WO2018127486A1 - Décorticage enzymatique de légumineuses - Google Patents

Décorticage enzymatique de légumineuses Download PDF

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Publication number
WO2018127486A1
WO2018127486A1 PCT/EP2018/050051 EP2018050051W WO2018127486A1 WO 2018127486 A1 WO2018127486 A1 WO 2018127486A1 EP 2018050051 W EP2018050051 W EP 2018050051W WO 2018127486 A1 WO2018127486 A1 WO 2018127486A1
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identity
pulse
dehusking
xylanase
enzyme composition
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PCT/EP2018/050051
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English (en)
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Suchitra TRIPATHY
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Novozymes A/S
Novozymes South Asia Pvt. Ltd.
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Publication of WO2018127486A1 publication Critical patent/WO2018127486A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/30—Removing undesirable substances, e.g. bitter substances
    • A23L11/33—Removing undesirable substances, e.g. bitter substances using enzymes; Enzymatic transformation of pulses or legumes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases

Definitions

  • the present invention relates to a method of dehusking pulses. More specifically, it relates to an enzymatic method for dehusking of pulses.
  • Pulses form a major source of protein component in a vegetarian diet. Economically, pulses are also one of the cheapest source of protein. Pulses are mainly consumed in the form of dehusked split pulses. Pulse milling involves conversion of the whole unhusked pulse grain into its dehusked and split form which is commonly termed as "dal”. It involves the following processes namely, loosening of husk, dehusking and splitting of pulses. Traditionally both dry and wet milling process are employed. In traditional wet milling process soaking with water is commonly used while in traditional dry milling process, coating with oil is used. Machineries in the form of carborundum emery rollers for dehusking and burr grinders for splitting are also employed.
  • Indian Patent application 657/DEL/2007 discloses an enzymatic process for preparation of de- hulled legume/dhals where xylanase and protease are used.
  • the invention relates to a method of dehusking a pulse comprising
  • pretreating the pulse with an enzyme composition comprising a GH1 1 or GH8 xy- lanase
  • the invention also relates to a method of dehusking a pulse comprising
  • pretreating the pulse with an enzyme composition comprising a GH5 endoglu- canase
  • the invention also relates to a method of dehusking a pulse comprising
  • the invention further discloses dehusked pulse produced according to the method of the in- vention.
  • the method of the invention provides for increased yield of the pulses.
  • the pulses so obtained also have an improved quality as judged by visual appearance.
  • Xylanase means a 1 ,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyses the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylans.
  • Xylanase activity can be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01 % TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C.
  • GH8 Xylanase A GH8 xylanase according to the invention is a xylanase enzyme conforming to the definition of the GH8 xylanase as set out by the Carbohydrate-active enzymes database (CAZy; http://www.cazy.org), a dedicated family classification system that correlate with the structure and molecular mechanism of CAZymes, developed by the Glycogenomics group at AFMB in Marseille, France, including any fragment derived therefrom, exhibiting xylanase ac- tivity.
  • CAZy Carbohydrate-active enzymes database
  • a GH 1 1 xylanase according to the invention is a xylanase enzyme conforming to the definition of the GH1 1 xylanase as set out by the Carbohydrate-active enzymes database (CAZy; http://www.cazy.org), a dedicated family classification system that correlate with the structure and molecular mechanism of CAZymes, developed by the Glycogenomics group at AFMB in Marseille, France, including any fragment derived therefrom, exhibiting xylanase activity.
  • CAZy Carbohydrate-active enzymes database
  • Endoglucanase means an endo-1 ,4-(1 ,3; 1 ,4)-beta-D-glucan 4- glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cel- lulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components.
  • Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481 ). Endoglucanase activity may be determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40 degrees centigrade Alternatively, the endoglucanase activity can also be determined using the procedure described in Example 8, 13 or 14 of WO13071883A1 .
  • CMC carboxymethyl cellulose
  • Cellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees.
  • Cellulose is a polymer of the simple sugar glucose covalently linked by beta-1 ,4- bonds.
  • Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta- glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it to attack by cellobiohydrolases.
  • Cellobiohydrolases sequentially release molecules of cellobiose from the ends of the cellulose polymer.
  • Cellobiose is a water-soluble beta-1 ,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobiose to glucose.
