阅读说明：本技术 生产富含鼠李半乳糖醛酸聚糖-i的果胶多糖分离物的方法 (Method for producing pectin polysaccharide isolate rich in rhamnogalacturonan-I ) 是由 路德·阿尔伯斯 玛丽亚·佐祖玛基 于 2018-10-23 设计创作，主要内容包括：一种生产富含鼠李半乳糖醛酸聚糖-1的水解果胶多糖分离物的方法,包括：提供在不使用有机溶剂从植物材料获得的富含果胶底物,富含果胶底物包含果胶多糖的至少3重量％的干物质；对富含果胶底物进行酶促处理以部分地水解果胶多糖,酶促处理包括使用选自果胶裂解酶(EC4.2.2.10)、果胶酸裂解酶(EC 4.2.2.2)、鼠李半乳糖醛酸聚糖半乳糖醛酸酶(EC 3.2.1.173)、内聚半乳糖醛酸酶(EC 3.2.1.15)、外聚半乳糖醛酸酶(EC 3.2.1.67和EC 3.2.1.82)的一或多种果胶酶；使用截留分子量在5至100kDa范围内的超滤膜对部分水解的果胶多糖进行超滤；及回收超滤渗余物。还涉及通过本方法获得的水解果胶多糖分离物,及制备选自营养制剂、食物产品、膳食补充剂、饮料或药物产品的产品的方法,方法包括添加上述水解果胶多糖分离物。(A process for the production of a rhamnogalacturonan-1 enriched hydrolysed pectin polysaccharide isolate comprising: providing a pectin-rich substrate obtained from a plant material without the use of an organic solvent, the pectin-rich substrate comprising at least 3 wt.% of dry matter of the pectic polysaccharide; subjecting the pectin-rich substrate to an enzymatic treatment to partially hydrolyze the pectic polysaccharide, the enzymatic treatment comprising the use of one or more pectinases selected from the group consisting of pectin lyase (EC4.2.2.10), pectate lyase (EC 4.2.2.2), rhamnogalacturonase (EC 3.2.1.173), polygalacturonase (EC 3.2.1.15), exopolygalacturonase (EC3.2.1.67 and EC 3.2.1.82); subjecting the partially hydrolysed pectic polysaccharide to ultrafiltration using an ultrafiltration membrane having a molecular weight cut-off in the range of 5 to 100 kDa; and recovering the ultrafiltration retentate. Also relates to a hydrolysed pectin polysaccharide isolate obtainable by the process, and to a process for preparing a product selected from a nutritional formulation, a food product, a dietary supplement, a beverage or a pharmaceutical product, the process comprising adding the above hydrolysed pectin polysaccharide isolate.)

1. A method for producing a rhamnogalacturonan-I rich hydrolysed pectin polysaccharide isolate, the method comprising the steps of:

providing a latex-rich substrate which has been obtained from plant material without the use of organic solvents, said latex-rich substrate comprising at least 3 wt% of dry matter of a pectin polysaccharide having a backbone consisting of galacturonic acid residues and rhamnose residues, the rhamnose residues being contained in the backbone consisting of galacturonic acid residues and rhamnose residues, the rhamnose residues being contained in α (1 → 4) -galacturonic acid- α (1 → 2) -rhamnose residues;

subjecting the pectin-rich substrate to an enzymatic treatment comprising the use of one or more pectinases selected from the group consisting of pectin lyase (EC4.2.2.10), pectate lyase (EC 4.2.2.2), rhamnogalacturonan galacturonase (EC 3.2.1.173), cohesive galacturonase (EC 3.2.1.15), extrinsic galacturonases (EC3.2.1.67 and EC 3.2.1.82) to partially hydrolyze the pectic polysaccharide;

ultrafiltration of the partially hydrolysed pectic polysaccharide using an ultrafiltration membrane having a molecular weight cut-off of 5 to 100 kDa; and

recovery of the ultrafiltration retentate.

2. The method of claim 1, wherein the pectin-rich substrate is selected from the group consisting of pomace, an aqueous extract of pomace, an oil cake, an aqueous extract of oil cake, and combinations thereof.

3. The method according to claim 1 or 2, wherein the pectin-rich substrate is obtained from one or more crops selected from the group consisting of fruit, carrot, olive, pea, beet, chicory, soybean, sunflower, rapeseed, corn.

4. The method of claim 3, wherein said pectin isolate is obtained from citrus, apple, pear, sugar beet, chicory, carrot.

5. The method according to any of the preceding claims, wherein the pectin-rich substrate comprises at least 10-80 wt.% of dry matter of a cellulosic material selected from the group consisting of cellulose, hemicellulose and combinations thereof.

6. The method according to claim 5, wherein at least 50 wt.% of the pectic polysaccharides present in the substrate are insoluble in distilled water at 45 ℃ and pH 5.5 at a pectic polysaccharide concentration of 1 g/l.

7. The process according to any one of the preceding claims, wherein the aqueous liquid comprising the partially hydrolysed pectic polysaccharides is subjected to solid-liquid separation after the enzymatic treatment and before ultrafiltration.

8. The method according to any one of claims 1-3, wherein the pectin-rich substrate is a pectin isolate comprising at least 10 wt.% of dry matter of the pectin polysaccharide.

9. The process according to any one of the preceding claims, wherein galacturonic acid residues and rhamnose residues are present in the recovered ultrafiltration retentate in a molar ratio of not more than 6: 1.

