WO2018099877A1 - Process for preparing soluble fibre - Google Patents

Process for preparing soluble fibre Download PDF

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Publication number
WO2018099877A1
WO2018099877A1 PCT/EP2017/080579 EP2017080579W WO2018099877A1 WO 2018099877 A1 WO2018099877 A1 WO 2018099877A1 EP 2017080579 W EP2017080579 W EP 2017080579W WO 2018099877 A1 WO2018099877 A1 WO 2018099877A1
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WIPO (PCT)
Prior art keywords
fruit
fibre
process according
vegetable material
enzyme
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PCT/EP2017/080579
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French (fr)
Inventor
Margot Elisabeth Francoise Schooneveld-Bergmans
Joanna Maria Josephina HEUVINK
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Dsm Ip Assets B.V.
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Publication of WO2018099877A1 publication Critical patent/WO2018099877A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01015—Polygalacturonase (3.2.1.15)

Definitions

  • the present invention relates to a process for preparing a soluble fibre.
  • soluble non-digestible fibres such as pectin-derived oligosaccharides.
  • Soluble nodigestible fibres are attracting increasing attention for their health benefits as a prebiotic.
  • soluble fibres like galacto-oligosaccharides (GOS), xylo-oligosaccharides and fructo-oligosaccharides (FOS) commercially available.
  • GOS galacto-oligosaccharides
  • POS pectic oligosaccharides
  • POS pectic oligosaccharides
  • POS are derived from pectin which is widely available and present in pectin-rich agro-industrial residues originating from dicotyledons, such as citrus peel, sugar beet pulp or cassava pulp.
  • Pectin can be isolated from fruit and vegetables by various methods known in the art.
  • US2014/030873 discloses a process for extracting pectin from raw material, which comprises mixing the raw material with a mild organic acid and a polygalacturonase-based enzyme.
  • US 5,350,593 discloses a process for the preparation of dietary fibre from tapioca pulp fibre by incubating the tapioca pulp fibre with an alpha-amylase, wherein a fibre is provided comprising at least 70% total dietary fibre of which at least 12 % is soluble fibre.
  • Babbar ef al. (2016), J. of Agric. Food Chem., 64, p. 268-276 discloses a process for the extraction of pectin from agricultural residues, such as berry pomace, onion hulls, pressed pumpkin, and sugar beet pulp, using enzymatic hydrolysis using cellulase or acid extraction with nitric acid, or chelator-based extraction, using ammonium oxalate or sodium hexametaphosphate.
  • the efficiency of the different extraction methods differed per agricultural residue and yielded pectic extracts with a different molecular weight.
  • Babbar ef al. (2016), Carbohydrate Polymers, 146, 245-252 discloses a process for the preparation of pectic oligosaccharides from onion skins, wherein the onion skins are first extracted with sodium hexametaphosphate at 95°C and subsequently the extract is treated with different enzyme compositions having pectinase activity. Incubation of the extract with an endopolygalacturonase yielded the highest amount of pectic oligosaccharides with a degree of polymerization of 3 and 4.
  • the present disclosure relates to a process for preparing a soluble fibre comprising galacturonan oligosaccharides having a degree of polymerization (DP) of 2 to 20 from a fruit or vegetable material comprising
  • step (b) incubating the fruit and / or vegetable material of step (a) at a pH of between 0.5 to 3 and a temperature of between 60 and 98°C,
  • step b) separating the material from step b) into a solid fraction and a liquid fraction
  • a high purity as used comprises a soluble fibre with a low caloric value.
  • a low caloric value as disclosed herein means that the soluble fibre comprises little or no glucose, such as a soluble fibre comprises less than 0.5 wt wt% of glucose on dry matter.
  • the present disclosure relates to a soluble fibre obtainable by a process as disclosed herein.
  • the present disclosure relates to a composition comprising a soluble fibre as disclosed herein.
  • the present invention relates to a process for preparing a soluble fibre comprising galacturonan oligosaccharides having a degree of polymerization (DP) of 2 to 20 from a fruit and / or vegetable material comprising
  • step a) incubating the fruit and / or vegetable material of step a) at a pH of between 0.5 to 3 and a temperature of between 60 and 98°C,
  • step b) separating the material from step b) into a solid fraction and a liquid fraction
  • a soluble fibre as disclosed herein is a fibre that is soluble in water.
  • the wording "fibre” and “fiber” is used interchangeably herein.
  • Incubating the fruit and / or vegetable material in step b) comprises mixing the fruit and / or vegetable material with an aqueous solution having a pH of between 0.5 and 3.
  • the aqueous solution comprises an acid, for instance hydrochloric acid (HCI), sulfuric acid (H2SO4) or nitric acid (HNO3).
  • the pH of the aqueous solution may be between 0.5 and 3, for instance a pH of between 1 and 2.5, for instance a pH of between 1.2 and 2.
  • incubating the fruit and / or vegetable material may be performed at a pH of between 0.5 and 3, for instance a pH of between 1 and 2.5, for instance a pH of between 1.2 and 2.
  • the temperature during incubating the fruit or vegetable material at a pH of between 0.5 and 3 may be between 60 and 98°C for instance between 65 and 95 °C, for instance between 70 and 90°C, for instance between 75 and 85°C.
  • Incubating in a process as disclosed herein may be performed during 30 to 300 min, such as 60 to 240 min, such as 90 to 200 min.
  • a process as disclosed herein comprises separating the material that has been incubated at a pH of between 0.5 and 3 and at a temperature of between 60 and 98°C into a solid fraction and a liquid fraction. Separating the incubated material may comprise a step of washing the solid fraction, for instance washing the solid fraction with water. Separating the incubated material may comprise centrifuging or filtering the incubated material.
  • Isolating fibre from the liquid fraction may be performed by any suitable method known to a person skilled in the art, for instance by adding an alcohol to the liquid fraction or filtering the liquid fraction through a membrane. Filtering the liquid fraction through a membrane is known to a person skilled in the art and comprises separating the low molecular weight fraction from higher molecular weight fractions, for instance as disclosed in Yapo et. al., (2007) Food Hydrocolloids 21 ; 245-255.
  • isolating the fibres comprises precipitating fibres.
  • Precipitating fibres as used herein comprises adding an alcohol, for instance ethanol or isopropanol, to the liquid fraction at a temperature of between 0 to 25°C, such as a temperature of between 1 to 15°C, for instance between 2 to 10°C, for instance between 3 to 5°C.
  • Ethanol that may be added to the liquid fraction may be a solution comprising at least 95 v/v%, 96 v/v%, 97 v/v%, 98 v/v%, or at least 99% v/v% of ethanol.