  • a GH5 endoglucanase according to the invention is a endoglucanase enzyme conforming to the definition of the GH5 endoglucanase as set out by the Carbohydrate-active enzymes database (CAZy; http://www.cazy.org), a dedicated family classification system that correlate with the structure and molecular mechanism of CAZymes, developed by the Glycogenomics group at AFMB in Marseille, France, including any fragment derived therefrom, exhibiting xylanase activity.
  • CAZy Carbohydrate-active enzymes database
  • Polygalacturonase (EC 3.2.1.15) means a pectinase that catalyze random hydrolysis of (1 ,4)-alpha-D- galactosiduronic linkages in pectate and other galac- turonans. They are also known as pectin depolymerase.
  • Pectin lyase The term "Pectin lyase” (EC 4.2.2.10) means pectinases that catalyze elimina- tive cleavage of (1 .4)-alpha-D- galacturonan methyl ester to give oligosaccharides with 4-de- oxy-6-0-methyl-alpha-D-galact-4- enuronosyl groups at their non-reducing ends. They are alternatively known as pectolyase, polymethylgalacturonic transeliminase, pectin methyl- transeliminase, pectin trans-eliminase, etc.
  • Pectin esterase (EC 3.1 .1 .1 1 ) means pectinases that hydrolyze pectin to methanol and pectate. They are alternatively known as pectin demethoxylase, pectin methoxylase, pectin methylesterase, etc. Pectin esterase catalyses the release of methanol from pectin with a resultant decrease in pH.
  • Pulse is annual leguminous crops yielding from one to 12 grains or seeds of variable size, shape and colour within a pod (as defined by FAO http://www.fao.Org/es/faodef/fdef04e.htm#4.01 ). The term pulse is limited to crops harvested solely for dry grain, thereby excluding crops harvested green for food or crops used mainly for oil extraction or leguminous crops that are used exclusively for sowing purposes. Sequence Identity: The relatedness between two amino acid sequences is described by the parameter "sequence identity”.
  • the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wun- sch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. Version 6.1 .0 was used.
  • the optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labelled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • Pulse forms the main source of protein in a vegetarian diet.
  • the United Nations Food and Agricultural organization (FAO) defines pulses as annual leguminous crops yielding from one to 12 grains or seeds of variable size, shape and colour within a pod. They are used for both food and feed. (see http://www.fao.Org/es/faodef/fdef04e.htm#4.01 ).
  • FAO also limits the term "pulse"(s) to crops harvested solely for dry grain, thereby excluding crops harvested green for food (green peas, green beans, etc.) which are classified as vegetable crops. Also excluded are those crops used mainly for oil extraction (e.g. soybean and groundnuts) and leguminous crops (e.g. seeds of clover and alfalfa) that are used exclusively for sowing purposes.
  • Adzuki bean Adzuki bean, azuki bean (Vigna angularis)
  • Tepary bean Phase acutifolius
  • Vetch common vetch (Vicia sativa) 10. Lupins ⁇ Lupinus spp.)
  • Minor pulses including:
  • Pulses are consumed in its dehusked and split form which, traditionally in India, is termed "Dal". Pulse milling is the process of conversion of pulse, most commonly whole unhusked pulse, to dal. It is accomplished in three major steps namely: loosening of husk, dehusking and splitting.
  • Pulses received at the mill need to be cleaned and size graded for yielding good quality dal with higher recovery. Even during dehusking operation, pulses are subjected to sieving to separate out husk, brokens, splits, gota (dehusked whole pulse) and whole unhusked pulses. Usually two types of cleaners are used: reciprocating air-screen cleaners and reel screen cleaners.
  • the reel screen cleaners consist of 2-4 cylindrical compartments.
  • the frame of the machine is made of wooden or mild steel sheet. In these compartments, different size perforation screens are fitted on a 5-7.5 mm diameter shaft. The machine is fitted at an inclination of 2-3 degrees.
  • the cylindrical screen drum rotates at 5-35 rpm.
  • Drying of pulses is necessary to ensure safe storage before milling as pulses received at mill have generally higher moisture content. After steeping of pulses for loosening of husk, it is also necessary to dry pulses. During splitting operation too, it is very much essential to dry the pulses to separate cotyledons.
  • Sun drying of pulses is an economical option for drying of pulses. The sun drying is done for a few hours to a few days depending on the season and type of pulses used. The pulses are spread over floor/roof in 5 to 7.5 cm thick layer which are intermittently stirred manually with the help of rakes or turning by foot. At night, the drying pulses are collected in heaps and covered with canvass sheet to preserve the heat.
