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EP1658359A1 - Traitement enzymatique d'une masse constituee d'olives et de constituants d'olives - Google Patents

Traitement enzymatique d'une masse constituee d'olives et de constituants d'olives

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Publication number
EP1658359A1
EP1658359A1 EP04764482A EP04764482A EP1658359A1 EP 1658359 A1 EP1658359 A1 EP 1658359A1 EP 04764482 A EP04764482 A EP 04764482A EP 04764482 A EP04764482 A EP 04764482A EP 1658359 A1 EP1658359 A1 EP 1658359A1
Authority
EP
European Patent Office
Prior art keywords
activity
oil
enzyme
olive
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP04764482A
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German (de)
English (en)
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EP1658359B1 (fr
Inventor
Björn-Oliver JACKISCH
Arno Cordes
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sued Chemie AG
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Sued Chemie AG
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Publication of EP1658359A1 publication Critical patent/EP1658359A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C11B1/025—Pretreatment by enzymes or microorganisms, living or dead

Definitions

  • Olive oil is obtained from the fruits of the olive tree (class: Dicotyle- doneae, order: Oleales, family: Oleaceae, type: Olea europaea).
  • the olive fruit (olive) has a rounded, oval cross-section and is a fleshy pome fruit with a bitter taste.
  • the physical and chemical properties of olive fruits depend on many factors, such as variety, ripeness, time of harvest, geographical location or growing conditions. Structurally, the olive fruit can be divided into two essential components, these are the pericarp and the endocarp.
  • the pericarp comprises the epicarp (fruit skin) and the me- socarp (pulp), which enclose the endocarp ' (wooden core), in which the seed is embedded.
  • the technology used and the type of process control determine, among other things. the yield of olive oil.
  • the olives are first chopped, e.g. by millstones or hammer mills, followed by a step in which the olive paste is incubated for a certain time (typically 30-90 minutes) with horizontal stirring, optionally with the addition of water, for further comminution and maceration.
  • the suspension obtained is then broken down into liquid and solid substance, typically by pressure (pressure) or by centrifugal force (two- or three-phase method).
  • the liquid obtained by pressing or by using the two-phase process in this process step is ideally an oil / water emulsion which is separated by a further centrifugation step.
  • the suspension is split into solids, water and oil in one process step.
  • the first isolation step described ie in the step into which chopped olives go directly, depending on the method, typically between 50 and 85% of the total oil contained is isolated.
  • the after the first isolation step Olive oil thus still has a significant residual oil content, the reduction of which can be brought about by repeating the first process step one or more times, possibly by changing certain process parameters such as temperature, applied centrifugal force, water addition etc.
  • the oil insulation can be carried out economically with mechanical-physical methods only up to a certain residual oil content.
  • chemical-extractive processes are used. For this purpose, the olive pulp is dried, extruded if necessary after optional removal of the stone remains and extracted with a solvent such as hexane.
  • the oil is obtained from the extract by removing the solvent, for example by evaporation.
  • the quality of the olive oil is also influenced by the type of process and the process control. It makes economic sense to select such processes that deliver high-quality oil, since the quality is positively correlated with the price to be achieved.
  • the mechanical-physical methods are particularly suitable for this. From environmental and safety aspects too, efforts are being made to avoid chemical-extractive processes as much as possible in favor of the mechanical-physical processes. For this reason, in addition to constructive improvements to the machines used, biochemical process aids have also been developed in the past, which can have a positive influence on the yield and quality of the oil obtained by means of mechanical-physical processes.
  • enzymes are functional polypeptides that are able to catalyze the cleavage of defined substrates or the synthesis of certain products.
  • those enzymes or enzyme mixtures which are capable have been described in the past are to have a degrading effect on certain cellular structures of the olive fruit.
  • Papain (EC 3.4.22.2), cellulases (EC 3.2.1.4), ⁇ -amylase (EC 3.2.1.1), proteases (EC 3.4.2x.yz), ß-glucosidases (EC 3.2.1.21), ß-galactosidases (EC 3.2.1.23), ⁇ -arabinosidases (EC 3.2.1.55), mannosidases (EC 3.2.1.24), ⁇ -xylosidases (EC 3.2.1.37), ⁇ -N-acetylglucoaminidase (EC 3.2.1.52), ⁇ - D-galactosidase (EC 3.3.1.22), pectin lyase (EC 4.2.2.10) and pectate lyase (EC 4.2.2.2) are known.
  • EP 0 616 024 AI describes a specific process for the production of olive oil from pitted olive fruits using a cellulase.
  • the object of the invention was therefore to provide an enzyme mixture in a process for extracting oil, with the aid of which the oil quantity and quality in the process of extracting olive oil are significantly noticeably improved and the process of oil separation can also be facilitated.
  • composition according to claim 1 This object is achieved by the composition according to claim 1.
  • Advantageous embodiments are specified in the subclaims.
  • the composition according to the invention initially contains a composition or mass of olives or olive components.
  • various components of the olive fruit can be present in any proportion.
  • Practical oil extraction will usually be an olive mass or olive pulp that contains the pericarp fractions of the olive fruit, i.e. contains the epicarp and the mesocarp.