  • ethanol is added to the liquid fraction until a concentration of 50 to 70 vol/vol % of ethanol is reached.
  • Precipitating may be performed during any suitable period of time, for instance during 1 to 30 hrs, for instance during 2 to 20 hrs, or during 4 to 15 hrs. Surprisingly, it was found that a step of precipitating fibres from the liquid fraction reduces the amount of glucose to less than 0.5 wt/wt% based on dry matter in the soluble fibre.
  • a process as disclosed herein may further comprise a step of bringing the material of step a) to a temperature of between 2 and 35 °C, for instance between 4 and 30 °C, for instance between 6 and 25 °C prior to separating the material into a solid fraction and a liquid fraction. It was found that bringing the material of step b) to a temperature of between 2 and 35°C increased the yield of the soluble fibre.
  • a process for preparing a soluble fibre from a fruit and / or a vegetable material as disclosed herein comprises bringing the fibre that has been precipitated into contact with an enzyme having endopolygalacturonase activity.
  • An endopolygalacturonase is an enzyme that hydrolyses the alpha-1 ,4-glycosidic bonds between the galacturonic acid residues in pectate and galacturonans.
  • An endopolygalacturonase is classified in classification number EC 3.2.1.15 and is also referred to as pectin depolymerase, pectinase, pectolase, pectin hydrolase, pectin polygalacturonase, endogalacturonase and poly- alpha-1 ,4-galacturonide glycanohydrolase.
  • Endopolygalacturonases may be derived from any suitable microorganism, for instance from bacteria of fungi.
  • Suitable bacteria may be from the genus Bacillus, for instance from the species Bacillus coagulans, Bacillus subtilis.
  • Suitable fungi may be from the genus Aspergillus, Penicillium, Trichoderma, Saccharomyces, or Kluyveromyces, for instance from the species A. niger, A. oryzae, A. tubigensis, P. chrysogenum, T. reesei, S. cerevisiae, or K. lactis.
  • the endopolygalacturonase is derived from Aspergillus tubigensis for instance as disclosed in WO00/17367.
  • An enzyme having endopolygalacturonase activity in a process as disclosed herein may be a pure enzyme.
  • a pure enzyme as used herein may be at least 50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 80% pure, at least 90% pure, or at least 95% pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% as determined by SDS-PAGE or any other analytical method suitable for this purpose and known to the person skilled in the art.
  • An enzyme having endopolygalacturonase activity may be an enzyme comprising less than 2 wt%, such as less than 1 wt% or less than 0.5 wt% of an enzyme having pectinmethylesterase activity, of the total weight of enzymes having endopolygalacturonase activity and pectinmethylesterase activity.
  • the amount of enzymes can be determined by SDS-PAGE and / or LC-MS methods or other analytical methods known to a person skilled in the art.
  • Pectinmethylesterase activity can be determined using methylated pectin, preferably pectin with a degree of methylation over 70%, and titrimetric analysis of the hydrolysis of the methyl-esters and the concomitantly produced acid, resulting in a number of micromoles of ester hydrolysed per minute, e.g. at pH 4.5 and 30°C.
  • an enzyme having endopolygalacturonase activity may be contacted with the fruit or vegetable material.
  • an enzyme having an endopolygalacturonase activity may be contacted with the fruit and / or vegetable material in an amount of 0.1 PGU - 100 PGU / g dry weight, such as 0.2 to 80 PGU / g dry weight, or 0.3 to 50 PGU / g dry weight, or 0.4 to 25 PGU / g dry weight, or 0.5 to 10 PGU / g dry weight, or 0.6 to 5 PGU / g dry weight. It was found that a lower amount of endopolygalacturonase resulted in a higher relative amount of soluble fibres with a degree of polymerization of DP2 to DP10.
  • a process as disclosed herein may further comprise a step of removing starch from the fruit and /or vegetable material.
  • a step of removing starch may be performed prior to incubating the fruit and / or vegetable material at a pH of between 0.5 and 3 and a temperature of between 60 and 98°C.
  • Removing starch from a fruit or vegetable material may comprise incubating the fruit and / or vegetable material with an enzyme having amylase activity, for instance amylase activity may comprise alpha amylase (EC 3.2.1 .1.), maltogenic alpha amylase (EC 3.2.1.133) and / or glucoamylase (EC 3.2.1 .3) activity.
  • An amylase may be a thermostable amylase.
  • Amylases may be obtained from any suitable origin, for instance from bacteria or fungi.
  • Incubating the fruit and / or vegetable material with an amylase may be performed at any suitable pH and temperature depending on the amylase used, which is known to a person skilled in the art. Incubating the fruit and / or vegetable material with an enzyme having amylase activity, usually further comprises a step of washing the fruit and / vegetable material that has been treated with an amylase and / or a step of separating the fruit and / or vegetable material that has been treated with an amylase into a fruit and / or vegetable material residue and a liquid phase. Separating is usually performed by centrifugation and / or filtration.
  • Removing starch from a fruit or vegetable material may comprise adding water to the fruit and / or vegetable material.
  • Removing starch may also comprise bringing the temperature of the fruit and / or vegetable material to a temperature of 55 to 85°, for instance a temperature of between 60 and 75°C that allows gelatinizing of starch, prior to incubating the fruit and / or vegetable with an enzyme having amylase activity.
  • a process as disclosed herein may further comprise a step of removing protein from the fruit and / or vegetable material.
  • a step of removing protein in a process as disclosed herein may be performed prior to incubating the fruit and / or vegetable material at a pH of between 0.5 and 3 and a temperature of between 60 and 98°C as described herein above.
  • Removing protein may comprise incubating the fruit and / or vegetable material with a protease.
  • protease There are many proteases available in the art. Proteases are enzymes that hydrolyse the peptide bonds between adjacent amino acids, and are classified in enzyme classification EC 3.4. [..], and may be derived from any suitable microorganism such as bacteria and fungi.
  • Incubating the fruit and / or vegetable material with a protease may be performed at any suitable pH and temperature depending on the protease used, which is known to a person skilled in the art.
  • a process as disclosed herein may comprise a step of drying the fruit and / or vegetable material and / or drying the precipitated fibre. Drying as used herein may be performed in any suitable way for instance by freeze drying known to a person skilled in the art.
  • a process for preparing a soluble fibre as disclosed herein may further comprise a step of inactivating the enzymatic activity of endopolygalacturonase, and / or inactivating the activity of amylase and / or protease.
  • Inactivating enzymatic activity may for instance be performed by heating the fruit and / or vegetable material and / or the fibre to a temperature suitable to inactivate the enzymatic activity, for instance a temperature of between 70°C and 100°C, such as a temperature of between 85°C and 98°C.