  • Mechani- cally heated air dryers either batch type or continuous flow type are also used by the millers. The temperature of heated air for drying varies from 60 degree Centigrade to 120 degree Centigrade (C).
  • husk is loosened by sequence of operations such as: oil smearing, water application, tempering and sun-drying. Cleaned and graded pulses are passed through roller de- husker in which scratches, cracks and dents are created on hard seed coat of pulses. This is known as "pitting" of pulses. The pitted pulse grains are then passed through the sieve cleaner to separate out the splits, husk and powder and later smeared with oil (100-500 gram per quintal of pulses) either manually or with auger mixer and stored for 1 -5 days.
  • oil 100-500 gram per quintal of pulses
  • Roller dehuskers coated with carborandum are used to dehusk the pulses.
  • Two types of rollers viz. cylindrical and tapered are available for dehusking. Tapered rollers are placed horizontally and the diameter of roller increases from feeding side to discharge side. The difference in diameter helps to gradually increase the pressure on pulse grains and thus helps in gradual dehusking.
  • the cylindrical rollers are installed at an angle of 10-15 degrees which enables forward movement of pulse grains inside the machine. Annular gap between rollers varies depending upon the type of pulses being dehusked. Inlet and outlet of the roller machine can be adjusted for regulation of grain flow and retention time respectively. Small dal mills use under run disc shellers or burr mills for dehusking operation in place of Roller mills.
  • Conditioned pulse grains subjected to mild abrasion inside the roller machine removes 10- 25% of husk in one pass. Shelled husk, cotyledon powder, brokens and splits are separated out by Air-screen cleaners after passing the grain lot once or twice through the roller machine. Depending upon adherence of husk to grain, the pulse grains are passed through mill for two to eight times.
  • Splitting operation involves loosening the bond between the cotyledons and splitting.
  • cotyledons loosening water at the rate of 1 -5 kg/quintal is applied to dehusked pulse grain (gota) and is stored for 2-12 hours and later sun-dried for 4-8 hours.
  • machines like under- run-disc sheller (URD), impact machine (Phatphatia), roller mill, and hitting the gota against the metal sheet at discharge side of bucket elevator are used. In this operation, the embryo attached to two cotyledons breaks away, thereby, causing a loss in dal recovery by 1.5 to 2%.
  • dal is imparted with a glazing appearance to improve its consumers' acceptance and market value.
  • different materials like water, oil, soap- stone powder are applied to dal surface.
  • removal of sticking powder from dal surface is considered sufficient to improve its surface glaze.
  • pretreating the pulse with an en- zyme composition comprising a GH5 endoglucanase prior to the dehusking process actually improves the yield of the pulse obtained after dehusking.
  • the invention relates to a method of dehusking a pulse comprising:
  • the invention relates to a method of dehusking a pulse comprising:
  • the invention relates to a method of dehusking a pulse comprising:
  • Xylans are hemicelluloses found in all land plants (Popper and Tuohy, Plant Physiology, 2010, 153:373-383). They are especially abundant in secondary cell walls and xylem cells.
  • Plant xylans have a ⁇ -1 ,4-linked xylopyranose backbone that can be substituted at the 02 or 03 position with arabinose, glucuronic acid and acetic acid in a species and tissue specific manner.
  • the known enzymes responsible for the hydrolysis of the xylan backbone are classified into enzyme families based on sequence similarity (www.cazy.org).
  • the enzymes with mainly enc/o-xylanase activity have previously been described in Glycoside hydrolase family (GH) 5, 8, 10, 1 1 , 30 and 98.
  • GH Glycoside hydrolase family
  • the enzymes within a family share some characteristics such as 3D fold and they usually share the same reaction mechanism.
  • Some GH families have narrow or mono-specific substrate specificities while otherfamilies have broad substrate specificities.
  • family 8 Of the various families of xylanases, family 8, and 1 1 are characterized below.
  • GH 8 xylanases Family 8 glycoside hydrolases or GH8 xylanases hydrolyse the beta-1 ,4 xylan chain. They cleave ⁇ -1 ,4 linkages of ⁇ -1 ,4 glucans, xylans (or xylooligosaccharides), chitosans, and lichenans (1 ,3-1 ,4-3-D-glucan). The majority of the enzymes are endo-acting enzymes, but one member has an exo-activity that releases ⁇ -D-xylose residues from the reducing end of xylooligosaccharides.
