ES2282889T3 - BIOTECHNOLOGICAL OPTIMIZATION OF THE PROCESS IN THE OBTAINING OF OIL. - Google Patents

Abstract

CLAIMS 1.- Procedure for the production of oil from masses of olives and / or components of olives, which covers the following steps: a.- making available a mass of olives and / or components of olives, without the separation of the bones from the olives; b.- adding an enzyme or a mixture of enzymes containing at least one pectin esterase, in liquid or solid form; c.- mixed mixing of the dough and enzyme mixture; d.- separation of the oil phase from the dough, with the help of a three phase decanter.

Description

Biotechnological optimization of the process in the Obtaining oil.

The present invention relates to a procedure for the production of oil from masses of olives and / or components of olives, using an enzyme or a mixture of enzymes.

Olive oil is obtained from the fruits of olive tree (tree of the class of dicotyledons, order of oleales, family of oleáceas, species: Olea europaea). The fruit of the olive tree (olive) has a round, sectional shape transverse oval, and is a fleshy fruit of bone with bitter taste. The physical and chemical properties of olive fruits depend of many factors, such as type or variety, grade of maturation, time of harvest, geographical location or growth conditions From the point of view structural, the olive fruit can be divided into two components essential, which are the pericarp and the endocarp. Pericarp It encompasses the epicarp (exocarp) and mesocarp (pulp), which surround the endocarp (wooden bone), in which, again, it is found including the seed.

The production of oil from the fruits Olive tree is of great economic importance. I agree with you data from the International Olive Oil Council (International Olive Oil Council, IOOC), for the production year 2003/2004 expects a total global amount of more than 2.8 million tons of olive oil This amount is made by more diverse technological methods, and encompasses various qualities. Is possible a subdivision of production methods on the basis of two principles, namely, on the one hand the technology mechanical-physics, such as pressing, percolation, as well as two or three phase procedures, and by other part, solvent based procedures in the form of chemical-extractive technology.

The technology used and the type of driving of the process, determine, among others, the oil yield of olive. For the elaboration in the procedures mechanic-physicists, we proceed first to crush or crush the olives, for example, using grinding wheels of mill or hammer mills, followed by a step in which incubate the olive paste for a while determined (typically for 30-90 minutes) under horizontal agitation, possibly under the addition of water, to greater shredding and maceration. This is followed by an obtaining of the oil from the suspension obtained, typically by pressure (pressing) and / or by centrifugal force.

In the first isolation step, described in the preceding, that is to say in the step in which the crushed olives enter directly, it is isolated, depending on the procedure, typically between 50 and 85% of the total oil contained. The mass of olives (pulp of olives) obtained after the first isolation step therefore still has a significant residual oil content, being possible to cause its reduction by the single or multiple repetition of the first step of the process, possibly under the modification of certain process parameters such as temperature, applied centrifugal force, water addition, etc. However, the isolation of the oil by mechanical-physical methods can be carried out in an economically convenient manner only up to a certain residual oil content. Therefore, in order to further reduce the residual oil content and thereby increase the oil yield, chemical extraction procedures are used. For this purpose, the olive pulp is dried, eventually extruded and extracted with a solvent such as hexane. The oil is obtained from the extract by solvent removal, for example by evaporation. In addition to performance, the quality of olive oil is also influenced by the type of procedure and the process. The choice of those procedures that provide high quality oil is economically convenient, since quality correlates positively with the price to be obtained. For this purpose, mechanical-physical procedures are especially suitable. Also for environmental and occupational safety reasons there is an effort to give up as much as possible chemical-extractive procedures, in favor of procedures
mechanical physicists

The use of enzymes is also known from The bibliography Enzymes are functional polypeptides that are in a position to catalyze the splitting of defined substrates or the synthesis of certain products. With reference to the process of olive oil production, in the past they have been described preferably those enzymes or mixtures of enzymes that are in conditions of having a degradation effect on certain cellular structures of the fruit of the olive tree.

The use of enzymes that degrade cell walls in olive oil production has already been described in 1954 by Soroa and Pineda (de Soroa and Pineda, JM (1954) Oleagineux 9, 865-868. Emploi des enzymes dans l 'extraction of l'huile d'olive). On the other hand, the use of endogenous or exogenous enzymes such as polygalacturonases (EC 3.2.1.15 ( endo -) or EC 3.2.1.67 ( exo -)), pectin esterases (EC 3.1.1.11), is known from the state. pepsin (EC 3.4.23.1), papain (EC 3.4.22.2), cellulases (EC 3.2.1.4), α-amylase (EC 3.2.1.1.), proteases (EC 3.4.2x.yz) , β-glucosidase (EC 3.2.1.21), β-galactosidase (EC 3.2.1.23), α-arabinosidases (EC 3.2.1.55), α-mannosidase ((EC 3.2.1.24), β-xylosidases (EC 3.2.1.37), β-N-acetylglucoamidase (EC 3.2.1.52), α-D-galactosidase (EC 3.3.1.22), pectin-liases (EC 4.2.2.10) and pectato-lyase (EC 4.2.2.2) The numbers of EC indicated refer to the nomenclature of enzymes according to the Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzyme- Catalysed Reactions (Nomencl Committee Recommendations Atura of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Catalyzed Reactions through Enzymes (www. chem.qmul.ac-uk / iubmb / enzyme). EP 0 616 024 describes a specific procedure for the production of olive oil from boneless olive fruits by using a cellulase.

