, T was measured₀(onset) and free enthalpy dH.

13. Measurement of viscosity

Inulin solution at 90 ℃: viscosity versus concentration:

the inulin amount was made up with the corresponding volume of distilled water (weight per volume) to produce an aqueous suspension of inulin. The resulting suspension was dissolved by heating in a water bath at 95 ℃ with constant stirring.

Measurements were made using a CVO120HR Bohlin/Malvern rheometer using an isothermal (90 ℃) viscosity measurement mode on the lamina system CP4 °/40 mm. The shear rate of 10/sec was used as pre-shear for 60 seconds, plus a relaxation time of 10 seconds. Shear is measured using a log scan type procedure of shear rate mode. The initial shear rate was 20/sec, the final shear rate was 30/sec, the rising waveform was 20 sec duration and 10 sec integration time. At 20s^-1Data are collected at the shear rate of (a).

14. Determination of gel Strength and viscoelastic Properties

The measuring cup MV of a Haake Rotovisco VT550 viscometer is charged with 70g of a 10-25% by weight aqueous inulin (distilled) suspension. Thereafter, a paddle stirrer was placed and set as a pre-heating (90 ℃, mantle) device. Then, the mixture was heated for 15 minutes under stirring at 128 rpm.

After 15 minutes, it was transferred into a container consisting of a base and two acrylic glass cylindrical rings (20 mm high each, 30mm diameter) placed one on top of the other and fixed with adhesive tape (width 19 mm). The sample was loaded in the container to a level below about 5mm up to without air bubbles. The container was then sealed with aluminum foil and left overnight at room temperature (23 ℃).

After storage at room temperature (23 ℃) for about 20 hours, the strength of the gel was measured with Texture Analyser TA XT 2. To measure gel strength on a smooth, non-drying surface, the adhesive tape that ties the two cylindrical surfaces of the container together was first removed. The gel was then cut with a blade along the space between the rings, thereby presenting a smooth surface on the lower portion of the gel.

Gel strength was measured by penetration of a Texture Analyser TA XT2 into the gel (depth 1mm) by a planar swivel (24.5mm diameter). The Texture Analyser is set as follows:

measurement principle: applying force in the direction of pressure

Front speed: 2 mm/sec

Testing speed: 2 mm/sec

Trigger value: 0.01N

Return speed: 2 mm/sec

Distance: 1mm

The maximum value of the Calotte one-time permeation is given in Newton.

Viscoelastic behaviour after cooling to 25 ℃

Aqueous inulin solutions of different concentrations were prepared according to the method already described in the viscosity determination (see above) and then cooled. The cooling rate was kept at 1K per minute at all times by automatic temperature control. The frequency sweep was started immediately after 25 ℃. The measurements were performed on the lamina system CP4 °/40mm using an isothermal (25 ℃) vibration mode of a CVO120HR Bohlin/Malvern rheometer. No pre-shear force is applied. The stress was measured with a 10 second duration logarithmic sweep type procedure. During the ramp, the starting frequency was 0.100Hz and the final frequency was 5,000Hz 0/sec. The stress was automatically adjusted from σ to 0.100Pa to γ to 0.010 (assuming deformation).

15. Fermentation study

Stool sample:

10 donors of stool samples were selected with reference to the following exclusion criteria:

lack of antibiotic treatment within the last 6 months

No continuous treatment with any kind of drug except for oral contraceptives

No discomfort for one week before donation

Absence of diarrheal symptoms one week before donation

Nutrition without special treatment meals

No special selection of food for weight loss purposes

Experimental stool samples were collected within 1 hour before the start of the experiment. Accurately weigh 4g of fecal sample, N₂/CO₂(80/20, v/v) aerated Wilkins-Chalgren anaerobic (WCA) medium 1:10 dilution. For homogenization, the sample is loaded into a Stomacher^TMLab Blender bag and Stomacher^TMMaximum speed processing of Lab Blender. The time required was within 1-10 minutes, depending on the consistency of the sample.