  • the fruit and / or vegetable material in a process for preparing soluble fibres comprises pectin.
  • the fruit and/ or vegetable material may comprise a husk, hull or peel from fruit and / or vegetable material.
  • the fruit and / or vegetable material may for instance be derived from cassava, potato, onion, apple and / or citrus fruits.
  • the fruit and / orvegetable material may comprise cassava pulp, potato pulp, onion hulls apple pomace and / or citrus peel.
  • a soluble fibre that is prepared in a process as disclosed herein comprises a galacturonan oligosaccharides having a degree of polymerization (DP) of between 2 and 20, for instance a DP of between 2 and 12, for instance a DP of 3 to 10, for instance a DP of 3 to 7.
  • Soluble fibre may comprise galacturonan monomer, i.e. galacturonan having a DP of 1.
  • a soluble fibre as disclosed herein may further comprise galactan, rhamnan and / or arabinan, for instance galactan, rhamnan, and / or arabinan having a DP of between 2 and 50, such as a DP between 4 and 25.
  • Degree of polymerization is defined as the number of monomeric units in an oligomer or polymer molecule.
  • a monomeric unit in a soluble fibre as disclosed herein may for instance be galacturonic acid, galactose, rhamnose, and / or arabinose.
  • a soluble fibre that is prepared in a process as disclosed herein comprises 0 to 0.5 wt/wt% of glucose on dry matter, for instance between 0.1 and 0.2 wt/wt% of glucose.
  • a soluble fibre may comprise 0 wt/wt% of glucose.
  • Glucose as used herein refers to glucose monomers and glucose dimers, maltodextrins, starch, and / or starch derivatives comprising alpha-(1 ,4)-glucose linkages.
  • the present invention relates to a soluble fibre obtainable by a process as disclosed herein. All the elements of a soluble fibre prepared by a process as described herein above are embodiments of a soluble fibre obtainable by a process as disclosed herein.
  • the present invention relates to a composition
  • a composition comprising a soluble fibre as disclosed herein.
  • a composition as disclosed herein may further comprise water, proteins, lipids and / or carbohydrates.
  • FIG. 1 Diagram of different soluble fibre extraction methods A, B and C
  • Cassava pulp the residue of the cassava tubers after removal of the starch at industrial scale (Corn products Co., Ltd, Thailand) with a dry matter content of 16% was used.
  • Polygalacturonic acid from oranges, rhamnose, arabinose, xylose, galactose, mannose, glucose, and galacturonic acid were purchased from Sigma.
  • Total sugar composition was determined based on NMR analysis of acid hydrolyzed sugars according the method published in A. Carvalho de Souza ef a/., Carbohydrate Polymers, 2013 (95), 657-663. Starch was determined after hydrolysis in 0.6M D2SO4 for 1.5 h at 100°C followed by released glucose quantification based on NMR (after addition of maleic acid as internal standard). Sugar Composition Analysis and Oligosaccharide Profiling of Soluble Materials
  • the protein content was determined according to the Kjeldahl method, as known in the art, and using a standard conversion factor of N x 6.25.
  • Rapidase ® EPG NS L was obtained from Aspergillus tubigensis endopolygalacturonase
  • DSM has an activity of 20,000 PGU / mL.
  • One PGU is the amount of enzyme per mL enzyme solution that creates a line with a slope of 0.068 when the ratio of the viscosity of a blank versus that of the test solution is plotted against the incubation time.
  • Conditions of the test are: 1.75% sodiumpolygalacturonate at pH 4.5 and 45°C. Viscosity reduction was determined using an Ubbelohde 1 C.
  • Delvolase bacterial protease > 580.000 DU/g was obtained from DSM.
  • One DU is defined as the amount of enzyme needed to liberate 7.59 ⁇ of para-nitroaniline per minute at pH 8.5 and 37°C, using 2.18 mg/ml of succinyl-Ala-Ala-Pro-Phe-para-nitroanilide as substrate.
  • TAU thermostable bacterial oamylase
  • One TAU is defined as the amount of enzyme converting 1 .0 mg starch per minute, having an equal absorbance at 620 nm to a reference solution (25 g C0CI2.H2O and 3.84 g of potassium dichromate in 100 mL of 0.01 N hydrochloric acid) after reaction with an iodine solution at pH 6.6 at 30°C.
  • the substrate solution contains 13.3 mg/ml starch EXAMPLE 1. COMPARISON OF SOLUBLE FIBRE EXTRACTION METHODS
  • cassava pulp was suspended in 100 mM citrate buffer of pH 4 at 2% (w/w) dry matter and soluble fibre was prepared by directly incubating the cassava pulp with Rapidase ® EPG NS L endopolygalacturonase at 2090 PGU / g dry matter (DM).
  • CCWM crude cell wall material
  • CCWM was prepared from cassava pulp as described in Example 2.1 , followed by the selective fibre extraction described in Example 2.2.
  • the fibre extract was suspended in 100 mM citrate buffer of pH 4 at 2% (w/w) and soluble fibre was prepared by incubating the fibre extract with Rapidase ® EPG NS L endopolygalacturonase (50 PGU / g dry matter (DM)).
  • soluble fibres Non-Starch Sugars (NSS) obtained in Methods A, B and C were determined as disclosed in the Materials and Methods section.
  • Non-Starch Sugars may comprise poly-, oligo and monosaccharides.
  • Table 1 Yield of soluble fibres (total dry matter NSS) and sugar composition of soluble fibres after extraction from cassava pulp using three different methods of isolation A, B, C
  • EXAMPLE 2 ISOLATION OF SOLUBLE FIBRE FROM CASSAVA WITH DIFFERENT RATIO OF GALACTURONAN -OLIGOSACCHARIDES
  • NSS non-starch sugars
  • NSS non-starch sugars
  • Table 2 Composition of the main fractions of cassava pulp, crude cell wall material (CCWM) and fibre extract, extracted from cassava pulp
  • NSS Non Starch Sugars
  • Fibre extract obtained in Example 2.2 was suspended in 100 mM citrate buffer of pH 4 at 2% (w/w) dry matter, and different doses of endopolygalacturonase as indicated in Table 1 were added and subsequently each mixture was incubated at 40°C for 1 h. The enzymatic reaction was stopped by boiling the incubation mixture for 5 min.
  • EXAMPLE 3 ISOLATION OF SOLUBLE FIBRE FROM CASSAVA WITH ADAPTED
  • Crude cell wall material was prepared from three separate cassava pulp batches of 500 g according to the procedure disclosed in Example 2.1 , with the exception that only one amylase incubation step was applied and the protease incubation was omitted.