  • endocarp parts can also be present. It is preferably a mass in which the olives or olive components have already been comminuted, in particular by mechanical processes such as grinding.
  • the method according to the invention is particularly well suited for obtaining oil from the mass from olives or olive components in which the kernels have not been removed or separated off. This enables a simplified process, whereby a very good oil yield and quality can be achieved.
  • a stored, pre-oiled olive pulp (sometimes also called "orrujo") is used, which has not been subjected to a further separation step in which the cores and pulp are separated, but which is directly subjected to a further deoiling step.
  • the enzyme (s) and olive mass can be mixed with all the usual means known to those skilled in the art (e.g. stirring), it also being possible to (further) comminute the olives (constituents) at the same time, e.g. in a mill.
  • composition according to the invention further contains an enzyme mixture, it having surprisingly been found that with a ratio of the activity of the pectin esterase (s) to the activity of the en o-polygalacturonase (s) of at least 0.13, and preferably a ratio of the activity of the pectin esterase (n ) for the activity of the exo-polygalacturonase (s) of at least 0.3 particularly advantageous properties can be obtained in the extraction of oil. It has unexpectedly been found that, if these conditions are observed in each case, both the release of the oil from the mass from olives or olive constituents is increased in quantity, and the release and separation of the aqueous portion (the aqueous phase) in the oil extraction process is facilitated. Overall, the oil yield can be increased surprisingly. An unexpected effect on the oil quality can also be observed and is explained in more detail below.
  • a pectin esterase is understood to mean an enzyme from the classification group EC 3.1.1.11.
  • the activity of this enzyme can in principle be determined using standard methods familiar to the person skilled in the art.
  • a method used according to the invention is given in the method part below.
  • the enzyme mixture used according to the invention thus contains at least one PEase (activity).
  • An endo-polygalacturonase (eiido-PGase, e.ndo-pectinase) is understood according to the invention to mean an enzyme from the classification group EC 3.2.1.15.
  • the activity of this enzyme can in principle be determined using standard methods familiar to the person skilled in the art.
  • the method used according to the invention is specified in the method part below (pectic acid (PGA) as substrate).
  • the enzyme mixture used according to the invention thus contains at least one e.ndo PGase (activity).
  • the ratio of the pectin esterase (PEase) to the activity of the eido-polygalacturonase is at least 0.15, preferably at least 0.16.
  • the enzyme mixture used according to the invention further contains at least one exo-polygalacturonase (exo-PGase, exo-pectinase), the ratio of the activity of the pectin esterase (s) to the activity of the exo-polygalacturonase (s) being at least 0.3.
  • An activity ratio of at least 0.33 is particularly preferred.
  • an exo-PGase is understood to be an enzyme from the classification group EC 3.2.1.67.
  • the activity of this enzyme can in principle be determined using standard methods familiar to the person skilled in the art. A method used according to the invention is given in the method part below.
  • exo-PGase catalyzes the release of D-galacturonate monomers from 1,4-alpha-D-galacturonide polymers by cleavage of terminal O-glycosidic bonds.
  • pectin is used as a substrate, it is necessary, as with endo-PGase, that any methyl acid ester of polygalacturonic acid is first converted into the free acid or its salt so that it is available to the exo-PGase as a substrate.
  • the setting of the (minimum) ratio according to the invention between pectin esterase and exo-PGase is apparently particularly advantageous in this context.
  • the cleavage of the acid esters by the pectin esterase along the polysaccharide chain probably occurs statistically.
  • the chain is made accessible to the e.ndo PGase as a substrate, which then breaks the chain into oligosaccharides. It is possible that the exo-PGase already performs a final cleavage here, but only until it again finds a methyl acid ester on the chain. A further terminal degradation of the chain could only take place if this methyl acid ester is converted into the acid.
  • the minimum ratio according to the invention between PEase and endo-PGase can ensure that the statistical cleavage of the methyl acid esters by the PEase is coordinated with the exo-PGase activity in such a way that it can work with particularly high performance without restricting the sales due to blocking methyl esters. Further favorable interactions between the above enzyme activities are also conceivable.
  • the ratio of the activity of the pectin esterase to the activity of the endo-polygalacturonase is particularly preferably not greater than about 1, in particular about 0.5, and the ratio of the activity of the pectin esterase to the activity of exo-polygalacturonase is not greater than about 1, in particular about 0.6.
  • the enzyme mixture contained in the composition according to the invention further contains at least one laminarinase ( ⁇ -1,3-glucanase). According to the invention, this is understood to mean an enzyme from the classification group EC 3.2.1.39.
  • the activity of this enzyme can in principle be determined using standard methods familiar to the person skilled in the art. A method used according to the invention is given in the method part below.
  • the ratio of the activity of the pectin esterase to the activity of the laminarinase is preferably at least 20, preferably at least 30.