1ml of diluted and homogenized fecal samples were inoculated into Hungate culture tubes respectively under sterile conditions,and mixed with a pre-filled medium. The tube contains 9ml of N₂/CO₂(80/20, v/v) aerated WCA medium, after addition of diluted fecal samples, to a final concentration of 10mM of carbohydrate to be evaluated, calculated as monomer, in the WCA medium. The cultures prepared as described above were incubated at 37 ℃ for 24 hours. Samples of 4ml were taken before the start of incubation and 24 hours after the following analysis.

Pretreatment of samples

Samples from the Hungate tube were dispensed into two 2ml reaction tubes and centrifuged at 8000 xg for 10 minutes. The supernatant was decanted and frozen at-20 ℃ until parameter evaluation. The settled cells were resuspended in 1.5ml of 1 XPBS, mixed vigorously with three 3mm glass beads and centrifuged at 300 Xg for 1 minute to remove coarse particles. Then, 1ml of the supernatant was mixed with 3ml of paraformaldehyde solution and incubated at 4 ℃ for 3 hours. Then, 1ml of the suspension was centrifuged at 8000 Xg for 3 minutes, the pellet was mixed with 300. mu.l of 1 XPBS and stored at-20 ℃ after addition of 300. mu.l of ethanol (pure).

Determination of cell Titers

Cell titers determined with FISH microscopy were analyzed according to the protocol of Thiel and Blaut (Thiel, r., Blaut, M. (2005) microbiol. meth.61, 369-reservoir 379).

Probes for determining cell number

The cells present in the sample are analyzed by means of an automated FISH microscope. For the determination of the total cell number, the probe mixture EUB-mix (EUB 338: Amann et al, 1990, appl. environ. Microbiol.56, 1919-. The lactobacilli determined with probe Lab158(Harmsen et al 1999 Microb. Ecol. health Dis.11: 3-12) were counted manually due to the expected low cell titers.

Detection of H ₂ -products of

Hydrogen was detected using an HP 6890 series type II gas chromatograph equipped with an HP-19091P-MS4 molecular sieve-5A capillary column (30 m. times.0.32 mm. times.12 μm thickness) and a thermal conductivity detector. Carrier gas N₂The flow rate of (2) was 1 ml/min. The temperature of the column furnace is 40 ℃ and the temperature of the detector is 205 ℃. The split was adjusted at 1:10 and the injection volume was 0.5 ml.

Determination of fructose

The fructose content of the samples was determined using a fructan assay kit (Megazyme order number: K-FRUC) and adjusted according to the sample volume provided. Fructose content is a measure of inulin residue in fermentation samples, since inulin is converted to fructose.

16. Food preparation

a)Low fat salad dressing

Recipe

Inulin type used	Artichoke, average DPw56	- - - (control)	CargillOliggo-fiber LC/HT	CargillOliggo-fiber F97	OraftiRaftilineHP
Composition (I)1)	Weight (g)	Weight (g)	Weight (g)	Weight (g)	Weight (g)
Oil phase:
						soybean oil	92.45	302.45	92.45	92.45	92.45
Egg, whole, pasteurized	23.00	23.00	23.00	23.00	23.00
						Yolk, pasteurizing	18.00	18.00	18.00	18.00	18.00

Water phase:
						fermented low-fat buttermilk	89.45	89.45	89.45	89.45	89.45
Water (W)	190.45	40.45	190.45	190.45	190.45
						Distilled white vinegar	8.00	8.00	8.00	8.00	8.00
Whole flake dehydrated parsley	0.50	0.50	0.50	0.50	0.50
						Potassium sorbate	0.10	0.10	0.10	0.10	0.10
						Dry mixture A
Salt (salt)	3.75	3.75	3.75	3.75	3.75
						Mustard powder	1.50	1.50	1.50	1.50	1.50
						Dry mixture B
Inulin powder	60.00	----	60.00	60.00	60.00
Granulated sugar	8.00	8.00	8.00	8.00	8.00
						Salt (salt)	2.25	2.25	2.25	2.25	2.25
Half-cooked black pepper	1.00	1.00	1.00	1.00	1.00
						Garlic powder	1.00	1.00	1.00	1.00	1.00
Onion powder	0.50	0.50	0.50	0.50	0.50
						Xanthan gum	0.05	0.05	0.05	0.05	0.05

1) Suppliers (all names of suppliers and names of products are registered trademarks);

soybean oil derived from AC Humko

Whole egg and yolk (10% salt) from Sysco

Vinegar (20% acidity) from Fleischmann' s

Whole parsley flakes and black pepper from McCormick

Potassium sorbate from ADM

The salt is from Morton

Mustard powder from French's (82841)

The granulated sugar is from C & H

The garlic powder and onion powder are from Conagra/Gilroy

Xanthan gum is from Kelco (Keltrol 521)

Preparation:

1. potassium sorbate is dissolved in water.