However, in this sense the systems described in the bibliography are not optimal, since they only raise relatively reduced oil yield in the Olive oil production. In addition, an optimization is missing biotechnology of the process of the production process of oil, especially in relation to olive masses previously unwanted.

Therefore, the mission of the invention consisted of make available an improved procedure for the production of oil under the use of an enzyme, or a mixture of enzymes, with whose help it is possible to raise significantly the performance of the oil production process and in addition to This will facilitate the process of oil separation.

Description of the invention

The mission is solved by the procedure according to claim 1. In the secondary claims Advantageous embodiments are indicated.

First, a composition or dough, olives or olive components. In this case, any mass of olives or components of usual olive for the expert person, usually being treated of a mass of olives or components of olives, previously crushed In this case, in principle they may be present various components of the olive fruit in arbitrary proportions.  In general, the practice of oil production is about of a mass of olives or an pulp of olives, which contains the pericardium portions of the olive fruit, that is the Epicarp and mesocarp. It is preferably a mass in which olives, or the components of olives, have already been crushed by mechanical procedures such as grinding. It has been surprisingly discovered that the procedure according to the invention is especially suitable and efficient for the production of oil from masses of olives or olives components, of which no removed or separated bones. Therefore, they are also found contained portions of endocarp. This enables a procedure.  simplified, being able to achieve performance and quality of Very good oil

On the other hand, it can be a mass of before or after the first deaerated (obtaining oil). Without However, it has been surprisingly proven that they are obtained especially good results in the framework of the present invention, when in the case of the dough it is a dough already previously unwanted, that is, it has already taken place so minus the first step of oil production. According to a especially preferred embodiment according to the invention, in this case an olive pulp is used previously unwanted, stored (which frequently also receives the denomination of "pomace"), which has not been exposed to a step additional separation in which bones and pulps are separated, but which is exposed directly to another step of unwanted.

To the mass of olives and / or components of olives, mentioned above, an enzyme or a Enzyme mixture containing at least one pectin esterase. The addition can take place in an arbitrary, liquid or solid. However, by virtue of the dosing it is recommended the preparation of an enzyme solution, that is, of a mixture liquid enzyme, before adding to the mass of olives or components of olives. In this case, it is possible to start from a concentrated stock solution of the enzyme mixture, which predilute shortly before adding to the dough. The addition of enzymes it can take place at an arbitrary moment; but nevertheless preferably before or during a step for intermingling and / or crushing the mass of olives or components of olives. An especially preferred addition is that which takes place in the mixing vessels with vertical stir bars or horizontal.

In general terms, the intermingling of the enzyme (s) and the mass of olives can be done with all the usual and known means of the person expert (for example, by agitation), and may also have Simultaneously place a (additional) crushing of the olives (from the components of olives), for example, in a windmill.

The optimal incubation time and temperature for each individual case, they can be determined by the person expert in a simple way through routine tests. For the In general, temperatures between 20 and 60 ° C, preferably 25-55ºC, especially 30-50ºC, and preferably incubation times between 5 and 120 min, preferably 10-100 min, especially 30-90 min, will lead to good results. Without However, durations can also be convenient essentially shorter or longer. It is also possible completely dispense with an incubation phase of its own.

Then, and as already stated in what above, the oil must be separated from the mixture. Obtaining the oil under separation of the oil phase can take place from any usual way for the expert, by referring also, for example, to the procedures indicated in the introduction of the present specification. It is about preferably from a mechanical-physical procedure, that is, unlike the procedure chemical-extractive, for the extraction of oil not no solvent agent is used such as hexane.

Within the scope of the invention it has been discovered now surprisingly, that thanks to employment according to the invention of the enzyme, or of the enzyme mixture that it contains at least one pectin esterase, a especially efficient oil production through the use of a three phase decanter.

In the so-called three-phase procedure, by using at least one three phase decanter, separates the dough introduced, for example, the olive pulp that contains oil (in a working step) in a liquid phase of oil, an aqueous phase (also often called "porridge") and a suspension (of solids) partially unwanted and partially dehydrated (which often receives also the name of "alperujo"). However, due to The limited separation capacity of the three-phase decanter is  conventionally finds a significant amount of oil in the porridge This oil is usually isolated from the porridge, in a subsequent step of the process. By the procedure according to the invention, the ability to separation of the three-phase decanter, in case it rises especially the oil yield and it is possible to reduce considerably the proportion of oil in the porridge. By way of surprising, with the help of the procedure according to the invention, it is possible to achieve in most cases that are superfluous the subsequent process steps for Isolate the oil from the porridge. In this regard, the decanters of three phases are known as such by the skilled person; they can use any models.

According to another aspect of the present invention, He has discovered that not only in the three-phase procedure, described above, but also in the use of others physical or mechanical-physical procedures in special so-called two-phase procedure, that is, in the which thanks to the use of two-phase decanters the pulp of oil separates in a suspension of strong solids content (which also receives the name "alperujo") and in a water / oil emulsion, and the oil is obtained by centrifugation at from this emulsion, by use according to the invention of enzymes, the interposition of kneaders can be made superfluous, that is, the usual agitation pools for the skilled person provided with horizontal stir bars, and / or pools of agitation, also usual for the expert, with bars vertical agitation In a particularly preferred way, according to the invention, it is possible to omit the kneaders in the process. In this case it is evident, without that the invention is limited to the accuracy of this assumption, which has place an improvement of coalescence due to enzymes only. Thanks to the possibility of skipping the kneaders, it is possible to significantly increase the economic profitability of process.