  • Table 4 shows that the yield of dry matter recovered from CCWM was 50%. This dry matter consisted for 57% of its dry matter of non-starch sugars, while protein content was similar as in Example 1 and residual starch content was higher.
  • the three isolated CCWM batches were pooled and split into two batches of 630 g, which contained 37.8 g dry matter each, were further used for fibre extraction and one batch of 680 g CCWM, which was not further used.
  • Example 3.1 To each batch of 630 g CCWM produced in Example 3.1. 120 mL of 3% HCI was added, and the pH was adjusted to 1.5 using 4 M NaOH. The suspensions were incubated at 82°C for 165 min. Washing of the residue and the ethanol precipitation was subsequently performed in a similar way as described in Example 2, while adapting the actual volumes to have a similar solid to liquid ratio. The finally isolated pectin pellet was freeze-dried. The dry matter recovery based on the starting material was 3.3%.
  • Table 4 an overview is given of the yield and the composition of the main fractions in the process of CCWM and fibre isolation.
  • Table 4 Yield and composition of the dry matter of cassava pulp, and crude cell wall material (CCWM) and fibre extract from cassava pulp
  • Example 3.2 The fibre extract obtained in Example 3.2 was incubated with endopolygalacturonase at a dose of 0.13 PGU / mL (6.5 PGU / g DM) of fibre extract solution at 5% dry matter content according to the method disclosed in Example 2.3.
  • the resulting relative amount of the galacturonan oligosaccharides was 9 : 10 : 7 : 4 : 3 : 2 for DP1 : DP2 : DP3 : DP4 : DP5 : DP6.

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Abstract

The present invention discloses a process for preparing a soluble fibre comprising galacturonan oligosaccharides having a degree of polymerization (DP) of 2 to 20 from a fruit and / or vegetable material comprising a) removing starch from the fruit and / or vegetable material, b) incubating the fruit or vegetable material of step a) at a pH of between 0.5 to 3 and a temperature of between 60 and 98°C, c) separating the material from step b) into a solid fraction and a liquid fraction, d) isolating fibre from the liquid fraction, and e) bringing the fibre from step d) into contact with an enzyme having endopolygalacturonase activity, and preparing the soluble fibre.

Description

PROCESS FOR PREPARING SOLUBLE FIBRE
FIELD
The present invention relates to a process for preparing a soluble fibre.
BACKGROUND
Fruits and vegetables, in particular their hulls, husk or peel, are a valuable source for the production of soluble non-digestible fibres, such as pectin-derived oligosaccharides. Soluble nodigestible fibres are attracting increasing attention for their health benefits as a prebiotic. There are only a few types of soluble fibres, like galacto-oligosaccharides (GOS), xylo-oligosaccharides and fructo-oligosaccharides (FOS) commercially available. However, pectic oligosaccharides (POS) have been identified as potentially higher performing and / or a lower cost prebiotic. POS are derived from pectin which is widely available and present in pectin-rich agro-industrial residues originating from dicotyledons, such as citrus peel, sugar beet pulp or cassava pulp.
Pectin can be isolated from fruit and vegetables by various methods known in the art. US2014/030873 for instance discloses a process for extracting pectin from raw material, which comprises mixing the raw material with a mild organic acid and a polygalacturonase-based enzyme.
Several processes have been disclosed for the extraction of water soluble fibres.
US 5,350,593 discloses a process for the preparation of dietary fibre from tapioca pulp fibre by incubating the tapioca pulp fibre with an alpha-amylase, wherein a fibre is provided comprising at least 70% total dietary fibre of which at least 12 % is soluble fibre.
Babbar ef al. (2016), J. of Agric. Food Chem., 64, p. 268-276 discloses a process for the extraction of pectin from agricultural residues, such as berry pomace, onion hulls, pressed pumpkin, and sugar beet pulp, using enzymatic hydrolysis using cellulase or acid extraction with nitric acid, or chelator-based extraction, using ammonium oxalate or sodium hexametaphosphate. The efficiency of the different extraction methods differed per agricultural residue and yielded pectic extracts with a different molecular weight.
Babbar ef al. (2016), Carbohydrate Polymers, 146, 245-252 discloses a process for the preparation of pectic oligosaccharides from onion skins, wherein the onion skins are first extracted with sodium hexametaphosphate at 95°C and subsequently the extract is treated with different enzyme compositions having pectinase activity. Incubation of the extract with an endopolygalacturonase yielded the highest amount of pectic oligosaccharides with a degree of polymerization of 3 and 4.
There is a need for an alternative process for the preparation of soluble fibres from fruits and vegetables. SUMMARY
The present disclosure relates to a process for preparing a soluble fibre comprising galacturonan oligosaccharides having a degree of polymerization (DP) of 2 to 20 from a fruit or vegetable material comprising
(a) removing starch from the fruit and / or vegetable material
(b) incubating the fruit and / or vegetable material of step (a) at a pH of between 0.5 to 3 and a temperature of between 60 and 98°C,
(c) separating the material from step b) into a solid fraction and a liquid fraction,
(d) isolating fibre from the liquid fraction, and
(e) bringing the fibre from step d) into contact with an enzyme having endopolygalacturonase activity,
and preparing soluble fibre.
Surprisingly, it was found that soluble fibre with a high purity was obtained in a process as disclosed herein. A high purity as used comprises a soluble fibre with a low caloric value. A low caloric value as disclosed herein means that the soluble fibre comprises little or no glucose, such as a soluble fibre comprises less than 0.5 wt wt% of glucose on dry matter.
In another aspect, the present disclosure relates to a soluble fibre obtainable by a process as disclosed herein.
In yet another aspect the present disclosure relates to a composition comprising a soluble fibre as disclosed herein.
DETAILED DESCRIPTION
In one aspect, the present invention relates to a process for preparing a soluble fibre comprising galacturonan oligosaccharides having a degree of polymerization (DP) of 2 to 20 from a fruit and / or vegetable material comprising
(a) removing starch from the fruit and / or vegetable material
(b) incubating the fruit and / or vegetable material of step a) at a pH of between 0.5 to 3 and a temperature of between 60 and 98°C,
(c) separating the material from step b) into a solid fraction and a liquid fraction,
(d) isolating fibre from the liquid fraction, and
(e) bringing the fibre from step d) into contact with an enzyme having endopolygalacturonase activity,
and preparing the soluble fibre.
A soluble fibre as disclosed herein is a fibre that is soluble in water. The wording "fibre" and "fiber" is used interchangeably herein.