2. Mixture B was added under shear.

3. The vinegar and buttermilk were added sequentially under shear.

4. Egg yolk and whole egg are added.

5. Dry mixture a was added under shear.

6. The oil mixture was added slowly under shear.

7. The parsley is stirred at low speed or by hand.

8. Kept under refrigeration.

b) White bread

Recipe

Inulin type used	Artichoke, average DPw56	- - - (control)	CargillOliggo-fiber LC/HT	CargillOliggo-fiber F97	OraftiRaftiline HP
Composition (I)1)	Weight (g)	Weight (g)	Weight (g)	Weight (g)	Weight (g)
						Bread flour, 12.6% protein	357.20	391.80	357.20	357.20	357.20
Water (W)	300.30	300.30	300.30	300.30	300.30
						Soft flour, 9.5% protein	135.80	148.90	135.80	135.80	135.80
Hydrogenated shortening	48.40	48.40	48.40	48.40	48.40
						Granulated sugar	48.40	48.40	48.40	48.40	48.40
Inulin powder	47.70	---	47.70	47.70	47.70
						Invert sugar	29.80	29.80	29.80	29.80	29.80
Skimmed milk powder, NthInstBulk	14.90	14.90	14.90	14.90	14.90
						Quick fermentation mother	8.60	8.60	8.60	8.60	8.60
Salt (salt)	6.00	6.00	6.00	6.00	6.00
						Distillation of monoglycerides	1.70	1.70	1.70	1.70	1.70
Emulsifier	1.10	1.10	1.10	1.10	1.10
						Bread enzyme	0.10	0.10	0.10	0.10	0.10

1) Supplier (all supplier names and product names are registered trademarks)

Bread flour and soft flour from General Mills (Superlative # 53521; Superlative #58431)

Shortening is from Loders Croaklaar (321)

The granulated sugar is from C & H

Inverted sugar derived from LSI (Nulomoline)

Skimmed milk powder from Kerry (I1532)

Rapid fermenting yeast from Fleischmann's (2139)

Salt (refined) from Morton

Distilled monoglycerides (Domodan PH 300K-A), emulsifiers (Panodan Datem 205K) and bread enzyme (GrindamylMax-Life U4) from Danisco

Preparation:

1. all dry matter except salt was weighed into a KitchenAid bowl.

2. Invert sugar, shortening and water were added and mixed for 1 minute at "stir" speed.

3. The salt was added and mixing continued for 1 minute at "stir" speed and for 6 minutes at "1" speed.

4. Proofed in a covered bowl at 29 ℃ for one and a half hours.

5. The dough is punched with a punch and the bread is formed and placed in a bread tray that is coated with oil.

6. Proofing takes about one and a half hours until the volume is doubled.

7. Baking in convection oven at 200 deg.C for 30 min until slight gold color and internal temperature of 96-100 deg.C.

8. The disc was rotated once during baking.

9. Cool on the shelf for 5 minutes, remove the bread tray and cool to room temperature.

Example 1: characterization of inulin from artichoke root

1. Cultivating artichoke plants

Variants of artichoke plant Nun9444 (also known as N9444) were grown in the vicinity of Valencia, spain. In June, seeds were sown in 104-notched nursery boxes (8X 13 wells, 4X 4cm) of a meshed plant greenhouse (mesh plant house). The plants were cultivated in nursery boxes for six weeks. Planted in the field at 10000 plants/hectare density in the beginning of the eight months. The whole plant was harvested in july of the second year. The roots were separated from the aerial parts and the attached soil was washed off by hand in water (under pressure). The roots were shade-dried on a fixture for 3 days. And then transported from spain to germany without refrigeration. The roots were stored at-80 ℃ before inulin extraction.