  • the enzyme mixture contained in the composition according to the invention further contains at least one cellulase, in particular a Cl cellulase.
  • the activity of this enzyme can in principle be determined using standard methods familiar to the person skilled in the art. A method used according to the invention is given in the method part below.
  • the ratio of the activity of the PEase to the activity of the Cl cellulase is particularly preferably at least 75, preferably at least 100.
  • the enzyme mixture contained in the composition according to the invention thus comprises at least six different individual activities, the preferred ratios of the individual activities having a surprisingly positive effect on the oil quantity and quality obtained and the phase separability.
  • the preferred enzyme activities are listed again below: exo-polygalacturonase (EC 3.2.1.67) endo-polygalacturonase (EC 3.2.1.15) pectin lyase (EC 4.2.2.10) pectin esterase (EC 3.1.1.11) Cl-cellulase (EC 3.2.1.91) laminarinase (EC 3.2.1.39)
  • cell or cell wall structure-degrading enzymes may be contained, such as, but not limited to, xylanase (s) (EC 3.2.1.8) or proteolytic enzymes (see introduction to the description and list of enzymes) before the method part; however, according to an embodiment according to the invention not contained in the enzyme mixture).
  • Further constituents in the composition according to the invention are also not excluded.
  • additives are known to the person skilled in the art and include, for example, salts, cofactors, inhibitors or activators for enzymes, stabilizers such as glycerol, etc.
  • the enzyme mixture contained in the composition according to the invention is particularly advantageously suitable for increasing the yield in the extraction of olive oil from olive fruits or their components, particularly in mechanical-physical extraction processes.
  • the following enzyme activities are particularly preferred and can be determined as indicated in the method part below: a. Pectin esterase: more than 300 U / ml, especially more than 320 U / ml b. exo-polygalacturonase: less than 1000 U / ml c. egg-do-polygalacturonase: between 1500 and 2500 U / ml, i.e. Laminarinase: less than 15 U / ml, especially less than 12 U / ml e. Cl cellulase: less than 3.3 U / ml f. Pectin lyase: between about 25,000 and 150,000 U / ml
  • the enzyme mixture according to the invention preferably contains no phospholipase C activity or lipase activity, and the detection can be carried out as indicated in the method section. If appropriate, a phospholipase C activity of not more than 5 U / ml, in particular not more than 1 U / ml, based in each case on the enzyme mixture, can be accepted, between about 50 and 400 ml of the enzyme mixture / t olive pulp being used.
  • the lipase activity should preferably be no more than 3 U / ml, in particular no more than 0.5 U / ml, determined as indicated in the method section and based on the enzyme mixture, using between about 50 and 400 ml enzyme mixture / t olive pulp ,
  • the individual enzymes are commercially available (for example, Sigma-Aldrich Chemie GmbH, Kunststoff, DE). Exemplary enzyme sources are given below before the method part. However, enzymes from other sources can of course also be used as long as they have the corresponding activity indicated above. According to a preferred embodiment, the enzymes are obtained from cultures or the culture supernatant from microorganisms, in particular from bacteria and fungi, including yeasts. A preferred source are cultures of Aspergillus, in particular Aspergillus niger strains, which have several or even all of the enzyme activities contained in the enzyme mixture according to the invention.
  • At least 50 ml in particular between approximately 50 and 400 ml, preferably between 100 and 300 ml, particularly preferably between 125 and 250 ml of the above-described enzyme mixture per ton of mass are particularly preferably used according to the invention.
  • preferably between about 50 and 250 ml, preferably between 100 and 175 ml, in particular between 125 and 150 ml of the above enzyme mixture per ton of mass are advantageous.
  • the protein or enzyme concentration in the enzyme mixture used is preferably between about 2 and 20 mg / 1, preferably 2 and 12 mg / ml, in particular 3 and 10 mg / 1.
  • the protein concentration can be determined using standard methods e.g. can be determined photometrically. From this, the preferred absolute total activities of the individual enzymes contained per ton of the mass of olives or olive components can easily be calculated.
  • the present invention relates to the use of an enzyme mixture as defined above for the treatment of masses from olives or olive components, in particular from pre-de-oiled masses when extracting oil.
  • the use in physical or mechanical-physical oil production has surprisingly been found to be particularly advantageous. As stated at the outset, higher-quality oils can be obtained with these processes, so that the oil yield increased according to the present invention is particularly worthwhile.
  • a parameter that defines the quality of an oil is e.g. B. the acidity.
  • the oil manufacturers strive to keep this as low as possible. It can also be observed that - as described above - the oil yield is increased by sharp centrifugation, but in this way undesirable by-products also enter the oil. These by-products slowly settle out as sediment. It can be stated that the oil manufacturers endeavor to keep the volume of this sediment as small as possible, the texture as uniform as possible and the color as neutral as possible.
  • the parameters mentioned are positively influenced with the aid of the present invention.
  • the present invention thus relates to the use of an enzyme mixture as defined herein for the treatment of masses from olives or olive constituents, in particular of pre-de-oiled masses in the oil production, preferably for improving the physical or mechanical-physical oil production.