In accordance with another aspect of the present invention, it has been found that the advantages, described herein, of the process for the production of the oil are especially great when the enzyme mixture has a certain composition. Thus, it has been surprisingly discovered that for a relationship between the activity of the pectin esterase (s) and the activity of the endo- polygalacturonase (s) of at least 0.13, especially properties are obtained advantageous in oil production. Furthermore, it has been unexpectedly discovered that a relationship between the activity of the pectin esterase (s) and the activity of the exo- polygalacturonase (s), of at least 0.3, is especially favorable. If these relationships are respected, the positive effects on the release of the oil from the mass of olives or components of olives, and on the release and separation of the aqueous ratio (from the aqueous phase) in the oil production process, They are specially marked. As a whole, it is possible to surprisingly increase the oil yield.

According to the invention, for a pectin esterase (PEase) means an enzyme of the Classification Group EC 3.1.1.11. In principle, it is possible to determine the activity of this enzyme by standard methods usual for the person expert In the next chapter, related to the methods, indicates a method used according to the invention. Thus, The enzyme mixture used according to the invention contains the minus one (activity of) PEase.

By an endo- polygalacturonase ( endo -PGase, endo -pectinase) is understood according to the invention an enzyme of Classification Group EC 3.2.1.15. In principle, the activity of this enzyme can be determined with the help of the usual standard methods for the skilled person. The method used according to the invention is indicated in the following chapter, related to the methods (pectinic acid (PGA) as a substrate). Accordingly, the enzyme mixture used in accordance with the invention contains at least one ( endo- PGase) activity.

According to an especially advantageous embodiment, the ratio between pectin esterase (PEase) and endo- polygalacturonase activity is at least 0.15, preferably at least 0.16.

The enzyme mixture used according to the invention also contains at least one exo- polygalacturonase ( exo -PGase, exo- kinase), the ratio between the activity of the pectin esterase (s) and the activity of the exo - polygalacturonase (s), at least equal to 0.3. A relationship between activities of at least 0.33 is especially preferred. According to the invention, exo -PGase means an enzyme of the EC Classification Group 3.2.1.67. In principle, it is possible to determine the activity of this enzyme with the help of the usual standard methods for the skilled person. In the next chapter, related to the methods, a method used according to the
invention.

It is assumed, without restricting the invention to this theoretical mechanism, because the proportion of pectin esterase relatively high, especially in the case of simultaneous presence of endo - and exo -PGasa (s) a disintegration is achieved especially efficient of polysaccharide structures of high molecular weight, in which the oil to be produced is "included". It is also assumed that due to the accessibility pectin esterase substrates is improved endo - and exo -PGasas as well as other enzymes that cleave polysaccharides. Therefore, it is manifestly possible to accelerate the decomposition of high molecular weight structures, with which the oil to be produced is associated. By definition, exo -PGase catalyzes the release of D-galactouronate monomers from 1,4-alpha-D-galacturonic polymers by splitting O-glycoside bonds in the extreme position. If pectin is used as a substrate, it will be necessary, as in the case of endo- PGase, that the possible methyl acid esters of polygalacturonic acids be converted firstly into the free acid, or salt thereof, so that they are available as a substrate for the exo -PGasa. In this context, the adjustment of the (minimum) ratio according to the invention between pectin esterase and exo -PGase is obviously especially advantageous. The splitting of the acid esters by the pectin esterases along the polysaccharide chain takes place in a more statistical manner. Thus, in the first step, the chain becomes accessible as a substrate for PGase, and the substrate then divides the chain into oligosaccharides. It is possible that in this case the exo -PGase already performs a split in the terminal position, but only until it meets again with an ester of methyl acid in the chain. Then, a new decomposition in the terminal position of the chain could only take place if this methyl acid ester is transformed into the acid. The minimum ratio, according to the invention, between PEase and endo- PGase can provide that the statistical cleavage of the methyl acid esters by the PEase is tuned to the exo- PGase activity, so that it can work with a especially high efficiency without a conversion restriction due to blocking methyl esters. It is also possible to imagine other favorable interactions between enzyme activities above
mentioned.

It is especially preferred that the relationship between the activity of pectin esterase and the activity of endo- polygalacturonase is not greater than about 1, especially not greater than about 0.5, and that the relationship between the activity of pectin esterase and the activity of exo- polygalacturonase is not greater than about 1, especially not greater than about 0.6.

According to another embodiment preferred, the enzyme mixture employed also contains at least a laminarinase (β-1,3-glucanase). According to the invention, by laminarinase is meant an enzyme of the Group of EC classification 3.2.1.39. In principle, it is possible to determine the activity of this enzyme by the usual standard methods for The expert person. In the next chapter of the methods, indicates a method used in accordance with the invention. It is preferable that the relationship between pectinerase activity and activity of the laminarinase is at least 20, preferably at least 30.

According to another embodiment preferred according to the invention, the enzyme mixture used It also contains at least one cellulase, especially a C1 cellulase. By cellulase C1 is understood according to the invention an enzyme of the EC Classification Group 3.2.1.91. In principle, it is possible determine the activity of this enzyme by the methods Usual standards for the skilled person. In the next chapter of the methods a method used according to the invention. It is especially preferable that the relationship between PEase activity and C1 cellulase activity is from at minus 20, preferably at least 30.