2. Inulin production from artichoke roots

For inulin production, roots of artichoke Madrigal (previously described Nun 9444) variety were thawed at room temperature and sliced. Inulin was extracted and purified as described in the aforementioned "inulin purification and fractionation" method. Purified inulin from most specimens was combined into samples.

3. The purity of the inulin produced was determined

The purity of the produced artichoke inulin was determined by measuring the fructan and water content of the freeze-dried material. The water content of artichoke was determined to be 5.4% (see method for "determining water content").

To determine the fructan/inulin content, 20mg of artichoke inulin were incubated in 2ml of double distilled water and shaken with a heating block at 95 ℃ for 1 hour. The fructan/inulin content was determined (1) using the "fructan assay protocol" kit (see method 2.1) and (2) by hydrolyzing inulin with exoinulase and then enzymatically determining the amount of glucose and fructose released (see method 2.2). (1) The sample of (2) was diluted 1:5 with double distilled water. The purity on a dry matter basis (DM) was determined in terms of fructan content and water content. Purity ═ fructan content × 100/(100-water content).

As can be seen from table 1, the average degree of purity of the produced artichoke inulin was 92.5% or 99.1% of Dry Matter (DM) according to the determination method.

Table 1: determining the purity of the prepared artichoke inulin

4. Determination of the molecular weight by GPC-RI-MALLS

A0.5% (w/v) aqueous solution was prepared from purified artichoke inulin, a commercially available reference sample of Raftiline HP (manufactured by Orafti, batch: HPBNO3DNO3) and dahlia tuber inulin (manufactured by Sigma, order No. I-3754, batch: 022K7045 or 75H7065) and the molar mass distribution of the inulin was determined by gel permeation chromatography (see method 4). This distribution is depicted in fig. 1, from which the calculated molar mass (anhydrofructose 162g/mol) and average chain length are compiled in table 2.

Analyzing molecular weight distribution with GPC-RI-MALIS system to obtain weight average molar mass M of artichoke inulin_wIs 9045g/mol and the number average molar mass M_nIs 7797 g/mol. The corresponding average chain length is DP_w56 and DP_n48. The average chain length of purified artichoke inulin is significantly longer than RaftilineHP (DP)_w＝25，DP_n23) and dahlia inulin (DP)_w＝29，DP_n26). This is also reflected in the significantly greater minimum and maximum molar masses of artichoke inulin.

Table 2: molar mass distribution of different inulins

Material	Mw[g/mol]	Mn[g/mol]	Polymer distribution (min-max) [ g/mol ]]	DPw	DPn	Degree of molecular dispersion
							Artichoke inulin	9045±45	7797±46	2650-28 630	56	48	1.17
Raftiline HP	4120±28	3673±45	1210-11 610	25	23	1.09
							Dahlia inulin	4734±68	4242±50	1590-12 300	29	26	1.11

Determination of molecular weight by hydrolysis with Perchloroacetic acid (PCA)

TABLE 2a

DPn	1. Measuring	2. Measuring
			Artichoke inulin Raftiline HP dahlia inulin	5127-	48-35

1. Measurement: 4.5% inulin final concentration, 0.5% TCA final concentration, 24 hours, 56 deg.C

2. Measurement: 2.5% Final concentration of inulin, 9% Final concentration of TCA, 4 hours, 37 deg.C

5. Results of glucose, fructose and sucrose measurements

To determine the proportion of glucose, fructose and sucrose in the purified artichoke inulin, 1% and 2% strength aqueous inulin solutions were prepared and incubated for 1 hour at 95 ℃. The sugars were determined photometrically as described in method 3 ("sugar determination").

As can be seen from table 3, the fructose content detectable in the purified artichoke inulin was only 0.1%. Under the above conditions, glucose and sucrose could not be detected by photometric detection methods.