Incubating the fruit and / or vegetable material in step b) comprises mixing the fruit and / or vegetable material with an aqueous solution having a pH of between 0.5 and 3. The aqueous solution comprises an acid, for instance hydrochloric acid (HCI), sulfuric acid (H2SO4) or nitric acid (HNO3). The pH of the aqueous solution may be between 0.5 and 3, for instance a pH of between 1 and 2.5, for instance a pH of between 1.2 and 2. Accordingly, incubating the fruit and / or vegetable material may be performed at a pH of between 0.5 and 3, for instance a pH of between 1 and 2.5, for instance a pH of between 1.2 and 2.
The temperature during incubating the fruit or vegetable material at a pH of between 0.5 and 3 may be between 60 and 98°C for instance between 65 and 95 °C, for instance between 70 and 90°C, for instance between 75 and 85°C.
Incubating in a process as disclosed herein may be performed during 30 to 300 min, such as 60 to 240 min, such as 90 to 200 min.
A process as disclosed herein comprises separating the material that has been incubated at a pH of between 0.5 and 3 and at a temperature of between 60 and 98°C into a solid fraction and a liquid fraction. Separating the incubated material may comprise a step of washing the solid fraction, for instance washing the solid fraction with water. Separating the incubated material may comprise centrifuging or filtering the incubated material.
Isolating fibre from the liquid fraction may be performed by any suitable method known to a person skilled in the art, for instance by adding an alcohol to the liquid fraction or filtering the liquid fraction through a membrane. Filtering the liquid fraction through a membrane is known to a person skilled in the art and comprises separating the low molecular weight fraction from higher molecular weight fractions, for instance as disclosed in Yapo et. al., (2007) Food Hydrocolloids 21 ; 245-255.
Advantageously, isolating the fibres comprises precipitating fibres. Precipitating fibres as used herein comprises adding an alcohol, for instance ethanol or isopropanol, to the liquid fraction at a temperature of between 0 to 25°C, such as a temperature of between 1 to 15°C, for instance between 2 to 10°C, for instance between 3 to 5°C. Ethanol that may be added to the liquid fraction may be a solution comprising at least 95 v/v%, 96 v/v%, 97 v/v%, 98 v/v%, or at least 99% v/v% of ethanol. Preferably, ethanol is added to the liquid fraction until a concentration of 50 to 70 vol/vol % of ethanol is reached. Precipitating may be performed during any suitable period of time, for instance during 1 to 30 hrs, for instance during 2 to 20 hrs, or during 4 to 15 hrs. Surprisingly, it was found that a step of precipitating fibres from the liquid fraction reduces the amount of glucose to less than 0.5 wt/wt% based on dry matter in the soluble fibre.
Usually, a process as disclosed herein may further comprise a step of bringing the material of step a) to a temperature of between 2 and 35 °C, for instance between 4 and 30 °C, for instance between 6 and 25 °C prior to separating the material into a solid fraction and a liquid fraction. It was found that bringing the material of step b) to a temperature of between 2 and 35°C increased the yield of the soluble fibre.
A process for preparing a soluble fibre from a fruit and / or a vegetable material as disclosed herein comprises bringing the fibre that has been precipitated into contact with an enzyme having endopolygalacturonase activity. An endopolygalacturonase is an enzyme that hydrolyses the alpha-1 ,4-glycosidic bonds between the galacturonic acid residues in pectate and galacturonans. An endopolygalacturonase is classified in classification number EC 3.2.1.15 and is also referred to as pectin depolymerase, pectinase, pectolase, pectin hydrolase, pectin polygalacturonase, endogalacturonase and poly- alpha-1 ,4-galacturonide glycanohydrolase.
Endopolygalacturonases may be derived from any suitable microorganism, for instance from bacteria of fungi. Suitable bacteria may be from the genus Bacillus, for instance from the species Bacillus coagulans, Bacillus subtilis. Suitable fungi may be from the genus Aspergillus, Penicillium, Trichoderma, Saccharomyces, or Kluyveromyces, for instance from the species A. niger, A. oryzae, A. tubigensis, P. chrysogenum, T. reesei, S. cerevisiae, or K. lactis. Advantageously the endopolygalacturonase is derived from Aspergillus tubigensis for instance as disclosed in WO00/17367.
An enzyme having endopolygalacturonase activity in a process as disclosed herein may be a pure enzyme. A pure enzyme as used herein may be at least 50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 80% pure, at least 90% pure, or at least 95% pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% as determined by SDS-PAGE or any other analytical method suitable for this purpose and known to the person skilled in the art.
An enzyme having endopolygalacturonase activity may be an enzyme comprising less than 2 wt%, such as less than 1 wt% or less than 0.5 wt% of an enzyme having pectinmethylesterase activity, of the total weight of enzymes having endopolygalacturonase activity and pectinmethylesterase activity. The amount of enzymes can be determined by SDS-PAGE and / or LC-MS methods or other analytical methods known to a person skilled in the art.
Pectinmethylesterase activity can be determined using methylated pectin, preferably pectin with a degree of methylation over 70%, and titrimetric analysis of the hydrolysis of the methyl-esters and the concomitantly produced acid, resulting in a number of micromoles of ester hydrolysed per minute, e.g. at pH 4.5 and 30°C.
Any suitable amount of an enzyme having endopolygalacturonase activity may be contacted with the fruit or vegetable material. For instance, an enzyme having an endopolygalacturonase activity may be contacted with the fruit and / or vegetable material in an amount of 0.1 PGU - 100 PGU / g dry weight, such as 0.2 to 80 PGU / g dry weight, or 0.3 to 50 PGU / g dry weight, or 0.4 to 25 PGU / g dry weight, or 0.5 to 10 PGU / g dry weight, or 0.6 to 5 PGU / g dry weight. It was found that a lower amount of endopolygalacturonase resulted in a higher relative amount of soluble fibres with a degree of polymerization of DP2 to DP10.
A process as disclosed herein may further comprise a step of removing starch from the fruit and /or vegetable material. A step of removing starch may be performed prior to incubating the fruit and / or vegetable material at a pH of between 0.5 and 3 and a temperature of between 60 and 98°C. Removing starch from a fruit or vegetable material may comprise incubating the fruit and / or vegetable material with an enzyme having amylase activity, for instance amylase activity may comprise alpha amylase (EC 3.2.1 .1.), maltogenic alpha amylase (EC 3.2.1.133) and / or glucoamylase (EC 3.2.1 .3) activity. An amylase may be a thermostable amylase. Amylases may be obtained from any suitable origin, for instance from bacteria or fungi.