  • a further preferred use according to the invention relates to improving the quality of the oil obtained, in particular reducing the acid content, reducing the concentration of conjugated dienes or trienes, reducing sterols or waxes and / or reducing the content of free fatty acids in the oil obtained ,
  • a further preferred use is characterized in that the oil is an olive oil and the treatment with the enzyme composition according to the invention has no negative influence on the content of chloro lesterol, brassicasterol, stigmasterol, ⁇ -stigmasterol and trilinolein.
  • the present invention relates to a method for extracting oil from olives or olive components in bulk, comprising the following steps:
  • any mass of olives or olive constituents familiar to the person skilled in the art can be used, which will generally be a mass of pre-shredded olives or olive constituents.
  • the enzyme mixtures can be added in liquid or solid form.
  • an enzyme solution ie a liquid enzyme mixture
  • a concentrated stock solution of the enzyme mixture can be assumed, which is pre-diluted shortly before addition to the mass.
  • the enzyme can be added at any time, but preferably before or during a step for mixing and / or crushing the mass of olives or olive components.
  • a particularly preferred addition is made in the mixing containers with vertical or horizontal stirring rods.
  • the mixing of the mass and the enzyme mixture can also be carried out in a conventional manner known to those skilled in the art, for example by stirring.
  • the optimum enzyme incubation time and temperature in the individual case can easily be determined by the person skilled in the art on the basis of routine tests. As a rule, temperatures between 20 and 60 ° C., preferably 25-55 ° C., in particular 30-50 ° C. and preferably incubation times between 5 to 120 minutes, preferably 10-100 minutes, in particular 30-90 minutes, will lead to good results , However, significantly shorter or longer times can also be useful. A separate incubation phase can also be omitted if necessary.
  • the oil must be separated from the mixture.
  • the oil can be obtained by separating off the oil phase in any manner known to the person skilled in the art, reference also being made to the methods mentioned in the introduction to the description by way of example. It is preferably a mechanical-physical method, i.e. In contrast to the chemical-extractive processes, no solvent such as e.g. Hexane added or used to extract the oil.
  • the use of the enzyme mixture according to the invention enables particularly efficient oil extraction using a three-phase decanter.
  • the mass introduced e.g. the oil-containing olive pulp (in one work step) is converted into a liquid oil phase, an aqueous phase (sometimes also called “papilla") and a partially de-oiled and partially dewatered (by using at least one three-phase decanter) Solid) suspension (sometimes also called “alperujo” called) separately.
  • papilla an aqueous phase
  • Solid Solid suspension
  • the method according to the invention now unexpectedly significantly increases the separation performance of the three-phase decanters, the oil yield in particular increasing and the oil content in the papilla being able to be significantly reduced. In most cases it is surprisingly possible with the aid of the method according to the invention to make any subsequent process steps for isolating the oil from the papilla unnecessary.
  • Three-phase decanters as such are known to the person skilled in the art; any models can be used.
  • the interposition of malaxers i.e. stirring basins with a horizontal stirring rod familiar to the person skilled in the art, and / or stirring basins with a vertical stirring rod also familiar to the person skilled in the art can even be made redundant.
  • the malaxer can particularly preferably be omitted in the process. Obviously, without the invention being restricted to the correctness of this assumption, an improvement in coalescence is brought about solely by the enzyme mixture. The process economy can be significantly increased by the possibility of omitting the malaxers.
  • pectin can also be used instead of pectic acid (PGA) in the method below for determining the activity of the endo-PGase as substrate.
  • PGA pectic acid
  • an overall activity of the pectin-degrading enzymes can be determined.
  • the present invention therefore also relates to a composition
  • a composition comprising a mass of olives or olive constituents and an enzyme mixture, in which the ratio of the PEase to the total activity of the pectin-degrading enzymes in the enzyme mixture, as determined above, in the enzyme mixture is at least 0.035, preferably at least 0.04 , in particular 0.05.
  • the total activity of the pectin-degrading enzymes is preferably less than 8000 U / ml, in particular less than 7000 U / ml.
  • the activity values of the other enzymes are preferably as indicated above.
  • the activity ratios of PEase to exo-PGase, from PEase to laminarinase and from PEase to Cl-cellulase are also preferably as stated above. The same amounts of enzyme mixture as described above can be used.
  • Another aspect of the invention relates to the use of an enzyme mixture as set out above in one of the methods described here for treating masses from olives or olive components or in a method for extracting oil.
  • the solid / liquid separation can be improved in the extraction of oil.
  • a preferred use here relates to the deoiling of a pre-deoiled mass from olives or olive components.
  • the solid / liquid separation will take place exclusively with the aid of at least one three-phase decanter.
  • the oil yield in the solid / liquid separation is increased, the oil content of the aqueous outlet from the three-phase decanter is reduced, the amount of water separated off in the aqueous outlet is increased and / or the oil and / or water content is reduced using of the solid phase obtained from the three-phase decanter.