10. The method according to any one of the preceding claims, wherein the pectin-rich substrate is enzymatically treated in the form of an aqueous liquid comprising the pectin-rich substrate and having a dry matter content of 0.5-40 wt.%.

11. The method according to any one of the preceding claims, wherein the enzymatic treatment is carried out at a pH range of 3.0 to 7.5.

12. The method according to any one of the preceding claims, wherein the one or more pectinases are selected from the group consisting of pectin lyase (EC4.2.2.10), pectate lyase (EC 4.2.2.2), cohesive galacturonase (EC 3.2.1.15) and exogalacturonase (EC3.2.1.67 and EC 3.2.1.82).

13. The process according to any one of the preceding claims, wherein the pectin-rich substrate is subjected to an enzymatic treatment comprising the use of one or more pectinesterase enzymes (EC3.1.1.11) prior to or simultaneously with the partial hydrolysis with the one or more pectinases.

14. The method according to any one of the preceding claims, wherein the partially hydrolysed pectic polysaccharide is subjected to ultrafiltration using an ultrafiltration membrane having a molecular weight cut-off of 6-50 kDa.

15. The process according to any one of the preceding claims, wherein the recovered ultrafiltration retentate is dried to a water content of less than 15 wt.%.

16. The process according to any one of the preceding claims, wherein the recovered ultrafiltration retentate comprises at least 20 wt.% of dry matter of pectin polysaccharides comprising on average at least 4 mol.% rhamnose residues, calculated on the total monosaccharide composition of the pectin polysaccharides.

17. A hydrolysed pectin polysaccharide isolate obtained by the method according to any one of the preceding claims.

18. The hydrolyzed pectin polysaccharide isolate of claim 17 wherein the galacturonic acid residues and rhamnose residues are present in the hydrolyzed pectin polysaccharide isolate in a molar ratio of not more than 6: 1.

19. The hydrolyzed pectin polysaccharide isolate of claim 17 or 18, wherein the isolate comprises less than 5% by weight saccharides having a molecular weight of less than 20 kDa.

20. The hydrolyzed pectin polysaccharide isolate of any one of claims 17-19, wherein the combination of galacturonic acid residues, rhamnose residues, arabinose residues and galactose residues constitute at least 50 mol.% of the monosaccharide residues present in the saccharides comprised in the hydrolyzed pectin polysaccharide isolate.

21. A method of manufacturing a product selected from a nutritional formulation, a food product, a dietary supplement, a beverage or a pharmaceutical product, the method comprising adding the hydrolysed pectin polysaccharide isolate according to any one of claims 17-20 at a concentration of at least 0.1 wt% dry matter in the final product.

Technical Field

The present invention relates to a process for the production of a pectin polysaccharide isolate rich in rhamnogalacturonan-I (RG-1) polysaccharide. More particularly, the present invention relates to a process for producing such polysaccharide isolates by enzymatic hydrolysis of a pectin-rich substrate, which is obtained from plant material without the use of organic solvents and which comprises a large amount of RG-1 polysaccharide, followed by ultrafiltration and recovery of the retentate.

Background

Pectin is a structural heteropolysaccharide present in the primary cell wall of terrestrial plants. The pectin polysaccharide composition and fine structure vary widely depending on the plant source and the extraction conditions applied. The biological activity of the pectic polysaccharides is highly dependent on their fine structure.

Pectin polysaccharides are a heterogeneous group of polysaccharides comprising different amounts of the following polysaccharide components:

(i) (ii) a homogalacturonic acid glycan (HG),

(ii) xylogalacturonan (XG),

(iii) celery galacturonic Acid Glycan (AG)

(iv) rhamnogalacturonan-I (RG-I), and

(v) rhamnogalacturonan-II (RG-II).

Figure 1 provides a schematic representation of the structure of a pectin polysaccharide, comprising the 4 polysaccharide components described above. Note that the polysaccharide components AG, XG and RG-II generally represent only a minor fraction of the pectin polysaccharides.

The polysaccharide components HG, XG and RG-II each comprise a backbone consisting of a linear chain of alpha- (1-4) -linked D-galacturonic acid monosaccharide units.

Only RG-I comprises a backbone consisting of a linear chain of disaccharide units repeated: 4) - α -D-galacturonic acid- (1,2) - α -L-rhamnose- (1). A schematic representation of the structure of RG-I is shown in fig. 2.

Homogalacturonan domains may be up to about 100 contiguous D-GalA residues in length. The RG-I domains containing side chains are commonly referred to as "branched" or "hairy" regions, whereas homogalacturonan domains (between the two RG-I domains) are not typically substituted by oligosaccharides.

The GalA residues in RG-I are linked to the Rha residue via the 1 and 4 positions, while the Rha residue is linked to the GalA residue via the anomeric and 2-OH positions. Generally, about 20-80% of the Rha residues are branched at the 4-OH position (depending on plant origin and isolation method), with neutral and acidic side chains. These side chains consist essentially of Ara residues and Gal residues linked in various ways, constituting polymers known as arabinogalactans I (AG-I) and/or AG-II. AG I consists of a β - (1,4) -linked D-Gal backbone which is an α -L-arabinosyl substituent at the 3-OH; the Gal skeleton may have spaced α (1,5) -L-Ara units. AG-II consists of highly branched galactans, with β (1,3) linked D-Gal mainly on the inside and short (1,6) linked chain substituents on the outside. The latter have additional attachments of (1,3) -and/or α (1,5) -linked L-Ara. The oligosaccharide side chains may be straight or branched chain, and some of these side chains may be terminated by α -L-fucoside, β -D-glucuronide and 4-O-methyl β -D-glucuronic acid residues.