According to one embodiment especially preferred, the enzyme mixture thus encompasses the minus six different individual activities, having the preferred relationships of individual activities so surprising a positive effect on oil performance obtained and on the separability of the phases. Then you give again a list of enzymatic activities preferably contained:

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) Cellulase-C1 (EC 3.2.1.91) Laminarinase (EC 3.2.1.39)

However, there may also be other components contained in the enzyme mixture according to the invention. In relation to the enzymatic components of the enzyme mixture there may be, for example, other contained enzymes that degrade cell structures, or cell wall structures, such as, but not limited to, xylanase (s) ( EC 3.2.1.8), or also proteolytic enzymes (cf. the introduction to the specification and the list of enzymes of the methods chapter; according to an embodiment according to the invention, not contained in the mixture of enzymes). Nor are other components excluded in the mixture of the mass of olives and the mixture of enzymes. Such additives are usual for the person skilled in the art, and include, for example, salts, cofactors, inhibitors or activators for enzymes, stabilizing agents such as
glycerin, etc.

As already mentioned, the enzyme mixture used in accordance with the invention is especially advantageous for the increase in yield in obtaining olive oil to from olive fruits or their component parts, especially in the mechanical-physical production processes.

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According to the invention, they are preferred especially the following enzyme activities, which can be determined as indicated in the following methods chapter:

to.-

Pectin esterase: more than 300 U / ml, in special, more than 320 U / ml

b.-

exo- polygalacturonase: less than 1,000 U / ml

C.-

endo- polygalacturonase: between 1,500 and 2,500 U / ml

d.-

laminarinase: less than 15 U / ml, in special, less than 12 U / ml

and.-

cellulase-C1: less than 3.3 U / ml

F.-

Pectin lyase: between approximately 25,000 and 150,000 U / ml.

Individual enzymes can be obtained commercially (for example, those provided by Sigma-Aldrich Chemie GmbH, Munich, Germany). Next, before the methods chapter, enzyme sources are indicated by way of example. However, it is of course also possible to use enzymes from other sources, with the proviso that they have the activity indicated above. According to a preferred embodiment, the enzymes are obtained from cultures, or from the supernatant material of cultures of microorganisms, especially bacteria and fungi, yeasts included. A preferred source consists of Aspergillus cultures, especially strains of Aspergillus Niger , which exhibit several of the enzyme activities contained in the enzyme mixture according to the invention, or even all such activities.

Referring to the mass of olives or component parts of olives, at least 50 ml, especially between about 50 and 400 ml, preferably between 100 and 300 ml, are used according to the invention. especially preferably, between 125 and 250 ml of the above-mentioned enzyme mixture, per tonne of dough. In many cases, between 100 and 175 ml, especially between 125 and 150 ml, of the enzyme mixture described above, per tonne of mass are preferably advantageous. In the present specification,
The term "enzyme mixture" should also be used in the case of the use of a single enzyme (PEase).

In this case, it is preferable that the concentration of proteins or enzymes in the enzyme mixture used be approximately 2 to 20 mg / l, preferably between 2 and 12 mg / ml, in special between 3 and 10 mg / l. It is possible to determine the concentration of proteins with the help of standard procedures, for example, by photometry From this it is possible to calculate so simple the preferred absolute joint activities of the individual enzymes contained, per tonne of olive mass or of component parts of olives.

According to another aspect, this invention relates to the use of a mixture of enzymes as defined above, for the treatment of masses of olives or components of olives, especially unwanted masses in the oil production. Surprisingly, it has turned out to be especially advantageous use in the case of production Physical or mechanical oil. As initially noted, it is possible to obtain high quality oils with these procedures, so the oil yield, increased In accordance with the present invention, it is presented as especially productive.

In the following method for the determination of endo- PGase activity, it is also possible to use pectin instead of pectinic acid (PGA) as a substrate. In this way it is possible to determine a total activity of the enzyme that degrades pectin. In this case, within the framework of an alternative embodiment of the present invention, it has been found that there are especially advantageous properties in the production of oil, when in the applied enzyme mixture the ratio between PEase and total activity, determined as described above, of the enzyme that degrades pectin, present in the enzyme mixture, is at least 0.035, preferably at least 0.04, especially at least 0.05. According to an alternative embodiment, therefore the present invention also relates to a composition containing a mass of olives or components of olives and a mixture of enzymes, wherein the ratio between PEase and total activity, determined as described above, of the enzyme that degrades pectin, present in the enzyme mixture, is at least 0.035, preferably at least 0.04, especially 0.05.

In this case, the joint activity of the enzyme that degrades pectin, preferably has a value of less than 8,000 U / ml, especially less than 7,000 U / ml. The activity values of the other enzymes are as indicated above. Also the relationships between the activities of PEase and exo -PGase, PEase and laminarinase, and PEase and C1 cellulose, are preferably as indicated in the foregoing. The same amounts of enzyme mixture can be applied as described above.

Another aspect according to the invention is that refers to the use of a mixture of enzymes as previously it was exposed in one of the procedures described in the present for the treatment of masses of olives or components of olives, or in a process for the production of oil. In This case, according to an embodiment of the invention, is possible to improve the separation of solids / liquids during oil production. Here, a preferred use is the one refers to the de-oiled of a previously de-oiled mass of olives or olives components.