  • niger g. EC 3.2.1.1 ⁇ -amylase e.g. Sigma-Aldrich, catalog no. A6211, from A. oryzae h. EC 3.4.2x.yz proteases, eg pepsin and papain, soi EC 3.2.1.21 ß-glucosidase, eg Sigma-Aldrich, catalog no. G6906, recombinant j. EC 3.2.1.22 ⁇ -galactosidase, e.g. Sigma-Aldrich, catalog no. G4408, from A. niger k. EC 3.2.1.23 ß-galactosidase, e.g. Sigma-Aldrich, catalog no.
  • exo-polygalacturonase cleaves galacturonic acid units from saponified citrus pectin, which can be detected photometrically due to the reducing aldehyde groups after reaction with 3,5-dinitrosalicylic acid (DNSS).
  • DNSS 3,5-dinitrosalicylic acid
  • 3-amino-5-nitrosalicylic acid is formed (Eq. 1).
  • a nitro group is reduced to the amino group, while the aldehyde group of the monosaccharide oxidizes to the carboxyl group (Ka tanninc and Vej delek, 1974).
  • Solution A Weigh 38.55 g K-Na-tartrate in a 200 ml beaker and in 125 ml dist. Dissolve water, 2.425 g NaOH (cookies) are dissolved in the solution.
  • Solution B 1.325 g of 3,5-dinitrosalicylic acid (C 7 H 4 N 2 0 7 ; 2-hydroxy-3, 5-dinitrobenzoic acid) in a brown screw-top bottle in 125 ml of dist. Dissolve water.
  • Solution C 1.05 g phenol in 12.5 ml dist. Dissolve water. Add 0.25 g NaOH (cookies) and 1.05 g Na 2 S0 4 in succession and dissolve with stirring.
  • a 250 ml bottle is set in a water bath at 35 ° C and stirrer with a stirrer motor fastened in it.
  • 2.2 g of 80% esterified citrus pectin (from Herbstreith and Fox KG, Pectin Classic CF 201, 00302026 from 30.04.03) are wetted with 4 ml of pure alcohol while slowly stirring. With a strong stirring movement, 50 ml of dist. Pour water into the middle of the stirring suction and stir for another 20 minutes. Make sure that a smooth, viscous solution without lumps is created. Thereafter, taking time to saponify from a dropping funnel 20th ml of 0.5 N sodium hydroxide solution was quickly added dropwise within 5 minutes.
  • the contents of the beaker are then transferred to a 100 ml measuring cylinder while rinsing with buffer solution. After the solution has cooled to 20 ° C., the volume is made up to 100 ml with citrate buffer pH 4.4. Before use, the finished pectic acid solution is filtered over glass wool.
  • a specific endo-PGase unit corresponds to the reciprocal of the amount of enzyme in g multiplied by 100 ml, which in 30 min lowers the viscosity (water value) of 1 liter of a 2.2% pectic acid solution *) by 3/5; (to rel. viscosity 0.40), at 30 ° C and at an optimum pH of 4.4.
  • Apparatus a) Ostwald viscometer 30 ml; Water value approx. 40 seconds in a water bath at 30 ° C. b) Ultra thermostat with water bath and hanging device for test tubes Water bath with immersion thermostat, stirrer motor with paddle stirrer, beakers 600 and 800 ml volumetric flasks 100 and 500 ml calibrated full pipette, 3, 20 and 30 ml test tubes 18 x 180 mm suction bottle, glass frit with rubber sleeve and glass wool, measuring cylinder 100 and 250 ml stopwatches, division 0.1 sec pH device and glass electrode and magnetic stirrer with sticks
  • Base solution 21.01 g citric acid monohydrate in a 1 liter volumetric flask in approx. 100 ml dist. Dissolve water. Pipette in 200 ml of 1N sodium hydroxide solution and mix well, fill up to the calibration mark.
  • Ready-to-use-solution About 350 ml of the basic solution are placed in an 800 ml beaker and a glass electrode with a connected pH device is immersed. 0.1 N hydrochloric acid is now slowly added with stirring (magnetic stirrer), until the pH reaches 4.4. b) Pectic acid solution (2.2%) 11 g of pectic acid are placed in a 600 ml beaker (flat form) and mixed with 20 ml of alcohol (ethanol pA). Let it soak for 30 minutes.
  • the beaker is then fixed in a water bath with immersion thermostat - set to 35 ° C - and a stirrer motor with paddle stirrer is attached in such a way that the paddle stirrer hangs as close as possible to the bottom of the beaker.
  • the finished pectic acid solution is filtered over glass wool.
  • the freshly prepared pectic acid solution turns into full pipettes, depending on the number of enzyme samples, 30 ml pipetted into test tubes and heated to 30 ° C for about 5 minutes before fermentation in an ultra-thermostat for test tubes.
  • the ferment addition of 3 ml (blow out the full pipette) to the 30 ml tempered pectic acid solution is carried out by pressing a stopwatch. In the case of several enzyme samples, the corresponding enzyme solutions are added every 4 minutes. After a fermentation time of less than 30 minutes at 30 ° C, the reaction sample is removed from the water bath and filled into the Ostwald viscometer, which is also heated to 30 ° C.
  • the solution in the viscometer is immediately sucked up, in such a way that it passes the top mark in the outlet exactly 30 minutes after the start of fermentation.
  • the throughput time between the calibration marks is stopped with a second stopwatch and registered as the final viscosity. In the case of several ferment samples, the interval of 4 min between each measurement must also be strictly observed.
  • the initial viscosity is determined in the same viscometer by eating the throughput time of 30 ml pectic acid solution at 30 ° C, but now adding 3 ml citrate buffer solution pH 4.4 instead of ferment solution.
  • the initial viscosity should be determined immediately before the final viscosity. If a whole series of enzyme samples is examined, the initial viscosity is determined immediately before and after the measurement series and the average value is taken.
  • the relative viscosity of the fermented solution corresponds to the ratio of final viscosity (water value) to initial viscosity (water value)
  • the value of the relative viscosity should be between 0.36 - 0.45. If this is not the case, the measurement must be repeated with a correspondingly changed amount of enzyme.
  • the activity can be estimated using the "calibration curve over a wide range" and the units calculated with it can be used as a guideline.