Khodaii and Karbource ("Extraction and structural characterization of rhamnogalacturonan I-type polysaccharides from cell wall". Foodchemistry,1:39(2013), page 617 and 623) describe the Extraction of galacturonan-rich rhamnogalacturonan I (RG-I) type pectic polysaccharides using alkaline (NaOH and KOH) and enzymatic (polygalacturonase from Aspergillus niger) methods.

Khodaii and Karbone ("Enzymatic extraction of galactan-richrhamnogalacturonan I from cell wall by-product". LWT-Food Science and technology, 57 (2014 edition), page 207-.

WO 2015/192247 describes a method of obtaining indigestible oligosaccharides from an extracted rhamnogalacturonan content by extracting the rhamnogalacturonan content from potato pulp, digesting the extracted rhamnogalacturonan content with a multi-enzymatic mixture; and a method for separating indigestible oligosaccharides from potato pulp.

WO 2016/132130 describes a method for preparing RG-I fragments from potato pulp and their use for providing immunomodulatory activity to a subject. The method comprises the following steps:

-providing RG-I obtained from enzymatic extraction of potatoes;

-preparing the RG-I fragment by selectively depolymerizing the RG-I to provide a fragment having an average molecular weight in the range of 5kDa to 30kDa, wherein the fragment has a monosaccharide composition comprising:

7 to 13 percent of arabinose,

5 to 10 percent of rhamnose,

0 to 1 percent of xylose,

galacturonic acid 20-40%, and

and 35-60% of galactose.

CN 102784193 a describes a method for separating polysaccharides from hedysarum polybotrys.

KR 2017/053144 a describes the extraction of polysaccharide fractions from barley leaves. The polysaccharide fraction is described as exhibiting immune function promoting activity.

Summary of The Invention

The inventors have found that polysaccharide isolates enriched in rhamnogalacturonan-I (RG-I) with high biological functionality can be obtained by a process comprising:

subjecting a pectin-rich substrate which has been obtained from plant material without the use of organic solvents and which comprises a large amount of RG-I polysaccharide to enzymatic hydrolysis to partially hydrolyze the RG-I polysaccharide;

ultrafiltration of the partially hydrolysed RG-I polysaccharide using an ultrafiltration membrane having a cut-off value in the range of 5 to 100 kDa;

and recovering the ultrafiltration retentate.

Enzymatic hydrolysis by means of one or more pectinases selected from the group consisting of pectin lyase (EC4.2.2.10), pectate lyase (EC 4.2.2.2), rhamnogalacturonan galacturonase (EC 3.2.1.173), cohesive galacturonase (EC 3.2.1.15), extrinsic galacturonases (EC3.2.1.67 and EC 3.2.1.82).

Accordingly, a first aspect of the present invention relates to a process for the production of a rhamnogalacturonan-1 rich hydrolysed pectin polysaccharide isolate, the process comprising the steps of:

providing a latex-rich substrate which has been obtained from plant material without the use of organic solvents, said latex-rich substrate comprising at least 3 wt% of dry matter of a pectin polysaccharide having a backbone consisting of galacturonic acid residues and rhamnose residues, the rhamnose residues being contained in the backbone consisting of galacturonic acid residues and rhamnose residues, the rhamnose residues being contained in α (1 → 4) -galacturonic acid- α (1 → 2) -rhamnose residues;

subjecting the pectin-rich substrate to an enzymatic treatment comprising the use of one or more pectinases selected from the group consisting of pectin lyase (EC4.2.2.10), pectate lyase (EC 4.2.2.2), rhamnogalacturonan galacturonase (EC 3.2.1.173), cohesive galacturonase (EC 3.2.1.15), extrinsic galacturonases (EC3.2.1.67 and EC 3.2.1.82) to partially hydrolyze the pectic polysaccharide;

ultrafiltration of the partially hydrolysed pectic polysaccharide using an ultrafiltration membrane having a molecular weight cut-off of 5 to 100 kDa; and

recovery of the ultrafiltration retentate.

The inventors have found that partially enzymatically hydrolyzing a pectic polysaccharide using one or more of the aforementioned pectinases, followed by removal of small molecule components including small molecules during hydrolysis, results in a hydrolyzed pectic polysaccharide isolate exhibiting exceptionally high biological function.

While the inventors do not wish to be bound by theory, it is believed that this high biological function is achieved by removing at least a portion of the homogalactose domain of the pectic polysaccharide contained in the substrate and by removing inactive components such as cellulose and hemicellulose. Removal of the homogalacturonan domains alters the physicochemical properties of the pectic polysaccharides, resulting in a three-dimensional molecular configuration that interacts more efficiently with so-called pattern recognition receptors in the gut and on human peripheral blood mononuclear cells. Removal of the homogalacturonic acid domain from the pectic polysaccharide increases the content of the RG-I domain. It is believed that the interaction of the RG-I polysaccharide domains with pattern recognition receptors expressed on intestinal cells and other immunocompetent cells may modulate their functional reactivity, which may improve resistance to infections in the intestinal tract and other sites in the body including the oral cavity, respiratory tract, urinary tract, vagina, and skin through the production of mediators and recirculation of immunocompetent cells.