  • Suitable GH8 xylanases include those of plant, archae, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful GH8 xylanases include xylanases from Achormobacter sp, Acidihalobacter sp, Acidithiobacillus sp, Acidiphilium sp., Acidobacterium sp, Actinoalloteichus sp, Actinoalloteichus sp, Aeromonas sp, Agrobac- terium sp; Aliivibrio sp; Alteromonas sp, Amphibacillus sp, Amycolatopsis sp; Anaerolinea sp; Anaeromyxobacter sp; Aquifex sp, Asaia sp; Aureimonas sp; Azospirillum sp, Azotobacter sp; Bacillus sp;
  • One preferred GH8 xylanase for use according to the invention is the GH8 xylanase having the sequence of SEQ ID NO: 1.
  • the GH8 xylanase is selected among xylanases having an amino acid sequence identity of at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity or 100% identity to the amino acid sequence of SEQ ID NO: 1 .
  • GH11 Xylanases Family 1 1 glycoside hydrolases or GH1 1 xylanases are endo- ⁇ -1 ,4-xy- lanases. They have the retaining mechanism and they belong to the GH-C clan have a beta- jelly roll 3D structure and generally are known to hydrolyze aryl ⁇ -glycosides of xylobiose and xylotriose at the aglyconic bond, but appear to be inactive on aryl cellobiosides.
  • Suitable GH1 1 xylanases include those of plant, archae, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful GH1 1 xylanases include xylanases from Halopiger sp; Halorhabdus so; Actinomadura sp; Actinoplanes sp;
  • Actinosynnema sp Aeromonas sp; Alteromonadaceae sp; Alteromonas sp; Amphibacillus sp;
  • the GH1 1 xylanase is selected among xylanases having an amino acid sequence identity of at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity or 100% sequence identity to the amino acid of SEQ ID NO: 2.
  • Endoglucanase means an endo-1 ,4-(1 ,3; 1 ,4)-beta-D-glucan 4- glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cel- lulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components.
  • Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481 ). Endoglucanase activity may be determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40 degrees centigrade Alternatively, the endoglucanase activity can also be determined using the procedure described in Example 8, 13 or 14 of WO13071883A1 .
  • CMC carboxymethyl cellulose
  • Cellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees.
  • Cellulose is a polymer of the simple sugar glucose covalently linked by beta-1 ,4- bonds.
  • Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta- glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it to attack by cellobiohydrolases.
  • Cellobiohydrolases sequentially release molecules of cellobiose from the ends of the cellulose polymer.
  • Cellobiose is a water-soluble beta-1 ,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobiose to glucose.
  • GH 5 endoglucanases Family 5 glycoside hydrolases or GH5 endoglucanases cause endo- hydrolysis of (1 ⁇ 4)-3-D-glucosidic linkages in cellulose, lichenin and cereal ⁇ -D-glucans. They will also hydrolyse 1 ,4-linkages in ⁇ -D-glucans also containing 1 ,3-linkages They belong to the GH-A clan, have a (beta/alpha)s 3D structure and are found to catalyse hydrolysis with retain- ing mechanism.
  • Suitable GH5 endoglucanases include those of plant, archae, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful GH5 endoglucanases include endoglucanases from Acidilobus sp., Caldivirga sp., Thaumarchaeota sp., Desulfurococcaceae sp., Halorhabdus sp., Halostagnicola sp., Haloterrigena sp., Ignisphaera sp., Picrophilus sp., Pyrococcus., Salinarchaeum sp., Staphylothermus sp., Sulfolobus sp., Thermococcus sp., Thermofilum sp., Thermoplasma sp., Thermoproteus sp., Vulcanisaeta sp.,
  • One preferred GH5 endoglucanase for use according to the invention is the GH5 endoglucanase having the sequence of SEQ ID NO: 4.
  • the GH5 endoglucanase is selected among endoglucanases having an amino acid sequence identity of at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity or 100% sequence identity to SEQ ID NO: 4
  • Pectin is a family of complex polysaccharides that contain 1 ,4-linked a-D- galactosyluronic acid residues. Three pectic polysaccharides have been isolated from plant primary cell walls and structurally characterized. These are:
  • Homogalacturonans are linear chains of a-( 1 -4 )-l inked D-galacturonic acid. Substituted galacturonans are characterized by the presence of saccharide appendant residues (such as D- xylose or D-apiose in the respective cases of xylogalacturonan and apiogalacturonan) bran- ching from a backbone of D-galacturonic acid residues.