  • the viscosity test is carried out with different concentrations of a standard enzyme (Panzym Combi 285/12, Boehringer Ingelheim).
  • the enzyme concentration which leads to a relative viscosity of 0.40, is equated with the curve value of 1.100.
  • apple pectin (Sigma, catalog no. P8471) was used in the above regulation instead of pectic acid (PGA). This enables a parallel viscosimetric activity determination of the following enzyme activities: pectin esterase, pectin lyase, endo- and exo-polygalacturonase.
  • apple pectin B citrus pectin from Herbstreith & Fox KG No. 00302026 from 30.04.03
  • 0.1 N citrate buffer from pH 5.0 to 100 ml and filtered (eg through a glass filter G2).
  • the increase in absorbance should be between 0.005 and 0.05 per minute at the start of the reaction, otherwise the measurement must be repeated with more or less diluted enzyme solution.
  • the lyase activity results from the measured extinction increase values per minute ( ⁇ Ext.) At the start of the reaction:
  • V F dilution factor
  • a specific pectin esterase unit corresponds to the amount of enzyme in g, which cleaves one micro equivalent of methyl ester groups in a pectin solution containing 550 mg of highly-esterified citrus pectin at 30 ° C and the pH optimum of 4.4 in one minute , Principle:
  • Alkali-metric titration of the pectin solution during the enzyme process at 30 ° C and at a constant pH value of 4.4 with 0.02 N sodium hydroxide solution. The first 5 minutes after adding the enzyme to the pectin solution are used to set the target pH. The actual titration of exactly 20 minutes begins after this time.
  • Apparatus pH device with glass electrode and titrator, water bath with immersion thermostat, stirrer motor with paddle stirrer, 2 micro burettes, 10 ml, automatic volumetric flask, 100 ml, 1 liter full pipette, 10 ml, 100 ml beakers, 200 ml, 1 liter stopwatch, 1 liter suction bottle; Glass frit with rubber sleeve glass wool or Microlith glass filter fleece type OR 80 N (Glastechnik Schuller, Westheim)
  • the finished pectin solution is filtered over glass wool.
  • a beaker (200 ml) is attached so that half of it is immersed in it. 20 ml of the pectin solution are added to the beaker and the temperature is controlled for exactly 15 minutes.
  • a glass electrode connected to a pH meter with a titrator is attached in such a way that it is immersed in the beaker in the pectin solution.
  • a paddle stirrer and the two leads of the car burettes with 0.1 N and 0.01 N NaOH were installed.
  • the amount of 0.01 N NaOH used should be between 1.6 and 4 ml. If this is not the case, the measurement must be repeated with a correspondingly changed amount of enzyme or dilution.
  • M molarity of the titration solution [ ⁇ mol / ml]
  • DNSS 3, 5-dinitrosalicylic acid
  • DNSS phenol reagent Solution A: Weigh 38.55 g K-Na-tartrate in a 200 ml beaker and in 125 ml dist. Dissolve water. Be in the solution then 2.425 g of NaOH (cookies) dissolved.
  • Working solution Solution A and solution C are poured into solution B without rinsing and homogenized for 10 min. Allow the solution to stand for at least one night before use and always keep it in the dark.
  • Buffer 0.1 M Na citrate buffer, pH 4.5
  • Laminarinase cleaves the ⁇ -1,3 bond between the glucose units within the laminarin. The released in the process
  • Glucose is detected photometrically with 3, 5-dinitrosalicylic acid (DNSS).
  • 3, 5-dinitrosalicylic acid (DNSS) is heated with reducing sugars in an alkaline medium, 3-amino-5-nitrosalicylic acid is formed (Eq. 1).
  • Eq. 1 3-amino-5-nitrosalicylic acid is formed (Eq. 1).
  • a nitro group is reduced to the amino group, while the aldehyde group of the monosaccharide oxidizes to the carboxyl group (Ka tanninc and Vejdelek, 1974).
  • DNSS phenol reagent Solution A: Weigh 38.55 g K-Na-tartrate in a 200 ml beaker and in 125 ml dist. Dissolve water. 2.425 g of NaOH (cookies) are then dissolved in the solution.
  • Solution B 1.325 g 3, 5-dinitrosalicylic acid (C 7 H 4 N 2 0 7 ; 2-hydroxy-3, 5-nitrobenzoic acid) in a brown screw-top bottle in 125 ml dist. Dissolve water.
  • Solution C 1.05 g phenol in 12.5 ml dist. Dissolve water. Add 0.25 g NaOH (cookies) and 1.05 g Na 2 S0 in succession and add Loosen stirring.
  • Working solution Solution A and solution C are poured into solution B without rinsing and homogenized for 10 min. Allow the solution to stand for at least one night before use and always keep it in the dark.
  • Buffer 0.1 M Na citrate buffer, pH 4.5
  • Standard solution 2.0 g / 1 glucose (glucose) in dist. Water.
  • Xylanase cleaves the ⁇ -1,4 bond between the xylose units within a xylan molecule.
  • the released xylos is detected photometrically with 3, 5-dinitrosalicylic acid (DNSS).
  • DNSS 3, 5-dinitrosalicylic acid
  • Eq. 1 3-amino-5-nitrosalicylic acid is formed (Eq. 1).
  • a nitro group is reduced to the amino group, while the aldehyde group of the monosaccharide oxidizes to the caboxyl group (Kakac and Vejdelek, 1974).
  • DNSS phenol reagent Solution A: Weigh 38.55 g K-Na-tartrate in a 200 ml beaker and in 125 ml dist. Dissolve water. 2.425 g of NaOH (cookies) are then dissolved in the solution.
  • Solution B 1.325 g 3, 5-dinitrosalicylic acid (C 7 H 4 N 2 0 7 ; 2-hydroxy-3, 5-nitrobenzoic acid) in a brown screw-top bottle in 125 ml dist. Dissolve water.
  • Solution C 1.05 g phenol in 12.5 ml dist. Dissolve water. Add 0.25 g NaOH (biscuit) and 1.05 g Na 2 S0 in succession and dissolve with stirring.
  • Working solution Solution A and solution C are poured into solution B without rinsing and homogenized for 10 min. Allow the solution to stand for at least one night before use and always keep it in the dark.
  • Buffer 0.1 M Na citrate buffer, pH 4.5
  • Lipase activity is determined using the pH-Stat method. Triolein is used as the substrate.