The present process further provides the advantage that the highly active hydrolysed pectin polysaccharides are obtained from pectin-rich substrates that have been produced without the use of organic solvents. Thus, unlike some prior art methods that utilize organic solvent precipitation to aid in the isolation of pectic polysaccharides, the present method employs a pectin-rich substrate that is not in contact with an organic solvent, and further processing of such substrate according to the present method does not require the use of an organic solvent.

The invention further relates to a hydrolysed pectin polysaccharide isolate obtainable by the process comprising the addition of the aforementioned hydrolysed pectin polysaccharide isolate and to the preparation of a product selected from a nutritional formulation, a food product, a dietary supplement, a beverage or a pharmaceutical product.

Detailed Description

Accordingly, a first aspect of the present invention relates to a method for providing a hydrolysed pectin polysaccharide enriched in rhamnogalacturonan-I, the method comprising the steps of:

providing a latex-rich substrate which has been obtained from plant material without the use of organic solvents, said latex-rich substrate comprising at least 3 wt% of dry matter of a pectin polysaccharide having a backbone consisting of galacturonic acid residues and rhamnose residues, the rhamnose residues being contained in the backbone consisting of galacturonic acid residues and rhamnose residues, the rhamnose residues being contained in α (1 → 4) -galacturonic acid- α (1 → 2) -rhamnose residues;

subjecting the pectin-rich substrate to an enzymatic treatment comprising the use of one or more pectinases selected from the group consisting of pectin lyase (EC4.2.2.10), pectate lyase (EC 4.2.2.2), rhamnogalacturonan galacturonase (EC 3.2.1.173), cohesive galacturonase (EC 3.2.1.15), extrinsic galacturonases (EC3.2.1.67 and EC 3.2.1.82) to partially hydrolyze the pectic polysaccharide;

ultrafiltration of the partially hydrolysed pectic polysaccharide using an ultrafiltration membrane having a molecular weight cut-off of 5 to 100 kDa; and

recovery of the ultrafiltration retentate.

The pectin-rich substrate used in the present method has been obtained from plant material without the use of an organic solvent, which means that the substrate is obtained from plant material without contacting the plant material with an organic solvent. Thus, pectin-rich substrates are not obtained by extraction or precipitation with organic solvents.

As used herein, the terms "backbone chain" and "backbone" are synonymous.

The term "saccharide" as used herein encompasses monosaccharides, double ponds, low sugars and polysaccharides.

The term "oligosaccharide" as used herein refers to a carbohydrate polymer comprising 3-10 monosaccharide residues.

The term "polysaccharide" as used herein, unless otherwise indicated, refers to a carbohydrate polymer comprising at least 11 monosaccharide residues.

The term "pectic polysaccharide" as used herein refers to an optionally branched polysaccharide having a molecular weight of at least 10kDa and comprising a backbone consisting of galacturonic acid residues and rhamnose residues, which rhamnose residues are comprised in α (1 → 4) -galacturonic acid- α (1 → 2) -rhamnose residues. The term "pectic polysaccharide" as used herein also encompasses hydrolyzed pectic polysaccharides having a molecular weight of at least 10kDa, unless otherwise indicated.

The term "branched polysaccharide" as used herein refers to a polysaccharide comprising a linear backbone chain of monosaccharide units joined together by glycosidic linkages, wherein at least one monosaccharide unit within the backbone chain carries a side chain of one or more glycosidically linked monosaccharide units.

The term "fragment" as used herein refers to the sequence of two glycoside-linked monosaccharide units within the polysaccharide backbone, excluding any side chains to which they are attached.

The term "domain" as used herein refers to a fragment plus any side chains attached to the fragment.

The term "rhamnogalacturonan-I fragment" or "RG-I fragment" refers to a fragment consisting of a galacturonic acid (GalA) and rhamnose (Rha) pair, wherein the GalA residue is linked to the Rha residue via the 1 and 4 positions, and the Rha residue is linked to the GalA residue via the anomeric and 2-OH positions, i.e. alternating α (1 → 4) -galacturonic acid- α (1 → 2) -rhamnose residues. The RG-I domain may include side chains such as, for example, galactan, arabinosan, and arabinogalactan side chains.

The term "rhamnogalacturonan-I" or "RG-I" polysaccharide refers to an optionally branched pectin polysaccharide comprising a backbone comprising one or more rhamnogalacturonan-I fragments.

The term "α (1,4) -linked galacturonic acid segment" refers to a segment consisting of α (1 → 4) -galacturonic acid residues.

In addition to the RG-I domain, the hydrolysed pectin polysaccharide isolate obtained by the method of the present invention may comprise one or more of the following domains:

homogalacturonan (HG),

xylogalacturonan (XG),

apigalacturonan (AG)

rhamnogalacturonan-II (RG-II).

The domains XG, AG and RG-II usually represent only a minor fraction of the RG-I polysaccharide.

The HG domain, XG domain, AG and RG-II domain optionally present in the RG-I polysaccharide of the present invention comprise a backbone consisting of a linear chain of two or more alpha- (1-4) -linked D-galacturonic acids.