  • Rhamnogalacturonan I pectins (RG-I) contain a backbone of the repeating disaccharide: 4)-a-D-galacturonic acid-(1 ,2)-a-L- rham- nose-(1 .
  • rhamnose residues From many of the rhamnose residues, side chains of various neutral sugars branch off.
  • the neutral sugars are mainly D-galactose, L-arabinose and D-xylose, the types and proportions of neutral sugars varying with the origin of pectin.
  • Another structural type of pectin is rhamnogalacturonan II (RG-I I), which is a less frequent complex, highly branched polysaccharide.
  • RG-I I rhamnogalacturonan II
  • Rhamnogalacturonan II is classified by some authors within the group of substituted galacturonans since the rhamnogalacturonan II backbone is made exclusively of D-galacturonic acid units.
  • Polygalacturonase (EC 3.2.1.15) Polygalacturonases are pectinases that catalyze random hy- drolysis of (1 ,4)-alpha-D- galactosiduronic linkages in pectate and other galacturonans. They are also known as pectin depolymerase. Polygalacturonase hydrolyses the alpha-1 ,4-glyco- sidic bonds in polygalacturonic acid with the resultant release of galacturonic acid. This reducing sugar reacted then with 3,5- dinitrosalicylic acid (DNS).
  • DNS 3,5- dinitrosalicylic acid
  • Suitable poygalacturonases include those of plant, archae, bacterial or fungal origin. Chemi- cally modified or protein engineered mutants are included. Examples of useful polygalacturonases include polygalacturonases from Acetobacter sp., Achaetomium sp., Achlya sp., Acholeplasma sp., Acidobacterium sp., Acorus sp., Actinidia sp., Actinoplanes sp., Acutalibac- ter sp., Adineta sp., Agrobacterium sp., Albugo sp., Algibacter sp., Alicyclobacillus sp., Alisma sp., Alistipes sp., Alkaliphilus sp., Alkalitalea sp., Alternaria sp., Alteromonas sp., Amborella sp.,
  • polygalacturonase for use according to the invention is the polygalacturonase having the sequence of SEQ ID NO: 5.
  • the polygalacturonase is selected among polygalacturonases having an amino acid sequence identity of at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity or 100% sequence identity to SEQ ID NO: 5.
  • the pulse is a whole unhusked pulse.
  • an unhusked pulse is a pulse which has not been dehusked.
  • the pulse is a split unhusked pulse.
  • a whole unhusked pulse is a pulse that is an unsplit grain covered with a husk.
  • a dehusked whole pulse is a pulse that is an unsplit grain and not covered with the husk.
  • a split unhusked pulse is a pulse that is split grain and covered with a husk.
  • the pulse for use in the invention can be of any quality - for example, it can be uncleaned or it can be variously cleaned and/or sorted and/or graded.
  • the pretreatment with the enzyme can be done any time prior to the dehusking step.
  • the pretreatment involves contacting the enzyme composition with the whole unhusked pulse or the split unhusked pulse.
  • the pretreatment with the enzyme is combined with the soaking step during the wet milling of the pulse.
  • the pretreatment is done separately prior to the soaking step. In another embodiment, the pretreatment is done immediately after the cleaning step.
  • the pretreatment involves mixing the enzyme with the water used for soaking of the whole unhusked pulse.
  • the pretreatment with the enzyme is done after the pitting step.
  • the pretreatment with the enzyme happens during the steeping step. In another embodiment, the pretreatment with the enzyme happens after the steeping step.
  • the pretreatment involves appropriate moisture adjustment required for enzymatic hydrolysis of pulses followed by addition of enzymes.
  • the pulse is Cicer arietinum variously called chickpea, garbanzo or bengal gram.
  • the chickpea can be of any type or variety namely Desi or Kabuli or others.
  • the pulse before the method of invention, is a pulse chosen from pulse(s) that are traditionally dehusked using the wet method.
  • the pulse is Pisum sativum, variously called pea or peas or garden peas or green peas or protein peas.