  • a substrate solution (30 ml) consisting of 2% w / v gum arabic, 5% v / v substrate in water is prepared and emulsified with a homogenizer. 1 ml of enzyme solution is added. The acid released in the reaction is back-titrated with 0.01 M sodium hydroxide solution in order to keep the pH constant. The amount of acid released and consequently the activity of the enzyme are calculated from the amount of NaOH.
  • the enzyme solution is replaced by distilled water. The measurements are carried out three times at 37 ° C and pH 7.5. The activity values obtained in U / ml are converted to the total composition of olives or olive constituents and enzyme mixture (ie the activity in the enzyme mixture already used for oil production).
  • the phospholipase C activity is carried out using p-nitrophenylphosphorylcholine as the test substance.
  • 50 ⁇ l of enzyme solution are added to 50 ⁇ l of a p-nitrophenylphosphorylcholine solution (100 mM in 50 mM Borax-HC1, pH 7.5).
  • the p-nitrophenol resulting from the hydrolysis of p-nitrophenylphosphorylcholine is measured at 410 nm on a photometer.
  • the enzyme solution is replaced by distilled water.
  • a calibration curve can be created by preparing suitable dilutions and measuring these solutions at 410 nm in the photometer. Each measurement is carried out three times. The activity values obtained in U / ml are converted to the enzyme mixture. Unless p-nitrophenylphosphorylcholine is cleaved by the enzyme solutions, there is no phospholipase C activity.
  • the yield was 1.11% (w / w) or 270% of the comparison sample, with the addition of 50 ⁇ l of the enzyme mixture according to the invention the yield was 1.14% (w / w) or 278% of the comparison sample, with the addition of 100 ⁇ l of the enzyme mixture according to the invention the yield was 1.26% (w / w) or 307% of the comparison sample and when 200 ⁇ l of the enzyme mixture according to the invention was added the yield was 1.45% (w / w) or 354% of the comparison sample.
  • the relative oil yield without enzyme addition was 2.5 + 0.1% (w / w), with the addition of 150 ⁇ l of the enzyme mixture according to the invention (see above) the relative yield was 4.0 + 0.2% ( w / w) or 163.6 + 4.2% (w / w) of the comparison sample, with the addition of 250 ⁇ l of the enzyme mixture according to the invention, the relative yield was 4.3 + 0.2% (w / w) or 177, 3 + 3.9% (w / w) of the comparative sample, with the addition of 350 ⁇ l of the enzyme mixture according to the invention, the relative yield was 4.8 ⁇ 0.1% (w / w) or 195.5 + 2.0% ( w / w) of the comparison sample and when 450 ⁇ l of the enzyme mixture according to the invention were added, the relative yield was 4.9 + 0.1% (w / w) or 200.0 + 2.0% (w / w) of the comparison sample.
  • the residual oil content was determined by Soxhlet extraction (Spanish standard UNE 55030), the moisture content was determined by drying at 110 ° C to constant weight.
  • the relative oil yield from the second pressing results from the calculation of the difference between the residual oil content in the raw mass after the first step and the equivalent residual oil content after the second step, divided by the residual oil content from the first step.
  • Example 4 (industrial test, industrial plant, "stored")
  • the oil was separated by solid / liquid in a horizontal two-phase decanter (model SPI7 Pieralisi, mass flow 2500-2725 kg / h) and liquid / liquid separation in a centrifuge (model P2000 Pieralisi) with the addition of 300 1 / h water with a Temperature of 38 ° C isolated.
  • the oil isolated in the parallel batches was then stored and the yield was determined by reading a level indicator in the storage containers, 1 cm level difference corresponding to an oil quantity of 95.26 kg.
  • PEase PEase: PEase:
  • the acidity of the oil is reduced by 12.3%.
  • the change in the ⁇ K value (according to EC 2568/91 in the last change of November 6, 2003 (EC 1989/2003)), a measure for the variation of the extinction coefficient at 270 + 4 nm and thus for the type of processing regime for the oil in question, can be neglected here.
  • the use of the enzyme mixture according to the invention has a positive effect (reduction) on the quality parameters of acidity and concentration of conjugated dienes or trienes of the oil obtained.
  • Example 7 Reduction in the content of free fatty acids
  • Fresh, simply industrially physically / mechanically pre-oiled olive pulp was removed in a second industrial process step (oil removal system Pieralisi type M-2) with the addition of 250 ml of the enzyme mixture according to the invention according to Example 1 or without adding the enzyme mixture according to the invention to the horizontal malaxer. oils.
  • the incubation time in the horizontal malaxer was 60 min at 40 ° C.
  • the oils obtained were analyzed with regard to their acid content, their spectroscopic data and their content of free fatty acids (according to EC 2568/91 in the last change of November 6, 2003 (EC 1989/2003)). The results are summarized in Table 2.
  • the use of the enzyme mixture according to the invention has a positive effect (reduction) on the parameters of acidity and concentration of conjugated dienes or trienes of the oil obtained. Furthermore, a positive influence (reduction) on the content of free fatty acids can be observed. To- In summary, a positive effect of the enzyme mixture according to the invention on the quality parameters of the olive oil shown here can be observed.
  • Example 8 Influence of the enzyme mixture according to the invention on further quality parameters
  • Fresh, simply industrially physically / mechanically de-oiled olive pulp was deoiled in a second industrial process step (oil removal system Pieralisi type M-2) with the addition of 250 ml of the enzyme mixture according to the invention according to Example 1 or without adding the enzyme mixture according to the invention to the horizontal malaxer.
  • the incubation time in the horizontal malaxer was 60 min at 40 ° C.
  • the oils obtained were analyzed with regard to the parameters listed in Table 3 (according to EC 2568/91 in the last change of November 6, 2003 (EC 1989/2003)).