Incubating the fruit and / or vegetable material with an amylase may be performed at any suitable pH and temperature depending on the amylase used, which is known to a person skilled in the art. Incubating the fruit and / or vegetable material with an enzyme having amylase activity, usually further comprises a step of washing the fruit and / vegetable material that has been treated with an amylase and / or a step of separating the fruit and / or vegetable material that has been treated with an amylase into a fruit and / or vegetable material residue and a liquid phase. Separating is usually performed by centrifugation and / or filtration.
Removing starch from a fruit or vegetable material may comprise adding water to the fruit and / or vegetable material. Removing starch may also comprise bringing the temperature of the fruit and / or vegetable material to a temperature of 55 to 85°, for instance a temperature of between 60 and 75°C that allows gelatinizing of starch, prior to incubating the fruit and / or vegetable with an enzyme having amylase activity.
A process as disclosed herein may further comprise a step of removing protein from the fruit and / or vegetable material. A step of removing protein in a process as disclosed herein may be performed prior to incubating the fruit and / or vegetable material at a pH of between 0.5 and 3 and a temperature of between 60 and 98°C as described herein above. Removing protein may comprise incubating the fruit and / or vegetable material with a protease. There are many proteases available in the art. Proteases are enzymes that hydrolyse the peptide bonds between adjacent amino acids, and are classified in enzyme classification EC 3.4. [..], and may be derived from any suitable microorganism such as bacteria and fungi. Incubating the fruit and / or vegetable material with a protease may be performed at any suitable pH and temperature depending on the protease used, which is known to a person skilled in the art.
A process as disclosed herein may comprise a step of drying the fruit and / or vegetable material and / or drying the precipitated fibre. Drying as used herein may be performed in any suitable way for instance by freeze drying known to a person skilled in the art.
A process for preparing a soluble fibre as disclosed herein may further comprise a step of inactivating the enzymatic activity of endopolygalacturonase, and / or inactivating the activity of amylase and / or protease. Inactivating enzymatic activity may for instance be performed by heating the fruit and / or vegetable material and / or the fibre to a temperature suitable to inactivate the enzymatic activity, for instance a temperature of between 70°C and 100°C, such as a temperature of between 85°C and 98°C.
The fruit and / or vegetable material in a process for preparing soluble fibres comprises pectin. The fruit and/ or vegetable material may comprise a husk, hull or peel from fruit and / or vegetable material. The fruit and / or vegetable material may for instance be derived from cassava, potato, onion, apple and / or citrus fruits. The fruit and / orvegetable material may comprise cassava pulp, potato pulp, onion hulls apple pomace and / or citrus peel. A soluble fibre that is prepared in a process as disclosed herein comprises a galacturonan oligosaccharides having a degree of polymerization (DP) of between 2 and 20, for instance a DP of between 2 and 12, for instance a DP of 3 to 10, for instance a DP of 3 to 7. Soluble fibre may comprise galacturonan monomer, i.e. galacturonan having a DP of 1.
A soluble fibre as disclosed herein may further comprise galactan, rhamnan and / or arabinan, for instance galactan, rhamnan, and / or arabinan having a DP of between 2 and 50, such as a DP between 4 and 25.
Degree of polymerization is defined as the number of monomeric units in an oligomer or polymer molecule. A monomeric unit in a soluble fibre as disclosed herein may for instance be galacturonic acid, galactose, rhamnose, and / or arabinose.
A soluble fibre that is prepared in a process as disclosed herein comprises 0 to 0.5 wt/wt% of glucose on dry matter, for instance between 0.1 and 0.2 wt/wt% of glucose. A soluble fibre may comprise 0 wt/wt% of glucose. Glucose as used herein refers to glucose monomers and glucose dimers, maltodextrins, starch, and / or starch derivatives comprising alpha-(1 ,4)-glucose linkages.
In one aspect, the present invention relates to a soluble fibre obtainable by a process as disclosed herein. All the elements of a soluble fibre prepared by a process as described herein above are embodiments of a soluble fibre obtainable by a process as disclosed herein.
In one other aspect, the present invention relates to a composition comprising a soluble fibre as disclosed herein. A composition as disclosed herein may further comprise water, proteins, lipids and / or carbohydrates.
FIGURES
Figure 1. Diagram of different soluble fibre extraction methods A, B and C
MATERIALS AND METHODS
Materials
Cassava pulp, the residue of the cassava tubers after removal of the starch at industrial scale (Corn products Co., Ltd, Thailand) with a dry matter content of 16% was used.
Polygalacturonic acid from oranges, rhamnose, arabinose, xylose, galactose, mannose, glucose, and galacturonic acid were purchased from Sigma.
Sugar Composition Analysis of Solid Materials
Total sugar composition was determined based on NMR analysis of acid hydrolyzed sugars according the method published in A. Carvalho de Souza ef a/., Carbohydrate Polymers, 2013 (95), 657-663. Starch was determined after hydrolysis in 0.6M D2SO4 for 1.5 h at 100°C followed by released glucose quantification based on NMR (after addition of maleic acid as internal standard). Sugar Composition Analysis and Oligosaccharide Profiling of Soluble Materials
Complete hydrolysis of soluble neutral polysaccharides and pectin was performed by hydrolyzing 1 - 5 mg of cassava pectin extract with 1 M H2SO4 at 99 °C for 3 hours. Analysis of the neutral monosaccharides was performed using high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). After removal of H2SO4, using Amberlite ion exchange resin, samples were separated on a CarboPac PA-20 column preceded by a CarboPac PA-20 guard-column. After injection, the sample was eluted for 19 minutes in 5 mM NaOH at a flow rate of 0.5 ml/min. Then, a 4 minutes gradient to 380 mM NaOH was used. After 6 min of washing with 380 mM NaOH, the column was equilibrated in 5 mM NaOH again. Rhamnose, arabinose, xylose, mannose, galactose and glucose were quantified using response factors of the pure compounds. Analysis of galacturonic acid was performed using HPAEC with PAD, using a different eluent composition and gradient. After injection, the sample was eluted for 2 minutes in 200 mM NaOAc in 100 mM NaOH at a flow rate of 0.5 ml/min. Then a 30min linear gradient of 200-700 mM NaOAc in 100 mM NaOH was used, followed by a 2 min gradient to 1000 mM NaOAc. After 5 min of washing with 1000 mM NaOAc in 100 mM NaOH the column was equilibrated in 200 mM NaOAc in 100 mM NaOH. Galacturonic acid monomer was quantified using the response factor of the pure compound. The profiling of the galacturonic acid oligosaccharides was performed using the same equipment and latter gradient, but the sample was not hydrolyzed with H2SO4. Protein Content
The protein content was determined according to the Kjeldahl method, as known in the art, and using a standard conversion factor of N x 6.25.