  • the pulse is Lens culinaris, variously called lens esculenta or lentils, adas flashmek, messer, masser or massur and heramame.
  • the reaction conditions such as concentration of enzymes to be used, pH, temperature and reaction time may in principle be determined using techniques known in the field for optimizing enzymatic reactions and is completely within the skills of the average practitioner.
  • the concentration of enzymes for the pretreatment is typically in the range of 1 to 10,000 milligram (mg), more preferred in the range of 10 to 1000 mg and most preferred around 40 to 200 mg per kg of pulses for xylanases.
  • the concentration of enzymes for the pretreatment is typically in the range of 1 to10,000 mg, more preferred in the range of 10 to1000 and most preferred around 40 to 500 mg per kg of pulses for endoglucanases.
  • the concentration of enzymes for the pretreatment is typically in the range of 1 to 10000 milli- gram (mg), more preferred in the range of 10 tol OOOmg and most preferred around 40 to 500 mg per kg of pulses for polygalacturonases.
  • the activity of enzyme used for pretreatment is typically in the range of 100 to 500 FXU-W/KG pulses or 9 to 47 NXU/Kg pulses for xylanases.
  • the activity of enzyme used is typically 200- 600 FBG /KG pulses for endoglucanases.
  • the activity of enzyme used for pretreatment is typically in the range of 50 to 3000 PGNU/KG pulses.
  • the pH in the reaction mixture should be selected in accordance with the pH preferences of the selected enzyme and is typically in the range of 3.0 to 9.0, preferably in the range of 4.0 to 8.0, more preferred in the range of 5.0 to 7.5, measured at 25°C.
  • the pH is regulated before the reaction is started whereas in other embodiments the pH is not regulated meaning that the pH in the reaction mixture is determined by the pH of the pulse substrate or water used.
  • the pretreatment temperature should be selected according to the temperature preferences and temperature stability of the selected enzyme. In general, a higher temperature is preferred to increase the reaction rate however, a higher temperature also provides for a higher inacti- vation rate for the enzyme, so the skilled person should select the temperature with due consideration of these factors.
  • the pretreatment temperature is typically in the range of 10°C to 80°C, preferably in the range of 20°C to 60°C, more preferred in the range of 25°C to 50°C.
  • the pretreatment should continue for a sufficient time to achieve desired degree of pretreatment in order to maximize yield and or other characteristics.
  • the reaction time is typically below 24 hours such as in the range of 5 minutes to 24 hours, preferably in the range of 2 hours to 16 hours, preferably in the range of 4 hours to 8 hours.
  • the GH1 1 or GH8 enzyme(s) may be added as pure enzyme, as an aqueous solution thereof or as an enzyme composition that comprises the one or more enzyme(s) that work in enzymatic dehusking. It is preferred to use enzyme compositions.
  • the GH5 endoglucanase enzyme(s) may be added as pure enzyme, as an aqueous solution thereof or as an enzyme composition that comprises the one or more enzyme(s) that work in enzymatic dehusking. It is preferred to use enzyme compositions.
  • the polygalacturonase enzyme(s) may be added as pure enzyme, as an aqueous solution thereof or as an enzyme composition that comprises the one or more enzyme(s) that work in enzymatic dehusking. It is preferred to use enzyme compositions.
  • the enzyme(s) may be added as pure enzyme, as an aqueous solution thereof or as an en- zyme composition that comprises the one or more enzyme(s) that work in enzymatic dehusking. It is preferred to use enzyme compositions.
  • An enzyme composition is the typical product wherein commercial enzymes are supplied and may in addition to the active enzyme comprise further enzymes, solvents, diluents, stabilizers, fillers, coloring agents etc.
  • the compositions may further comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of pectinases, phytase, xylanase, galactanase, alpha-galactosidase, protease, phospholipase A1 , phospholipase A2, lysophos- pholipase, phospholipase C, phospholipase D, amylase, lysozyme, arabinofuranosidase, beta- xylosidase, acetyl xylan esterase, feruloyi esterase, cellulase, cellobiohydrolases, beta-gluco- sidase, pullul
  • the enzyme composition further contains a glucanase.
  • the enzyme composition further contains a xylanase.
  • the enzyme composition further contains a polygalacturonase.
  • the enzyme composition further contains a GH8 or GH1 1 xylanase. In another embodiment, the enzyme composition further contains a GH5 endoglucanase.