According to one embodiment especially preferred, the solids / liquids separation has exclusively place with the help of at least one three decanter phases

According to another aspect of using the present invention, the oil yield is raised during solids / liquids separation, the oil content of the aqueous drain from the three phase decanter, the amount of water separated in the aqueous drain, and / or the oil and / or water content of the solid phase obtained with the three phase decanter support.

According to another aspect of using the present invention, it becomes possible to reduce the temperature of the process during oil production, keeping at least Equal oil yield.

In the process of oil production of olive there is also the effort to produce the maximum amount possible of high quality oil, in order to obtain in this way a price as high as possible in the market. In the system of applied production there is a correlation in the first approximation between oil quantity and quality: if an installation is operated with a separation efficiency as high as possible, that is, if the quantity of oil is optimized, the quality is reduced, and inversely. By using the enzyme mixture according to the invention it is possible to surprisingly achieve a High quality oil along with high performance.

One of the parameters that defines the quality of an oil, is for example the acid content. The producers of Oil strive to keep it as low as possible. In addition, it should be noted, as described above, that although by an energetic centrifugation increases the oil yield of this way also enter unwanted by-products in the oil. These secondary products are slowly deposited in sediment form. It may be taken for granted that the producers of oil strive to keep the volume of this sediment as much as small as possible, the texture as uniform as possible, and the colors the most neutral possible. With the help of the present invention, positively influences the mentioned parameters.

Therefore, according to another aspect, the The present invention relates to the use of a mixture of enzymes as defined herein in the process according to the invention described herein, for the improvement of quality of the oil obtained, especially the reduction of the content of acids, reducing the concentration of dienes or triphenes conjugates, reduction of peroxy substances, reduction of sterols or waxes and / or reduced fatty acid content free in the oil obtained. Another preferred use is characterized because due to the treatment with the composition of enzymes according to the invention, no influence takes place negative on the content of cholesterol, brassicasterol, stigmaesterol, Δ-stigmaesterol and trilinolein

Enzyme Suppliers and Sources

to. EC 3.2.1.15

endo -PGasa, for example, the Sigma-Aldrich Firm, Catalog- No. P4716, of A. Niger

b. EC 3.2.1.67

exo -PGasa (Kobayashi, T., Higaki, N., Yajima, N., Suzumatsu, A., Hagibara, H., Kawai, H., and Ito, S. (2001), Biosci. Biotechnol. Biochem . 65 , Pp. 842-848) or from ASA Spezialenzyme GmBH, Wolfenbüttel, Germany, in the form of enzyme solutions containing exo -PGase.

C. EC 3.1.1.11

pectin esterases, for example, from the Firm Sigma-Aldrich, Catalog - No. 5400, from Orange peels

d. EC 3.4.23.1

pepsin, for example from the Firm Sigma-Aldrich, Catalog No. P7012, from mucosa gastric pig

and. EC 3.4.22.2

Papain, for example, from the Firm Sigma-Aldrich, Catalog No. P4762, from papaya

F. EC 3.2.1.4

cellulase, for example from the Sigma-Aldrich Firm, Catalog No. C1184, from A. niger

g. EC 3.2.1.1

α-amylase, for example from Sigma-Aldrich, Catalog No. A6211, from A. oryzae

h. EC 3.4.2x.yz

proteases, for example pepsin and papain, above mentioned

i. EC 3.2.1.21

β-glucosidase, for example of the Sigma-Aldrich Firm, Catalog No. G6906, recombinant

j. EC 3.2.1. 22

α-galactosidase, for example from Sigma-Aldrich, Catalog No. G4408, from A. Niger

k. EC 3.2.1.23

β -g alactosidase, for example from Sigma-Aldrich, Catalog No. G3522, from A. niger

l. EC 3.2.1.55

α -a rabinosidase

m. EC 3.2.1.24

α-mannosidase, for example, of the Sigma Firm, Catalog Nº M7257, from beans

n. EC 3.2.1.37

β-xylosidase, for example, from Sigma, catalog No. X3501, from A. Niger

or. EC 3.2.1.52

β-N-acetylglucosaminidase, for example, from the Sigma Firm, Catalog Nº A2264, from broad beans.

p. EC 4.2.2.10

pectin-lyase, for example, from Sigma, Catalog No. PP7052, from A. niger

q. EC 3.2.1.6

laminarinase, for example, from the Firm Sigma-Aldrich, Catalog No. L5272, from Trichoderma spec.

r. EC 3.2.1.8

Xylanase, for example, from the Firm Sigma-Aldrich, Catalog N` X3876, by Trichoderma veride

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Methods: Determination of enzyme activities (according to Methods of the Firm ASA Spezialenzyme GmbH, Wolfenbüttel, Germany)

Unless otherwise indicated, within the framework of the invention the activities of enzymes are determined in accordance with the procedures indicated in the following Chapter of Methods

1.- Determination of the activity of exo- polygalacturonase- ( exo -PGase)

(Sugar measurement reducer)

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Trial Principle

The exo- polygalacturonase ( exo -PGase) is split from saponified citrus pectin galacturonic acid units, which due to the aldehyde reducing groups could be detected by photometry after the reaction with 3,5-dinitrosalicylic acid (DNSS).