Table 3: the content of glucose, fructose and sucrose in purified artichoke inulin

Material	Glucose (g/100g inulin powder)	Fructose (g/100g inulin powder)	Sucrose (g/100g inulin powder)
				Artichoke inulin	Can not detect	0.1	Can not detect

6. The percentage content of oligofructans with a chain length between DP-3 and DP-10 in the total inulin was determined by HPAEC-PAD

To calculate the percentage content of oligofructans with chain length DP ═ 3 to DP ═ 10 in the total inulin, a 2% (w/v) aqueous solution of the purified artichoke inulin was prepared and fractionated with HPAEC-PAD (method 5), the proportion of oligofructans being calculated with method 6. The values obtained are as follows

Inulin oligomer	Content (g/100g powder)
		DP3	0.007
DP4	0.016
		DP5	0.012
DP6	0.013
		DP7	0.024
DP8	0.029
		DP9	0.044
DP10	0.072
		Total amount of	0.217

Example 2: characterization of the inulin paste

1. Determination of the stability of the inulin paste

Analysis of the stability of the inulin paste (protocol: see methods) showed that the stability behaviour of artichoke inulin was significantly altered compared to inulin from chicory (Raftiline HP) and dahlia (see table 4). At the shear rate used herein, artichoke inulin showed the highest stability, exceeding 90% of the initial value. Analysis of the viscosity stability (acid stability) of the inulin paste in acidic medium also showed that the artichoke inulin showed a minimum change after 2 weeks of storage compared to the initial value after 24 hours, with an increase in viscosity of only 6.7%. In contrast, the viscosity of dahlia inulin paste increased by more than 15% in acidic medium, whereas Raftiline HP increased by more than 32%. This post-thickening is undesirable in many applications in the food sector. The melting temperature of the artichoke inulin paste was 73 ℃ which is significantly higher than the 62.3 ℃ of the reference substance, namely Raftline HP (manufactured by Orafti, batch: HPBNO3DNO3) and 65.5 ℃ of dahlia inulin (manufactured by Sigma, order number I-3754, batch: 75H 7065). The high thermal stability of inulin pastes is a great advantage in many heat treatments in the food sector.

TABLE 4

Material	Shear stability [% ]]	Acid stability [% ]]	Melting temperature [ deg.C]
				Artichoke inulin	90.5	106.7	73.0
Raftiline HP	49.8	132.1	62.3
				Dahlia inulin	81.0	115.9	65.5

2. Differential scanning calorimetry study on inulin

Differential scanning calorimetry analysis (protocol: see method) of inulin showed clear differences in melting behaviour between the different materials (see table 5). Although T of all inulin samples_onset(To) are all very similar, but the difference in melting enthalpy is very large. The content of artichoke inulin is more than 30J/g, the content of Raftiline HP is only 21.3J/g, and the content of dahlia inulin is 22.9J/g. Furthermore, the melting curve shape of artichoke inulin is clearly different from that of the other two inulins (see fig. 2). The melting curve of artichoke inulin is shallower than the course of the other two inulins at the beginning, but then rises higher than Raftiline HP or dahlia inulin (fig. 2). This increased thermal stability of artichoke inulin is an important advantage in certain heat treatments in the food sector, since artichoke inulin is significantly less sensitive to high temperatures than Raftiline HP or dahlia inulin.

TABLE 5

Material	To[℃]	Melting enthalpy dH [ J/g ]]
			Artichoke inulin	51.6	30.2
Raftiline HP	51.2	21.3
			Dahlia inulin	51.1	22.9

Example 3: viscosity, gelation behavior, solubility

a)Viscosity of the oil

Table 6: comparison of the kinetic viscosities of chicory and artichoke inulin in water as a function of concentration (T. 90 ℃ C.)

As can be seen from the table above, concentrations of up to 22.5% were achieved, and both inulins exhibited very low viscosities at 90 ℃ (1 mPas). The inulin of the invention started to become viscous when the concentration reached 25% (w/v), while Raftiline HP was still very similar to water at 30%. At 30%, the inulin of the invention showed an unexpectedly high viscosity.

b)Gelation behavior

Two types of analyses were performed on the gelation behavior of inulin after heat solubilization. Inulin is that described in table 2 and example 2. First, artichoke inulin (of the invention), Raftiline HP and dahlia inulin at different concentrations were heated to 90 ℃ and then cooled at room temperature for 20 hours. The gel formed exhibited a particle type characteristic ("particle gel"). Gel strength was quantified by texture analysis and the values are listed in the table below. At a given concentration of 20% (w/v), the gel produced by artichoke inulin was very strong, whereas no formation of the gel by Raftiline HP was observed, with only a pudding-like consistency being obtained for dahlia inulin.