  • a method for dehusking a pulse comprising:
  • pretreating the pulse with an enzyme composition comprising a GH1 1 or GH8 xylanase
  • a method for dehusking a pulse comprising:
  • pretreating the pulse with an enzyme composition comprising a GH5 endoglucanase
  • a method for dehusking a pulse comprising:
  • pretreating the pulse with an enzyme composition comprising a polygalacturonase
  • xylanase is selected among xylanases having an amino acid sequence identity of at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity or 100% identity to SEQ ID NO: 2
  • endoglucanase is selected among endoglucanases having an amino acid sequence identity of at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity or 100% sequence identity to SEQ ID NO: 4.
  • polygalacturonase is selected among polygalacturonase having an amino acid sequence identity of at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity or 100% sequence identity to SEQ ID NO: 5.
  • enzyme composition further comprises a pectinase.
  • the xylanase is a xylanase having at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, prefer- ably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity or 100% identity to SEQ ID NO: 1 or 2
  • the endoglucanase is an endoglucanase having an aminoacid sequence having at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity or 100% identity to SEQ ID NO: 4
  • polygalacturonase is a polygalacturonase having an aminoacid sequence having at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity or 100% identity to SEQ ID NO: 5
  • An enzyme composition comprising a GH8 or GH1 1 xylanase and a GH 5 endoglucanase for dehusking of pulse.
  • An enzyme composition comprising a GH8 or GH1 1 xylanase and a polygalacturonase for dehusking of pulse.
  • An enzyme composition comprising a GH5 endoglucanase and a polygalacturonase for dehusking of pulse.
  • Water used was ground water or water supplied by the municipal corporation and the pH was found to be between 6.5 to 8.0.
  • GH8 xylanase of SEQ ID no 1 was prepared as described in WO201 1070101 -A1 .
  • GH1 1 xylanase of SEQ ID no 2 was prepared as described in WO2005059084-A1 .
  • GH10 xylanase of SEQ ID no 3 was prepared as described in WO2009074650-A2.
  • GH5 endoglucanase of SEQ ID no 4 was prepared as described in WO2012122518-A1.
  • Polygalacturonase of SEQ ID no 5 was prepared using standard recombinant methods as described in for example in WO201 1070101 -A1 .
  • Pectinesterase of SEQ ID no 6 was prepared as described in WO201 1 103812-A1 .
  • Pectin lyase of SEQ ID no 7 was prepared as described in EP353188-A. Methods for analysis Assay for enzyme activity:
  • FXU-W is defined as the endoxylanase activity using Bio-feed Wheat (Novozymes A S) as a standard. In other words, The FXU-W endoxylanase activity is determined relative to Bio-feed Wheat (Novozymes A/S) as a standard.
  • NXU is defined as the endoxylanase activity using Panzea (Novozymes A/S) as a standard. In other words, The NXU endoxylanase activity is determined relative to Panzea (Novozymes A/S) as a standard.
  • Betaglucanase activity was assayed using Betaglucan as substrate at 50 degrees C and pH 5. This reaction is stopped by an alkaline reagent including PAHBAH and Bi3+, which complexes with reducing sugar, producing color that is detected at 405 nm. The color is proportional to the betaglucanase activity.
  • One FBG unit is the amount of enzyme that produces reducing carbohydrate equivalent to 1 ⁇ of glucose per minute at 50 degrees C and pH 5. The activity is determined relative to Energex (Novozymes A/S) as enzyme standard.
  • Polygalacturonase hydrolyses the alpha-1 ,4-glycosidic bonds in polygalacturonic acid with the resultant release of reducing carbohydrate. This reaction is stopped by an alkaline reagent including p-hydroxybenzoic acid hydrazide (PAHBAH) and Bi3+, which complexes with reducing sugar, producing colour, detected at 405 nm. The produced colour is proportional to the polygalacturonase activity.
  • PAHBAH p-hydroxybenzoic acid hydrazide
  • Bi3+ p-hydroxybenzoic acid hydrazide
  • the produced colour is proportional to the polygalacturonase activity.
  • One PGNU(PL) is defined as the amount of enzyme which produces reducing carbohydrates equivalent to 1 mg of galacturonic acid sodium salt under standard conditions (50 degrees C and 4.5 pH).