When heated acid 3,5-Dinitrosalicylic (DNSS) with reducing sugars in alkaline medium, acid is formed 3-amino-5-nitro-salicylic (Equation 1). In this case a nitro group in the form of amino group, while the aldehyde group of the monosaccharide is oxidized in the form of a carboxyl group (Kakác and Vejdelek, 1974).

one

Different sugars result in different colorations whose maximum absorption is between 500 and 550 nm. Thus, with this test it is also possible to detect mono-, di-, oligo- and polysaccharides as well as methylpentoses and O-methylsaccharides.

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Reagents

DNSS-phenol reagent:

- Solution to:

Weigh 38.55 g of tartrate K and Na in a 200 ml glass cup, and dissolve in 125 ml of water distilled; 2,425 g of NaOH are dissolved in the solution (cookie).

- Solution B:

Dissolve 1,325 g of acid 3,5-dinitrosalicylic (C 7 H 4 N 2 O 7; acid 2-hydroxy-3,5-dinitrobenzoic acid) in a bottle of brown thread, in 125 ml of distilled water.

- Solution C:

Dissolve 1.05 g of phenol in 12.5 ml of water distilled Add 0.25 g of NaOH (biscuit) and 1.05 g consecutively of Na 2 SO 4, and dissolve under stirring.

- Usage Solution:

Solution A and solution B are poured (without further rinse) in solution C, and homogenize for 10 min. Leave the solution at rest for at least one night before of use, and always keep in the dark.

- Substrate solution:

Saponified Citrus Pectin Solution 2.2% in 0.1 M Na citrate, pH 4.4; saponification method: protocol according to Boehringer Ingelheim, page 3.

- Standard solution:

12.0 g / l galacturonic acid (GA) in water distilled

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Realization

Samples:

Mix 0.9 ml of substrate solution with 0.1 ml of enzyme solution, incubate at 30 ° C for 30 min, and then immediately place in the ice bath. Add 2.0 ml of DNSS-phenol reagent and boil for 5 min. Cool the mixture for 5 min in the ice bath, and measure the extinction at room temperature and at 492 nm.

Default values:

Mix 0.9 ml of substrate solution with 0.1 ml of water, add 2.0 ml of DNSS-phenol reagent, and boil for 5 min. Cool the mixture for 5 min in the bath ice, and measure the extinction at room temperature and at 492 nm.

Standard:

Mix 1.0 ml of diluted standard solution with 2.0 ml of DNSS-phenol reagent and boil for 5 min. Cool the mixture for 5 min in the ice bath, and measure the extinction at room temperature and at 492 nm.

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Calibration

Dilutions of the standard solution for the plotting the calibration lines:

C (GA) in g / l Standard solution in \ mul Distilled water in \ mul 0.6 fifty 950 1.2 100 900 1.8 150 850 3.0 250 750 4,5 375 625 6.0 500 500

Evaluation

Exo -PGase activity [U / ml] = \ frac {\ Delta c (GA)} {M (GA)} \ cdot \ frac {V_ {solution \ reactive}} {V_ {enzymes} \ cdot t_ {reaction} } \ cdot Diluc \ cdot

M (GA) = 194 g / mol

T_ {reaction} = 30 min

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Preparation of the saponified solution of 2.2% pectin (pectinic acid solution)

A 250 ml bottle is adjusted to 35 ° C in a water bath, and an agitator with motor is installed in it agitation. In this, 2.2 g of citrus pectin, esterified in more 80% (provided by Herbstreith und Fox KG, Pektin Classic CF 201, 00302026 of 30.04.03) are moistened with 4 ml of low pure alcohol slow agitation Under a more intensive agitation movement, 50 ml of distilled water are poured on it in the middle of the swirling stirring, and stirring is continued for another 20 min. It should be borne in mind that in this case a smooth viscous solution without lumps. Then, and taking note of the time for saponification, by means of a drip funnel is added quickly drop by drop 20 ml of a 0.5 N soda lye in a 5 minute interval. After a total saponification time for exactly 6 min at 35 ° C under constant stirring, the reaction by adding 10% citric acid from a rod pipette By adding 3 ml of acid solution Citric pH is adjusted to 4.4. It cannot be corrected later pH value by the subsequent addition of bleach.

Then the content of the glass cup under rinse with buffer solution, to a test tube 100 ml graduated. After cooling the solution to 20 ° C, it is complete the volume with citrate buffer pH 4.4, until obtaining a total volume of 100 ml. Before use the solution is filtered Finished pectinic acid on glass wool.

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2.- Determination of the activity of endo- polygalacturonase ( endo -PGase) Definition

A specific unit of endo -PGase corresponds to the reciprocal value, multiplied with 100 ml, of the amount of enzymes in g, which in 30 min reduces the viscosity (- equivalent in water) of 1 liter of a solution of pectinic acid to 2, 2% *) in 3/5; (referred to a relative viscosity of 0.4), at 30 ° C and for an optimum pH of 4.4.

*) Pure Pectinic Acid

Fluka AG, Chemische Fabrik, CH-9470 Buchs SG

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Beginning

Measurement of viscosity degradation of Fluka's fermented pectinic acid solution in the Ostwald viscometer.