Enzymes
Aspergillus tubigensis endopolygalacturonase, Rapidase® EPG NS L, was obtained from
DSM and has an activity of 20,000 PGU / mL. One PGU is the amount of enzyme per mL enzyme solution that creates a line with a slope of 0.068 when the ratio of the viscosity of a blank versus that of the test solution is plotted against the incubation time. Conditions of the test are: 1.75% sodiumpolygalacturonate at pH 4.5 and 45°C. Viscosity reduction was determined using an Ubbelohde 1 C.
Delvolase, bacterial protease > 580.000 DU/g was obtained from DSM. One DU is defined as the amount of enzyme needed to liberate 7.59 μιηοΐβ of para-nitroaniline per minute at pH 8.5 and 37°C, using 2.18 mg/ml of succinyl-Ala-Ala-Pro-Phe-para-nitroanilide as substrate.
MATS L Classic, thermostable bacterial oamylase (> 7400 TAU/g), was obtained from DSM. One TAU is defined as the amount of enzyme converting 1 .0 mg starch per minute, having an equal absorbance at 620 nm to a reference solution (25 g C0CI2.H2O and 3.84 g of potassium dichromate in 100 mL of 0.01 N hydrochloric acid) after reaction with an iodine solution at pH 6.6 at 30°C. The substrate solution contains 13.3 mg/ml starch EXAMPLE 1. COMPARISON OF SOLUBLE FIBRE EXTRACTION METHODS
Three different methods for isolation of soluble fibre from cassava pulp were compared. The three methods are indicated as methods A, B and C and presented in Figure 1.
In Method A, cassava pulp was suspended in 100 mM citrate buffer of pH 4 at 2% (w/w) dry matter and soluble fibre was prepared by directly incubating the cassava pulp with Rapidase® EPG NS L endopolygalacturonase at 2090 PGU / g dry matter (DM).
In Method B crude cell wall material (CCWM) was first prepared from cassava pulp as described in Example 2.1. Subsequently, CCWM was suspended in 100 mM citrate buffer of pH 4 at 2% (w/w) dry matter and soluble fibre was prepared by incubating the CCWM with Rapidase® EPG NS L endopolygalacturonase at 2090 PGU / g dry matter (DM).
In Method C, CCWM was prepared from cassava pulp as described in Example 2.1 , followed by the selective fibre extraction described in Example 2.2. The fibre extract was suspended in 100 mM citrate buffer of pH 4 at 2% (w/w) and soluble fibre was prepared by incubating the fibre extract with Rapidase® EPG NS L endopolygalacturonase (50 PGU / g dry matter (DM)).
The soluble fibres (Non-Starch Sugars (NSS)) obtained in Methods A, B and C were determined as disclosed in the Materials and Methods section.
In Table 1 , the yield and compositions of the soluble fibres isolated from cassava pulp using the three different methods of isolation A, B and C are given. Soluble fibre prepared according to Method C, contained the highest percentage of non-starch sugars (NSS).
Non-Starch Sugars may comprise poly-, oligo and monosaccharides.
Table 1 : Yield of soluble fibres (total dry matter NSS) and sugar composition of soluble fibres after extraction from cassava pulp using three different methods of isolation A, B, C
Figure imgf000010_0001
a expressed as % on dry matter of cassava pulp
b expressed as % (w/w) on dry matter per fraction
c expressed as % (w/w) based on polymer base, which means excluding the molar weight of water
d calculated by deduction of protein, starch and NSS; it is expected to contain lignin, salts, fat
EXAMPLE 2: ISOLATION OF SOLUBLE FIBRE FROM CASSAVA WITH DIFFERENT RATIO OF GALACTURONAN -OLIGOSACCHARIDES
2.1. Isolation of Crude Cell Wall Material (CCWM)
500 gram of cassava pulp wet material was mixed with 500 mL water to which 30 ppm calcium was added and the pH was adjusted to pH 6.5, with 4N NaOH. 0.5 mL of thermostable bacterial oamylase was added and the suspension was incubated in a water bath of 75°C for 3.5 h, while shaking at 60 rpm. Additionally, the suspension was shaken more rigorously at intervals of 30 minutes.
After cooling to 25°C the suspension was centrifuged and the supernatant was removed. The residue was resuspended in water to a total volume of 800 mL, and the amylase incubation was repeated. The final residue was washed twice with 200 mL water. This residue was resuspended in water to a volume of approximately 800 mL. The pH was adjusted to pH 7.6 with 4 N NaOH and 2 mL of bacterial protease was added and the suspension was incubated at 60°C in a shaking incubator (60 rpm) for 3.5 hours, with rigorous shaking at 30 minutes intervals. The protease was denatured by heating the suspension to 75-80°C during 1 h. After cooling to 25°C the residue was separated from the liquid by centrifugation, and freeze-dried.
Table 2 shows that the isolation of CCWM from industrial cassava pulp resulted in enrichment of the non-starch sugars (NSS) fraction from 31 % of the original dry matter to 63% in the CCWM dry matter. NSS may comprise polysaccharides, oligosaccharides and / or monosaccharides.
The removal of starch was nearly complete, with only 2% of the CCWM dry matter being starch versus 53% of the original pulp dry matter. The protein content was only 2% of the pulp dry matter, and ending up at a similar level in the isolated CCWM.
2.2. Selective Fibre Extraction
10 gram of freeze-dried cassava CCWM was dispersed in 200 mL 0.5% HCI, and pH was adjusted to pH 1.8, using 4N NaOH. The dispersion was incubated in a water bath of 82°C, while shaking (50 rpm) for 105 minutes. After cooling to 25°C the supernatant was collected by centrifugation. The residue was washed with a total amount of 50 ml of 0.5% HCI. The final volume of the extract and wash liquid was 200 mL. 400 mL of absolute ethanol of 4°C was added to the crude extract and after mixing, the slurry was left to precipitate at room temperature for 1 hour. The clear supernatant was discarded after centrifugation, and the residue was washed with 75 mL of acidic 70% ethanol, subsequently with 70% ethanol until pH was 5.5, and finally with absolute ethanol. The washed pectin pellet was freeze-dried.
After the selective fibre extraction and ethanol precipitation, a slightly off-white non-starch polymer gel was obtained. Table 2 shows that the non-starch sugars (NSS) comprised galacturonic acid and galactose as main sugars, and minor amounts of rhamnose, arabinose and xylose.