The HG domain does not comprise any side chains. The carboxyl group of the galacturonic acid residue within the backbone of the HG domain may be esterified. The esterified galacturonic acid can be present as methyl or acetyl esters.

The backbone of the XG domain comprises one or more side chains in the form of D-xylose.

The backbone of the AG domain comprises one or more side chains consisting of one or more D-apiose residues.

The backbone of RG-II contains one or more side chains that are not composed solely of D-xylose or D-apiose. The carboxyl group of the galacturonic acid residue within the backbone of the RG-II domain may be esterified. The esterified galacturonic acid can be present as methyl or acetyl esters.

The term "degree of acetylation" refers to the number of acetyl residues per galacturonic acid residue, expressed as a percentage.

The term "degree of methylation" refers to the number of methyl residues per galacturonic acid residue, expressed as a percentage.

The concentration of the different polysaccharides and their monosaccharide composition can be determined by analytical techniques known to the skilled person. After methane hydrolysis, the monosaccharide composition can be suitably determined by high performance anion exchange chromatography (HPAEC-PAD) combined with pulsed amperometric detection.

Molecular size distribution can be determined by high performance exclusion chromatography (using Refractive Index (RI) detection (concentration), light scattering detection (molecular mass detection), UV detection (indicating the presence of protein), and differential pressure detection (intrinsic viscosity detection).

The above analytical methods are described in the following journal: analytical Biochemistry, volume 207, phase 1, 1992 edition, page 176 (for methyl decomposition and neutral sugar analysis); and mol.nutr.food res, vol 61, phase 1, 2017 edition, 1600243 (for galacturonic acid analysis and molecular size distribution).

The term "pomace" or "cake" as used herein refers to the optionally dried residue of fruit, vegetables or seeds after juicing or pressing. The term "oil cake" refers to a cake obtained after removal of oil from seeds, fruits or vegetables.

All percentages mentioned herein refer to weight percentages unless otherwise stated.

The process preferably does not employ any organic solvent. In other words, the pectin-rich substrate obtained from the plant material without the use of organic solvents is further treated (enzymatic treatment and ultrafiltration) without the use of organic solvents to produce the desired hydrolyzed pectin polysaccharide isolate.

The pectin-rich substrate used in the present method preferably comprises at least 4 wt.% of dry matter of the pectic polysaccharide, more preferably at least 6 wt.% of dry matter, even more preferably less than 8 wt.% of dry matter.

Preferably, the pectic polysaccharides in the pectin-rich substrate are characterized by a mass weighted average molecular weight of at least 100kDa, more preferably at least 150kDa, most preferably at least 200kDa,

the pectic polysaccharides in the pectin-rich substrate typically have an average rhamnose content of at least 0.1 mol.%, more preferably of at least 0.5 mol.%, even more preferably of at least 1 mol.%, and most preferably of at least 2 mol.%, calculated on the total monosaccharide composition of the pectic polysaccharides.

The pectin-rich substrate used in the present method may be obtained from different crops. In a preferred embodiment, the pectin-rich substrate is obtained from one or more crops selected from the group consisting of fruits (including tomato), carrots, olives, peas, sugar beets, chicory, soybeans, sunflowers, rapeseed and corn. More preferably, the pectin-rich substrate is obtained from one or more crops selected from the group consisting of apple, pear, citrus, carrot, beet and chicory. More preferably, the pectin-rich substrate is obtained from one or more crops selected from apple, pear, carrot and chicory. According to a particularly preferred embodiment, the pectin-rich substrate is obtained from carrots and/or apples.

Pectin-rich crop material may be used as a substrate in the present method, such as pectin-rich. This crop material should be comminuted to produce a pulp or juice in order for the pectinase to break down the pectic polysaccharides in the substrate. The comminuted crop material may be dried or concentrated prior to use in the present process.

Preferably, the pectin-rich substrate is obtained as a side stream from a production process using crop material as starting material, and wherein no organic solvent or chemical reaction is employed until the process stage of generating the side stream. According to a particularly preferred embodiment of the present process, the pectin-rich substrate is selected from the group consisting of pomace, an aqueous extract of pomace, oil cake, an aqueous extract of oil cake, screeds, skins, pericarp, stone, seeds, and combinations thereof. Most preferably, the pectin-rich substrate is selected from the group consisting of pomace, an aqueous extract of pomace, and combinations thereof.

The pectin-rich substrate employed in the present method is suitably obtained by a procedure comprising grinding, heating, microwaving, drying or alternative techniques that disrupt cell wall structure to promote enzymatic hydrolysis of the pectic polysaccharide.

The pectin-rich substrate preferably has a moisture content of no more than 15 wt.%, more preferably no more than 10 wt.%, and most preferably no more than 8 wt.%.

The pectin-rich substrate is preferably enzymatically treated in the form of an aqueous liquid comprising the pectin-rich substrate and having a dry matter content of 0.5-40 wt.%, more preferably 1-30 wt.%, even more preferably 2-20 wt.%, most preferably 3-15 wt.%. Such an aqueous liquid may suitably be prepared by mixing a pectin-rich substrate with water.

The pectin-rich substrate preferably constitutes at least 50 wt.%, more preferably at least 75 wt.%, and most preferably at least 85 wt.% of the dry matter comprised in the aqueous liquid subjected to the enzymatic treatment.