  • Pectin Lyase activity is assayed by an enzymatic reaction that consists of splitting alpha 1 -4 galacturonosidyl bond producing unsaturated delta 4,5 uronide.
  • the double bond with carbonyl function in C6 has an absorption in UV Optical density at 235 nm assays the pectin lyase activity.
  • One Pectin lyase unit (PELU) is the quantity of enzyme that catalyses the split of bound endo alpha 1 -4 galacturonosidyl (C6 methyl ester) forming one micromole of delta 4,5 unsaturated product in one minute, at conditions of 45°C and pH 5.5.
  • Pectin esterase catalyses the release of methanol from pectin with a resultant decrease in pH.
  • Sodium hydroxide is added to maintain the pH at 4.5.
  • the amount of sodium hydroxide con- sumed is an indication of the enzyme activity.
  • One unit of PE activity is that amount of enzyme which consumes 1 micro equivalent of sodium hydroxide per minute under standard conditions (30°C, pH 4.5).
  • Chickpea grains were cleaned and pitted to about 50% using mini dal mill roller.
  • the pitted grains were mixed with about 2-3% water to increase the moisture content of the grain to about 13.5 to 14%.
  • Enzyme was incorporated to the pitted grains through the required amount of water used for adjusting the moisture content of the grains. All the grains were mixed for uniformly in water distribution through all the grains. The grains were incubated at about 30 to 35 deg C for 15 hours. Post incubation the moisture content of the grains was readjusted to 10 to 1 1 % using hot air dryer/oven. Dehusking of the pulses were carried out using Random orbit sander from Bosch model no
  • the sander was fitted with 40 no emery coated disc and 10g of pitted grains were kept on 120no emery coated paper and dehusking was done by placing the sander on the grain and running at speed 1 for 1 :30 minutes.
  • the seeds were passed through classifier to separate the whole grains (husked and dehusked), dehusked splits, husk, broken and powdered samples.
  • the dehusking efficiency (DE) of the pulses was calculated using the following formula.
  • EXAMPLE 1 ENZYMATIC DEHUSKING OF PULSE USING GH 1 1 XYLANASES
  • EXAMPLE 3 ENZYMATIC DEHUSKING OF PULSE USING GH 10 XYLANASES.
  • EXAMPLE 5 ENZYMATIC DEHUSKING OF CHICKPEA USING PECTIN LYASE TABLE 5.1 : CONCENTRATION OF PECTIN LYASE for CHICKPEA DEHUSKING
  • EXAMPLE 6 ENZYMATIC DEHUSKING OF CHICKPEA USING POLYGALACTURONASE TABLE 6.1 : CONCENTRATION OF POLYGALACTURONASE FOR CHICKPEA DEHUSKING
  • EXAMPLE 7 ENZYMATIC DEHUSKING USING COMBINATION OF POLYGALACTURONASE, PECTIN METHYL ESTERASE IN CHICKPEA DEHUSKING

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Abstract

L'invention concerne un procédé de décorticage de légumineuses par prétraitement de celles-ci avec une composition enzymatique comprenant une xylanase GH11 ou GH8 ou une endoglucanase GH5 ou une polygalacturonase préalablement au décorticage.
PCT/EP2018/050051 2017-01-03 2018-01-02 Décorticage enzymatique de légumineuses WO2018127486A1 (fr)

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WO2020206058A1 (fr) 2019-04-02 2020-10-08 Novozymes A/S Procédé de production d'un produit de fermentation
CN113512572A (zh) * 2021-04-15 2021-10-19 苏州朗邦营养科技有限公司 一种发酵法高效制备β-葡聚糖的方法、剑菌及其筛选方法
CN114958688A (zh) * 2022-06-29 2022-08-30 北京大学 一种产酶菌
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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WO2020206058A1 (fr) 2019-04-02 2020-10-08 Novozymes A/S Procédé de production d'un produit de fermentation
CN111394375A (zh) * 2020-04-27 2020-07-10 广西大学 一种编码β-葡萄糖苷酶的基因mg163及其应用
CN113512572A (zh) * 2021-04-15 2021-10-19 苏州朗邦营养科技有限公司 一种发酵法高效制备β-葡聚糖的方法、剑菌及其筛选方法
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes
CN114958688A (zh) * 2022-06-29 2022-08-30 北京大学 一种产酶菌
CN114958688B (zh) * 2022-06-29 2023-06-09 北京大学 一种产酶菌

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