Table 2: Composition of the main fractions of cassava pulp, crude cell wall material (CCWM) and fibre extract, extracted from cassava pulp
Figure imgf000012_0001
a Non Starch Sugars (NSS) expressed as % (w/w) based on polymer base, which means excluding the molar weight of water
b calculated by deduction of protein, starch and NSS; it is expected to contain lignin, salts, fat
2.3. Enzymatic Galacturonan Oligosaccharide Production
Fibre extract obtained in Example 2.2 was suspended in 100 mM citrate buffer of pH 4 at 2% (w/w) dry matter, and different doses of endopolygalacturonase as indicated in Table 1 were added and subsequently each mixture was incubated at 40°C for 1 h. The enzymatic reaction was stopped by boiling the incubation mixture for 5 min.
The results in Table 3 show that enzyme dosages in the range of 0.8 to 50 PGU / g dry matter (1.6- 10"2 to 1.0 PGU / mL) resulted in galacturonan-oligosaccharides with a different ratio of the degree of polymerization (DP). The relative amounts of oligosaccharides of DP 7-9 (not shown) were lower than for DP6, but they increased slightly with lower enzyme dose. The results in the table also show slight increases in levels of DP > 4 and decreases in levels of DP < 3, when a lower enzyme dose is applied. Table 3: Relative amounts of galacturonan saccharides with a degree of polymerization (DP) of 1 to 6 obtained after enzymatic hydrolysis of fibre extract derived from cassava pulp, with different doses of enzyme (PGU / g dry matter (DM)
Figure imgf000013_0001
EXAMPLE 3: ISOLATION OF SOLUBLE FIBRE FROM CASSAVA WITH ADAPTED
PROTOCOLS
3.1. Isolation of Crude Cell Wall Material (CCWM)
Crude cell wall material was prepared from three separate cassava pulp batches of 500 g according to the procedure disclosed in Example 2.1 , with the exception that only one amylase incubation step was applied and the protease incubation was omitted.
Table 4 shows that the yield of dry matter recovered from CCWM was 50%. This dry matter consisted for 57% of its dry matter of non-starch sugars, while protein content was similar as in Example 1 and residual starch content was higher.
The three isolated CCWM batches were pooled and split into two batches of 630 g, which contained 37.8 g dry matter each, were further used for fibre extraction and one batch of 680 g CCWM, which was not further used.
3.2. Selective Fibre Extraction
To each batch of 630 g CCWM produced in Example 3.1. 120 mL of 3% HCI was added, and the pH was adjusted to 1.5 using 4 M NaOH. The suspensions were incubated at 82°C for 165 min. Washing of the residue and the ethanol precipitation was subsequently performed in a similar way as described in Example 2, while adapting the actual volumes to have a similar solid to liquid ratio. The finally isolated pectin pellet was freeze-dried. The dry matter recovery based on the starting material was 3.3%.
In Table 4 an overview is given of the yield and the composition of the main fractions in the process of CCWM and fibre isolation. Table 4: Yield and composition of the dry matter of cassava pulp, and crude cell wall material (CCWM) and fibre extract from cassava pulp
Figure imgf000014_0001
a expressed as % on dry matter of cassava pulp
b expressed as % (w/w) on dry matter per fraction
c expressed as % (w/w) based on polymer base, which means excluding the molar weight of water
d calculated by deduction of protein, starch and NSS; it is expected to contain lignin, salts, fat
The results in Table 4 show that more starch is present in the CCWM in Example 3.1 than in the CCWM isolated in Example 2.1. However, no remaining starch in the final fibre extract was detected.
3.3. Enzymatic Galacturonan Oligosaccharide Production
The fibre extract obtained in Example 3.2 was incubated with endopolygalacturonase at a dose of 0.13 PGU / mL (6.5 PGU / g DM) of fibre extract solution at 5% dry matter content according to the method disclosed in Example 2.3. The resulting relative amount of the galacturonan oligosaccharides was 9 : 10 : 7 : 4 : 3 : 2 for DP1 : DP2 : DP3 : DP4 : DP5 : DP6.

Claims

1. A process for preparing a soluble fibre comprising galacturonan oligosaccharide having a degree of polymerization (DP) of 2 to 20 from a fruit and / or vegetable material comprising
(a) removing starch from the fruit and / or vegetable material,
(b) incubating the fruit and / or vegetable material of step a) at a pH of between 0.5 to 3 and a temperature of between 60 and 98°C,
(c) separating the material from step b) into a solid fraction and a liquid fraction, (d) isolating fibre from the liquid fraction, and
(e) bringing the fibre from step d) into contact with an enzyme having endopolygalacturonase activity,
and preparing the soluble fibre.
2. The process according to claim 1 , wherein incubating in step b) is performed during 30 to 300 min.
3. The process according to claim 1 or 2, wherein isolating fibres comprises adding ethanol to the liquid fraction at a temperature of between 0 to 25°C.
4. The process according to any one of the claims 1 to 3, wherein the process further comprises a step of bringing the material of step a) to a temperature of between 2 and 35 °C prior to step b).
5. The process according to any one of the claims 1 to 4, wherein the process further comprises a step of removing protein from the fruit and / or vegetable material.
6. The process according to any one of the claims 1 to 5, wherein the enzyme having endopolygalacturonase activity comprises less than 2 wt% of an enzyme having exopolygalacturonase activity and/ or an enzyme having pectinmethylesterase activity, of the total weight of enzymes having endopolygalacturonase activity, exopolygalacturonase and / or pectinmethylesterase activity.
7. The process according to any one of the claims 1 to 6, wherein the enzyme having endopolygalacturonase activity is contacted with the fruit and / or vegetable material in an amount of 0.1 PGU - 100 PGU / g dry weight.
8. The process according to any one of the claims 1 to 7, wherein the fruit or vegetable material comprises pectin.
9. The process according to any one of the claims 1 to 8, wherein the fruit or vegetable material is derived from cassava, onion, apple and / or citrus fruits.
10. The process according to any one of the claims 1 to 9, wherein the soluble fibre further comprises galacturonan monomers.
1 1. The process according to any one of the claims 1 to 10, wherein the soluble fibre further comprises a galactan, rhamnan and / or arabinan oligomers.
12. The process according to any one of the claims 1 to 1 1 , wherein the soluble fibre comprises 0 to 0.5 wt/wt% of glucose.
13. The soluble fibre obtainable by a process according to any one of the claims 1 to 12.
14. A composition comprising a soluble fibre according to claim 13.
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CN117512032A (en) * 2023-11-07 2024-02-06 北京市农林科学院 Preparation method and conformational characterization of low molecular weight HG pectin
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