The enzymatic treatment according to the process of the invention is preferably carried out at a pH in the range of 3.0 to 7.5, more preferably in the range of 4 to 7.5, even more preferably in the range of 4.5 to 7, most preferably in the range of 5 to 7.

The treatment for partially hydrolyzing the pectin polysaccharides preferably employs one or more pectinases selected from the group consisting of pectin lyase (EC4.2.2.10), pectate lyase (EC 4.2.2.2), cohesive galacturonase (EC 3.2.1.15) and exo-polygalacturonase (EC3.2.1.67 and EC 3.2.1.82).

According to a preferred embodiment, the pectic polysaccharide is partially hydrolysed using one or more pectinases selected from the group consisting of cohesive galacturonases (EC 3.2.1.15) and extrinsic galacturonases (EC3.2.1.67 and EC 3.2.1.82).

According to another preferred embodiment, the pectic polysaccharide is partially hydrolyzed using one or more pectinases selected from the group consisting of pectin lyase (EC4.2.2.10) and pectate lyase (EC 4.2.2.2). Hydrolysis of the pectin polysaccharides by these lyases inevitably produces polysaccharide fragments comprising terminally unsaturated non-reducing galacturonic acid residues.

In the process of the present invention, the pectic polysaccharide is preferably hydrolysed using a combination of one or more pectinases and one or more pectinesterases (EC3.1.1.11) as described above. The one or more pectinesterase enzymes may be employed prior to or simultaneously with the one or more pectinase enzymes. The use of a combination of pectinase and pectinesterase is particularly advantageous in case the pectinase employed is selected from the group consisting of polygalacturonase, exopolygalacturonase and combinations thereof. The combined use of pectinases and pectinesterases generally results in partially hydrolyzed pectin polysaccharides with a reduced degree of methylation and an increased acetylation/methylation ratio.

According to a particularly preferred embodiment, the pectin-rich substrate is subjected to an enzymatic treatment, comprising the use of one or more pectinesterases (EC3.1.1.11), prior to or simultaneously with the partial hydrolysis with one or more pectinases. Most preferably, the enzymatic treatment with pectin esterase and the enzymatic treatment with one or more pectinases are performed simultaneously.

In the present method, the pectin-rich substrate may suitably be treated with cellulase and/or hemicellulase (EC 3.2.1.4, EC 3.2.1.176, EC3.2.1.203) prior to or as part of the enzymatic treatment with pectinase. Treatment with cellulase and/or hemicellulase disrupts the plant cell walls in the substrate, thereby inducing the release of the pectins contained within these cell walls, thus making these pectins more readily available for use in pectinases.

The enzymatic treatment of the pectin-rich substrate with one or more pectinases is preferably performed at a temperature of 15 to 70 ℃, more preferably 25 to 55 ℃. The duration of the enzymatic treatment is preferably at least 10 minutes, more preferably 20 minutes to 3 hours.

The present method suitably uses a pectin-rich substrate containing a relatively large amount of more or less intact plant cell wall material. Typically, such pectin-rich substrates comprise at least 40 wt.% of dry matter of cellulosic material selected from the group consisting of cellulose, hemicellulose and combinations thereof. Typically, most of these substrates are water-insoluble, i.e., the pectic polysaccharides are entrapped in the cellulose/hemicellulose matrix of the plant cell wall (debris). Preferably, at least 50 wt.% of the pectic polysaccharides present in the substrate are insoluble in distilled water at 45 ℃ and pH 5.5 at a pectic polysaccharide concentration of 1 g/l.

According to a particularly preferred embodiment of the process, the water-soluble pectic polysaccharides are separated from the water-insoluble solids by subjecting the aqueous liquor comprising the partially hydrolysed pectic polysaccharides to a solid-liquid separation after the enzymatic treatment and before the ultrafiltration. Examples of solid-liquid separation techniques that may be employed include settling, decanting, centrifuging, hydrocyclone and/or filtration. This particular embodiment provides the advantage that a high yield of the desired pectic polysaccharide can be achieved.

In one embodiment of the method, the pectin-rich substrate comprises 10-80 wt.% of dry matter of the cell wall polysaccharide selected from pectin, cellulose, hemicellulose and combinations thereof. More preferably, the substrate comprises 20-70 wt% dry matter of the cell wall polysaccharide. Most preferably, the substrate comprises 30-60 wt% dry matter of the cell wall polysaccharide. Pomace is an example of a pectin-rich substrate that can be used in the method of the invention and comprises a relatively large amount of cell wall polysaccharides.

The pomace typically comprises 2-50 wt% dry matter, preferably 5-40 wt% dry matter, more preferably 10-30 wt% dry matter of carbohydrates having a molecular weight of less than 20 kDa.

The protein content of the pomace is typically in the range of 0-20 wt% protein on dry matter, more preferably in the range of 1-18 wt% protein on dry matter, even more preferably in the range of 2-16 wt% protein on dry matter.

In embodiments of the present process in which the pectin-rich substrate is pomace or oil cake, undissolved material (particle size > 5 μm) is preferably removed from the enzymatically treated pomace or enzymatically treated oil cake prior to ultrafiltration. The undissolved material is preferably removed by solid-liquid separation, more preferably by decantation, centrifugation and/or filtration.