Table 7: strength of gel of heat-dissolved inulin

Gel formation and stability are more finely characterized with a kinetic rheometer. Raftiline HP and artichoke inulin of the invention (DP) at different concentrations (w/v)_w56) was soluble in water at 90 c and cooled to 25 c at a rate of 1 c/min。

TABLE 8

The results in the above table show that at 20% to 22.5%, Raftiline HP does not exhibit gelation behavior ("G" > G; viscosity modulus > elastic modulus). In contrast, the artichoke inulin of the invention already forms a very firm gel at the same concentration.

c)Solubility in water

In another experiment, a 20% (w/v) solution of Raftiline, dahlia inulin and artichoke inulin was prepared at 98 ℃ and then stored in a refrigerator. The amount of inulin still in solution was then quantified. The inulin used was the inulin described in table 2 and example 2.

Table 9: solubility of different inulin

	Chicory	Globe artichoke (Cynara scolymus L.)	Dahlia (dahlia flower)
				Solubility [% ]]	97.2	14.2	88

As shown in the above table, it is evident that Raftiline dissolves best, followed by dahlia inulin, whereas artichoke inulin only dissolves in very small amounts.

The above observations suggest that at moderate (below 60 ℃) or lower temperatures, it is possible to incorporate larger amounts of artichoke inulin into food products (e.g. beverages, condiments) without affecting the viscosity, compared to other commercially available inulins. This is a great advantage for food applications.

Example 4: fermentation study

a)Fermentation rate

By generation of H₂The yield of gas indicates the fermentation of artichoke inulin from DP58 (batches a and B) and Raftilin HP by human fecal bacteria compared to controls without inulin.

The table below shows the percentage of inulin content (residual inulin + standard error) in the amount of inulin initially used after 24 hours of fermentation (average for each material produced by independent fermentation, n-10).

Watch 10

Sample (I)	Residual inulin (%)	P value
			Raftiline HP	2.6+/-1
Artichoke inulin batch a	9.0+/-3	0.00002
			Artichoke inulin batch B	10.0+/-4	0.00010

^＊Significance of difference compared to Raftiline HP (T-test of paired Point samples)

The table shows the samples of the invention (artichoke inulin, average DP after 24 hours of fermentation_w58) Is more than that in the Raftiline HP sampleMuch residual inulin. Thus fecal bacteria fermented DP58 artichoke inulin slower than commercial Raftiline HP. Longer-chain inulin, which ferments more slowly than commercial inulin, such as the inulin of the invention, can be expected to exert metabolic and prebiotic effects at the more distal end of the colon. Fermentation in the distal colon is beneficial because many intestinal diseases (e.g., ulcerative colitis, diverticular disease) are primarily distributed there.

b) Probiotic effect

The table below shows the increase in the number of lactobacilli (Lab) due to fermentation after 24 hours, corrected for the percentage of blank samples (without inulin), and the ratio of lactobacilli to eubacteria (Eub) after 24 hours of fermentation (average of the independent fermentation preparations of each substrate, n-10).

TABLE 11

Sample (I)	Increase in Lactobacillus (%)	Lab/Eub
			Raftiline HP	81	0.62
Artichoke inulin batch a	119	0.87
			Artichoke inulin batch B	100	0.81

The invented artichoke inulin stimulates the number of lactobacilli to a higher level compared to Raftiline HP. The advantageous effect of artichoke inulin over Raftiline HP in stimulating lactobacilli becomes more pronounced if the ratio lactobacilli/eubacteria is taken into account. This value is more suitable for expressing a specific stimulatory effect on the desired lactobacillus.

Example 5: food applications

a)Low fat salad dressing

TABLE 12

And (3) making adhesion:

according to what appears in the table above, the dressing using inulin of the invention is slightly viscous than the control, but still smooth and pourable. Performs better in visual consistency than the dressing using inulin of the comparative example, forming a smooth, thick pourable typical Ranch dressing. Use of Cargill Oliggo-Fiber^TMThe flavor of the LC/HT inulin was closest in consistency to the inulin of the invention and the control, but the consistency was slightly thin. OraftiHP exhibits a much more viscous consistency, much like cream cheese, and Cargill Oliggo-Fiber was used^TMThe F-97 flavor was thinner than the control and isolated after overnight storage.