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Appliances

to)

viscometer according to Ostwald 30 ml; value of water approximately 40 sec in 30 ° C bath.

b)

thermostat-Ultra with water bath and suspension devices for pipes test

C)

bathroom of water with suspension thermostat

d)

engine stirrer with paddle shaker

and)

cups 600 and 800 ml glass

F)

100 and 500 graduated flasks ml

g)

3, 20 and 30 graduated pipettes ml

h)

pipes test, 18 x 180 mm

i)

suction bottle, fried glass with rubber sleeve and glass wool, graduated test tube of 100 and 250 ml

j)

stopwatches, division 0.1 sec.

k)

pH device and glass electrode, Y

l)

magnetic stirrer with rods

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Reagents

-

acid pure scallop (Fluka AG, Chem. Fabrik, Buchs, SG)

-

acid citric monohydrate, DAB 6

-

bleach Soda 1N, 0.5 N, 0.1 N

-

acid 0.1 N hydrochloric

-

Absolute alcohol p.A.

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Solutions

a) citrate buffer solution pH 4.4 (0.1 cool)

Basic Solution:

Dissolve 21.01 g of citric acid monohydrate in a 1 liter graduated flask, in approximately 100 ml of water distilled

To this add 200 ml of bleach by pipette 1 N soda and mix well, complete until graduation mark

Use of the solution:

From the base solution are introduced approximately 350 ml in an 800 ml glass cup, and immersed  a glass electrode with a coupled pH device. Low Stirring (magnetic stirrer) is now slowly added acid 0.1 N hydrochloric until pH = 4.4 is achieved.

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b) pectinic acid solution (2.2%)

11 g of pectinic acid are placed in a cup 600 ml glass (flat form), and mixed with 20 ml of alcohol (ethanol p.A.). Let it swell for 30 minutes. Then it fix the glass cup in a water bath with thermostat suspension regulated at 35 ° C, and on this a motor of stirring with paddle shaker so that the stirrer of pallets are hung as close as possible to the bottom of the glass cup

Under a strong stirring movement it pour 240 ml of distilled water into the glass cup, and then stirring is continued for exactly 20 min, maintaining a constant and strong stirring speed.

It should be borne in mind that a smooth viscous solution without lumps. Then the value is adjusted of the pH of the solution with sodium hydroxide solution (first 0.5 N and then 0.1 N) at exactly pH = 4.4 (pH meter). It should not be captioned, ¡Since with pH values higher than pH = 4.4 lumps are formed by gel formation! After adjusting the pH the glass cup content under a good final wash with the citrate buffer solution pH 4.4, to a 500 ml graduated flask, and after cooling to 20 ° C it is completed with citrate buffer until the brand.

Before using the solution is filtered Finished pectinic acid, on glass wool.

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Making the determination

From the fresh pectinic acid solution prepared are introduced by volumetric pipette, depending on the amount of enzyme samples, each time 30 ml in glasses glass, and before fermentation in the Ultra thermostat for test tubes regulated at 30 ° C - heated for approximately 5 min at 30 ° C. The addition of the ferment in each case 3 ml (blow off the volumetric pipette) to the solution 30 ml of hardened pectinic acid takes place registering the times for oppression of a stopwatch. In the case of having several Enzyme samples are added enzyme solutions corresponding to intervals of in each case 4 min. After a fermentation time of at least 30 min at 30 ° C, the water bath reaction sample and with it the Ostwald viscometer, also tempered at 30 ° C. The solution in the viscometer rises immediately by suction, specifically from such that at the exit of the liquid passes through the upper mark exactly 30 min after the start of fermentation. He Pass time between graduation marks is taken with a second stopwatch, and is recorded as final viscosity. In this case, if there are several fermentation samples, you should also respect exactly the separation of in each case 4 min between Each of the measurements.

The initial viscosity is determined therein. viscometer, measuring the run time of 30 ml of solution of pectinic acid at 30 ° C, but now being added, instead of ferment solution, 3 ml citrate buffer solution pH 4.4. Should determine the initial viscosity immediately before final viscosity If a complete series of samples of enzymes, the initial viscosity is determined before and after the measurement series, adopting the average value.

Evaluation

Measurement in it viscometer Equivalent value in Water Water pass time distilled \ rightarrow W Viscosity initial Pass time of the solution Pectinic acid + 3 ml buffer pH 4.4 \ rightarrow TO Final viscosity Weather in passing of the fermented solution \ rightarrow AND

The relative viscosity of the fermented solution corresponds to the ratio between the final viscosity (value equivalent in water) and the initial viscosity (-value equivalent in Water).

Viscosity relative = \ frac {E - W} {A - W}

The relative viscosity value should be between 0.36 and 0.45. If this requirement is not met, it must be repeated measurement with an applied amount of enzyme, correspondingly modified. For the determination of the new past you can undertake an assessment of the activity with the help of the "calibration curve over a wide range", and apply the units thus calculated as normative value.

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Activity Calculation

Endo- polygalacturonase activity [U / ml] = \ frac {k \ cdot x \ cdot V_ {T} \ cdot x \ cdot V_ {F}} {V_ {E}}

k = 1,100; curve value of the calibration curve under a relative viscosity of 0.40 (obtained with an enzyme standard)

V_ {T} = test volume

V_ {F = dilution factor

V_ {= volume of the enzyme solution [ml]

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Obtaining the calibration curve

The viscosity test is carried out with various concentrations of a standard enzyme (Panzym Combi 285/12, Boehringer Ingelheim). The enzyme concentration, which leads to a relative viscosity of 0.40, equals the value of 1,100 curve.