In another embodiment of the method, the pectin-rich substrate is a pectin isolate comprising at least 10 wt.% of dry matter of the pectin polysaccharides, preferably at least 20 wt.% of dry matter, more preferably at least 40 wt.% of dry matter, and less than 20 wt.% of dry matter of the cell wall polysaccharides selected from the group consisting of cellulose, hemicellulose and combinations thereof. More preferably, the pectin isolate comprises cell wall polysaccharides in a concentration of less than 10 wt.% dry matter, even more preferably less than 5 wt.% dry matter. Aqueous extracts of pomace and aqueous extracts of oil cake are examples of pectin isolates that can be used as a pectin-rich substrate in the present method.

Preferably, the aqueous extract of pomace is obtained by a series of aqueous extraction steps, more preferably at least two aqueous extraction steps. Thus, the first extraction step preferably comprises mixing the marc with water, followed by solid-liquid separation. The second step preferably comprises mixing the recovered solid portion with water, followed by another solid-liquid separation. Suitable solid-liquid separation methods are, for example, decantation, centrifugation and filtration.

Preferably, the aqueous extraction is performed at a pH in the range of 2.0 to 8.5, more preferably in the range of 4.5 to 7.5, even more preferably in the range of 6.0 to 7.0.

In a particularly preferred embodiment of the process, the partially hydrolysed pectic polysaccharides are subjected to ultrafiltration using an ultrafiltration membrane having a molecular weight cut-off in the range of 6-50kDa, preferably in the range of 7-40kDa, more preferably in the range of 8-30 kDa.

The recovered ultrafiltration retentate may suitably be dried using one or more drying techniques selected from the group consisting of spray drying, freeze drying, air drying, roll drying, plate drying, belt drying and drum drying.

Preferably, the recovered ultrafiltration retentate is dried to a water content of less than 15 wt.%, more preferably less than 13 wt.%, even more preferably less than 11 wt.%, and most preferably less than 9 wt.%.

The ultrafiltration retentate recovered preferably comprises galacturonic acid residues and rhamnose residues in a molar ratio of no more than 6:1, more preferably no more than 5:1, even more preferably no more than 4:1, and most preferably no more than 3.5: 1.

The pectic polysaccharides present in the recovered ultrafiltration retentate preferably have an average rhamnose content, calculated on the total monosaccharide composition of the pectic polysaccharides, of at least 4%, more preferably of from 5 to 50%, even more preferably of from 6 to 45%, still more preferably of from 6.5 to 40%, and most preferably of from 7 to 30%.

The ultrafiltration retentate recovered preferably comprises at least 5 wt.% of dry matter, preferably at least 15 wt.% of dry matter, more preferably at least 25 wt.% of dry matter, most preferably at least 50 wt.% of dry matter of pectin polysaccharides having an average rhamnose content of at least 4 mol.%, calculated on the total monosaccharide composition of the pectin polysaccharides.

According to another preferred embodiment, the pectic polysaccharides in the recovered retentate have a weight-weighted average molecular weight of more than 20kDa, more preferably more than 30kDa, even more preferably more than 40kDa, most preferably more than 50 kDa.

The process of the present invention preferably results in a hydrolyzed pectin polysaccharide isolate as specified below.

A second aspect of the invention relates to a hydrolysed pectin polysaccharide isolate obtainable by a method according to the invention.

The hydrolysed pectin polysaccharide isolate typically has a dry matter content of at least 85 wt%, more preferably at least 90 wt%, and most preferably at least 95 wt%.

Preferably, the hydrolysed pectin polysaccharide isolate comprises at least 10 wt% dry matter, more preferably at least 20 wt% dry matter, even more preferably at least 40 wt% dry matter of pectin having an average rhamnose content of at least 4 mol.%, calculated on the total monosaccharide composition of the pectin polysaccharide.

According to a particularly preferred embodiment, the hydrolyzed pectin polysaccharide has a mass weighted average molecular weight of more than 20kDa, more preferably more than 30kDa, even more preferably more than 40kDa, most preferably more than 50 kDa.

In a particular embodiment, the hydrolyzed pectin polysaccharide isolate comprises less than 15 wt.%, less than 10 wt.%, more preferably less than 5 wt.%, most preferably less than 1 wt.% of saccharides (polysaccharides, oligosaccharides, disaccharides and monosaccharides) having a molecular weight of less than 2.5 kDa.

Preferably, the hydrolysed pectin polysaccharide comprises less than 30 wt%, more preferably less than 20 wt%, most preferably less than 10 wt% of the material having a molecular weight of less than 20 kDa.

Typically, the hydrolysed pectin polysaccharide isolate comprises less than 15 wt% dry matter, preferably less than 10 wt% dry matter, more preferably less than 5 wt%, most preferably less than 1 wt% dry matter of insoluble polysaccharides selected from cellulose, hemicellulose, lignin and starch.

The protein content of the hydrolysed pectin polysaccharide isolate is preferably not more than 15 wt% of dry matter, more preferably in the range of 0.5-10 wt% of dry matter, and most preferably in the range of 1-5 wt% of dry matter.

Preferably, the hydrolysed pectin polysaccharide isolate comprises at least 10 wt% dry matter, preferably at least 20 wt% dry matter, more preferably at least 30 wt% dry matter of pectin polysaccharides having an average rhamnose content of at least 5 mol%, more preferably 6%, calculated on the total monosaccharide composition of the pectin polysaccharides.