Emulsification of fat replacement:

flavoring using inulin of the invention and Cargill Oliggo-Fiber^TMLC/HT and OraftiHP was similar in emulsifying capacity to that of CargillOliggo-Fiber^TMF-97 is more preferred, the latter being isolated after overnight storage.

b) White bread

Watch 13

The inulin of the present invention binds more water and forms a tighter dough than the control and competing inulins. Thus, the dough produced from the inulin of the invention has a higher binding capacity, resulting in a tighter and firmer dough, resulting in a more equal breading.

Doughs using the inulin of the invention can be baked to a better height and color than the control. CargillOliggo-Fiber^TMThe dough and bread of LC/HT were similar to the control. OraftiThe dough of HP was similar to the control, but the baked bread had a coarser but more equal crumb. CargillOliggo-Fiber^TMF-97 does not have sufficient binding properties resulting in a dough that does not initiate or bake well.

The inulin of the invention produced the most equal crumb appearance and a tighter texture. Control and Cargill Oliggo-Fiber^TMLC/HT has an unequal crumb appearance, OraftiThe HP dough had a rougher breadcrumb appearance, whereas Cargill Oliggo-Fiber^TMF-97 had a more crispy crumb appearance.

As a result, the inulin of the invention is more preferable in appearance and breadcrumb generation than competing inulins.

Claims (19)

1. Inulin having an average degree of polymerization DPw of between 54 and 61.

2. Inulin as claimed in claim 1, which has an average degree of polymerization DP_wBetween 55 and 60.

3. Inulin as claimed in claim 1, which has an average degree of polymerization DP_wBetween 56 and 57.

4. Inulin as claimed in any of claims 1 to 3, wherein the glucose content is less than 2%.

5. Inulin as claimed in claim 4, wherein the glucose content is less than 1%.

6. Inulin as claimed in any of claims 1 to 5, wherein the fructose content is less than 2.5%.

7. Inulin as claimed in claim 6, wherein the fructose content is less than 1.5%.

8. Inulin as claimed in any of claims 1 to 7, wherein the content of fructooligosaccharides with a DP of 3 to 10 is less than 3%.

9. Inulin as claimed in any of claims 1 to 8, wherein the content of fructooligosaccharides with a DP of 3 to 10 is less than 1.5%.

10. Inulin as claimed in any of claims 1 to 8, wherein the content of fructooligosaccharides with a DP of 3 to 10 is less than 0.7%.

11. Inulin according to any of claims 1-10, characterized in that the aqueous inulin paste has an increase in viscosity of less than 10% after storage at pH4 and room temperature for 2 weeks, compared with the initial value of the viscosity.

12. Inulin according to any of claims 1-10, characterized in that the shear rate is 20s^-1Next, inulin at a concentration of 30% w/v in water at 90 ℃ has a viscosity of 300-1000 mPas.

13. Food product comprising inulin according to any of claims 1-12.

14. The food product according to claim 13, selected from the group consisting of dairy, yogurt, ice cream, dairy-based smoothie, whipped cream, pudding, milkshake, custard, cheese, nutritional bars, energy bars, breakfast bars, confection, baked bread, crackers, cookies, biscuits, cereal chips, assorted dried fruits, ice tea granules, fruit juice smoothies, weight control beverages, ready-to-drink beverages, sports beverages, endurance beverages, supplement powder mixes, infant formula, high calcium orange juice, bread, croissants, cereals, pasta, bread spreads, sugar-free candies and chocolates, calcium candy chews, meat products, mayonnaise, salad dressings, nut butter, frozen entrees, sauces, soups, and ready-to-eat meals.

15. Food product according to claim 13 or 14, characterized by an extruded product.

16. A dietary supplement comprising inulin according to any one of claims 1-12.

17. Cosmetic preparation comprising inulin according to any of claims 1 to 12.

18. Use of inulin according to any one of claims 1-12 as a food additive.

19. Use of inulin according to any one of claims 1-12 as an additive in cosmetic preparations.