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Microbiology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Edible Oils And Fats (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Fats And Perfumes (AREA)

Abstract

L'invention concerne une composition qui contient une masse constituée d'olives ou de constituants d'olives ainsi qu'un mélange enzymatique comprenant au moins une pectine estérase, au moins une endopolygalacturonase et au moins une exopolygalacturonase. Dans cette composition, le rapport de l'activité de la pectine estérase à l'activité de l'endopolygalacturonase est égal à au moins 0,13 et le rapport de l'activité de la pectine estérase à l'activité de l'exopolygalacturonase est égal à au moins 0,3. L'invention concerne également l'utilisation d'un mélange enzymatique correspondant lors de l'extraction d'huile, ainsi qu'un procédé correspondant.
EP04764482A 2003-08-25 2004-08-25 Traitement enzymatique d'une masse constituee d'olives et de constituants d'olives Expired - Lifetime EP1658359B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2003139010 DE10339010A1 (de) 2003-08-25 2003-08-25 Enzymatische Behandlung einer Masse aus Oliven oder Olivenbestandteilen
PCT/EP2004/009506 WO2005021694A1 (fr) 2003-08-25 2004-08-25 Traitement enzymatique d'une masse constituee d'olives et de constituants d'olives

Publications (2)

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EP1658359A1 true EP1658359A1 (fr) 2006-05-24
EP1658359B1 EP1658359B1 (fr) 2007-04-11

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EP04764482A Expired - Lifetime EP1658359B1 (fr) 2003-08-25 2004-08-25 Traitement enzymatique d'une masse constituee d'olives et de constituants d'olives

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ES (2) ES2281824T3 (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010070146A1 (fr) 2008-12-19 2010-06-24 Danisco A/S Procédé pour la production d'un produit de type enzyme

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8898018B2 (en) 2007-03-06 2014-11-25 Schlumberger Technology Corporation Methods and systems for hydrocarbon production
FR2951461B1 (fr) * 2009-10-16 2011-11-25 Lorraine Inst Nat Polytech Procede d'extraction enzymatique en milieu aqueux d'huiles et de proteines a partir de matiere vegetale
DE102011053527A1 (de) 2011-09-12 2013-03-14 Gea Mechanical Equipment Gmbh Verfahren und Anlage zur Aufarbeitung von Alpeorujo
DE102012023136A1 (de) * 2012-11-27 2014-05-28 Pur'oliv Gbr (Vertretungsberechtigter Gesellschafter: Prof. Dr. Dr. Reinhold Carle, 72657 Altenriet) Olivenpaste
EP4108747B1 (fr) * 2021-06-23 2024-05-08 DSM IP Assets B.V. Procédé de production d'huile d'olive
RU2761654C1 (ru) * 2021-07-12 2021-12-13 Общество с ограниченной ответственностью "Маслоэкстракционный завод Юг Руси" Способ переработки высокобелкового растительного сырья

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DE2234263C3 (de) * 1972-07-12 1980-07-31 Maizena Gmbh, 2000 Hamburg Verfahren zur ölgewinnung aus ölhaltigen Getreidekeimen
GB2127425A (en) * 1982-09-28 1984-04-11 Imp Biotechnology Extraction of vegetable oils
CH675730A5 (fr) * 1988-03-30 1990-10-31 Bucher Guyer Ag Masch
DE3843027A1 (de) * 1988-12-21 1990-06-28 Battelle Institut E V Biotechnisches verfahren zur gewinnung von oel und ggf. fettsaeuren aus oelhaltigen pflanzen
DD290912A5 (de) * 1989-09-05 1991-06-13 Adw Zi Fuer Ernaehrung,De Verfahren zur gewinnung von oelen und fetten
JPH0559390A (ja) * 1991-01-16 1993-03-09 Nisshin Plant Eng Kk オリーブ油の抽出法
DE4431394C1 (de) * 1994-08-25 1996-02-15 Heilscher Karl Prof Dr Sc Verfahren zur Kaltgewinnung von Klarsaft, Trub und Öl aus Sanddornbeeren und ihre Verwendung

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010070146A1 (fr) 2008-12-19 2010-06-24 Danisco A/S Procédé pour la production d'un produit de type enzyme

Also Published As

Publication number Publication date
ES2282889T3 (es) 2007-10-16
EP1658359B1 (fr) 2007-04-11
MA28013A1 (fr) 2006-07-03
WO2005021695A1 (fr) 2005-03-10
DE502004003787D1 (de) 2007-06-21
MA27956A1 (fr) 2006-06-01
ES2281824T3 (es) 2007-10-01
WO2005021694A1 (fr) 2005-03-10
DE502004003491D1 (de) 2007-05-24
EP1658360A1 (fr) 2006-05-24
DE10339010A1 (de) 2005-03-24
EP1658360B1 (fr) 2007-05-09

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