Typically, the combination of galacturonic acid residues, rhamnose residues, arabinose residues and galactose residues constitutes at least 50 mol.% of the monosaccharide residues present in the saccharides comprised in the hydrolyzed pectin polysaccharide isolate. More preferably, this combination constitutes at least 60 mol.%, even more preferably at least 65 mol.%, still more preferably at least 70 mol.%, and most preferably at least 75 mol.% of monosaccharide residues in the saccharides comprised in the hydrolyzed pectin polysaccharide isolate.

In a particularly preferred embodiment of the hydrolysed pectin polysaccharide isolate, the combination of galacturonic acid residues and rhamnose residues represents at least 15 mol.%, more preferably 17-70 mol.%, most preferably 18-60 mol.% of the monosaccharide residues present in the saccharides comprised in the hydrolysed pectin polysaccharide isolate.

The galacturonic acid residues and rhamnose residues are preferably present in the hydrolysed pectin polysaccharide isolate in a molar ratio of not more than 6:1, more preferably not more than 5:1, even more preferably not more than 4:1, most preferably not more than 3.5: 1.

Rhamnose residues typically represent 5-50%, more preferably 6-45%, even more preferably 6.5-40%, and most preferably 7-30% of all monosaccharide residues comprised in the pectin polysaccharide present in the hydrolysed pectin polysaccharide isolate.

The galacturonic acid residues typically represent 5-80%, more preferably 8-60%, and most preferably 10-50% of all monosaccharide residues comprised in the pectic polysaccharide present in the hydrolysed pectin polysaccharide isolate.

The pectic polysaccharides in the isolate preferably have a mass weighted average molecular weight of no more than 800 kDa. More preferably, the pectic polysaccharide has a mass weighted average molecular weight of from 22kDa to 500kDa, most preferably from 25kDa to 250 kDa.

In a particular embodiment, the pectic polysaccharides in the isolate of the invention preferably have an average degree of acetylation of at least 10%, more preferably 20-110%, even more preferably 25-100%, and most preferably 30-80%.

In another particular embodiment, the pectic polysaccharides in the isolate have an average degree of methylation of preferably not more than 60%, more preferably not more than 50%, and most preferably 5-30%.

The backbone of the pectic polysaccharide in the isolate may comprise one or more side chains. These side chains may contain arabinose and/or galactose residues, as well as minor amounts of monomeric fucose, glucose, glucuronic acid, xylose and/or uronic acid residues. The one or more side chains are preferably selected from galactan side chains, arabinosan side chains and arabinogalactan side chains.

The arabinoside chains comprise at least one or more alpha (1,5) -linked arabinose residues and are substituted in the 4-OH position of the rhamnose residue in the RG-I domain. The arabinoglycan side chains may be linear or branched. If the side chain is linear, the side chain consists of α (1,5) -linked arabinose residues. In case the arabinosan side chains are branched, one or more alpha-arabinose residues are linked to the 2-OH and/or 3-OH of the alpha (1,5) -linked arabinose.

The galactan side chains contain at least one or more β (1,4) -linked galactose residues and are substituted at the 4-OH position of rhamnose residues in the RG-I domain.

The arabinogalactan side chains are substituted at the 4-OH position of the rhamnose residues in the RG-I domain and may be either type I Arabinogalactan (AGI) or type II Arabinogalactan (AGII). AGI is composed of a (1 → 4) - β -D-Galp backbone, on which substitution of monomeric Galp units at the O-6 or O-3 position can occur. AGI is further substituted by α -L-Araf-p residues and/or (1 → 5) - α -L-Araf short side chains. AGII consists of an α (1 → 3) - β -D-Galp backbone decorated with (1 → 6) - β -D-Galp secondary strands, which are arabinosylated.

Arabinose residues typically represent 0-50%, more preferably 3-48% and most preferably 5-46% of all monosaccharide residues comprised in the pectin polysaccharides present in the hydrolyzed pectin polysaccharide isolate.

Galactose residues typically represent 0-50%, more preferably 3-35%, and most preferably 5-25% of all monosaccharide residues comprised in the pectin polysaccharide present in the hydrolyzed pectin polysaccharide isolate.

The galactose residues and rhamnose residues are preferably present in the pectin polysaccharide in a molar ratio of less than 4:1, more preferably less than 3:1, most preferably less than 2: 1.

A third aspect of the invention relates to a method for the manufacture of a product alternative to an autotrophic formulation, a food product, a dietary supplement, a beverage or a pharmaceutical product, said method comprising the addition of a hydrolysed pectin polysaccharide isolate according to the invention in a concentration of at least 0.1 wt.% of dry matter comprised in the final product.

The hydrolysed pectin polysaccharide isolate is preferably added at a concentration of at least 0.2%, more preferably 0.3-95%, most preferably 1-80% by weight of dry matter comprised in the final product.

The product of the invention may comprise traces of one or more pectinases for use in the obtaining method of hydrolysing a pectic polysaccharide isolate. These pectinases may be present in the product in active and/or inactive form.

In a particularly preferred embodiment, the pectic polysaccharides of the hydrolyzed pectic polysaccharide isolate represent at least 20 wt.%, more preferably at least 30 wt.%, even more preferably 60 wt.%, most preferably at least 80 wt.% of the total amount of pectic polysaccharides present in the final product.

The invention is further illustrated by the following non-limiting examples.

Examples