According to the alternative embodiment of the invention, in the preceding protocol, apple pectin (Sigma, Catalog No. P8471) was used instead of pectinic acid (PGA). This allows a parallel determination of the viscosity of the following enzyme activities: pectin esterase, pectin lyase, endo- and exo -polygalacturonase.

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3.- Determination of pectin activity liasa Trial Principle

During the splitting of the pectin by the pectin lyase, a double bond is formed between the C -4 atoms and C-5 of the non-reducing limb, newly formed, of the chain. The double bond leads to light absorption at 235 mm The speed, with which the extinction increases, serves as Measurement of lyase activity.

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Substrate Solution

0.41 g of apple pectin B (pectin from citrus, from Herbstreith & Fox KG No. 00302026 of 04/30.03) Dissolve over 100 ml with 0.1 n citrate buffer, pH 5.0, and filter (for example, through a G2 glass filter).

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Measurement completion

In the cuvette with a layer thickness of 1 cm it Pipette 2.9 ml of substrate solution (warm to 30 ° C) and 0.1 ml of enzyme dilution at 30 ° C and at 235 nm. The increase in extinction over time compared to a zero sample of 2.9 ml of substrate and 0.1 ml of dilution of enzyme previously deactivated by heat.

At the beginning of the reaction, the increase in extinction should be in the range between 0.005 and 0.05 per minute; otherwise, the measurement must be repeated with a enzyme diluted to a greater or lesser degree.

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Activity Calculation

The activity of the lyase results from the values measured increment of extinction per minute (ΔExt.), at start of reaction:

Activity of the U / ml pectin lyase = \ frac {\ Delta Ext \ cdot x \ cdot 1. 000 \ cdot x \ cdot 30 \ cdot x \ cdot V_ {F}} {5.5}

V_ {F = dilution factor

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4.- Determination of pectin esterase activity (PEase) Definition

A specific unit of pectin esterase (PEase-E) corresponds to the amount of enzyme in g, which in a minute unfolds at 30 ° C and with the optimum pH of 4.4, a microequivalent methyl ester groups in a pectin solution It contains 550 mg of highly esterified citrus pectin.

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Beginning

Alkalometric titration of the solution pectin during the enzymatic process at 30 ° C and maintained constant the pH value of 4.4 with a 0.02 N soda bleach. first 5 minutes after the addition of the enzyme to the solution of pectin, they are used to adjust the theoretical pH value. The proper degree of approximately 20 minutes begins after this time

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Appliances

to)

pH device with glass electrode and titration team

b)

bathroom of water with suspension thermostat

C)

engine stirrer with paddle shaker ...

d)

two 10 ml microburettes, automatic

and)

100 ml graduated flasks, 1 liter

F)

10 ml volumetric pipettes, 100 ml

g)

cups 200 ml glass, 1 liter

h)

chronometer

i)

1 liter suction bottle; fried glass with rubber sleeve

j)

wool glass or fleece Microlith glass filter type OR 80 N (Glazier Schuller, Westheim)

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Reagents

-

dried citrus pectin, esterified in approximately 74% (Pemosin, Frankfurtt / M)

-

bleach 0.1 N soda, Tritisol

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Solutions

a) 0.01 N soda bleach

2 x 100 ml of 0.1 N soda lye are introduced by pipette in a 1 liter graduated flask, and is completed with distilled water to the mark.

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b) pectin solution (0.55%)

A 100 ml glass cup is fixed in a bathroom of water with suspension thermostat -adjusted at 30ºC-, and on the a stirring motor with vane stirrer is installed so that the paddle agitator hangs as close as possible to on the bottom of the glass cup.

Approximately 700 ml of water is introduced distilled (H2O distilled) in the glass cup, and then 5.5 g of dry citrus pectin are introduced highly esterified under strong stirring for approximately 2 min in the medium of the stirring swirl. Then the agitation speed, and for all investigations further stirring is carried out under a r.p.m. constant for exactly 30 min.

It should be noted that in this case a smooth viscous solution without lumps.

The content of the glass cup is transferred under final wash along with a small amount of distilled water in a 1 liter graduated flask, and is completed at 20 ° C until brand.

Before use, the glass wool is filtered finished pectin solution.

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Making the determination

In a water bath with suspension thermostat - set at 30 ° C - a glass cup (200 ml) of way that is submerged in it halfway. 20 are added ml of the pectin solution in the glass cup, and warm for exactly 15 min.

Within this time interval a glass electrode, coupled to a pH meter with equipment titration, so that it is immersed in the pectin solution in The glass cup. A paddle shaker is also installed and the two feed pipes of the autoburettes with 0.1N and 0.01 NaOH N.

After the 15 minutes have elapsed, they are added by pipette 2 ml of enzyme solution in the solution of pectin, and kept under constant agitation.

Now switch to the autoburette with 0.01 N NaOH, at the same time the stopwatch is started and also keeps the pH value constant at pH = 4.4 by adding, drop by drop of 0.04 N NaOH. After exactly 10 min after from the time taken, the amount consumed is determined by reading 0.01 N NaOH.

The amount of 0.01 N NaOH consumed should be between 1.6 and 4 ml. If this is not the case, you should repeat the measurement with a corresponding amount modified from the amount, or dilution, of the enzyme.

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Evaluation

Activity of the pectin esterase [U / ml] = \ frac {A \ cdot x \ cdot M \ cdot x \ cdot V_ {F}} {V_ {E} \ cdot x \ cdot t}

A = 0.01 N NaOH consumption [ml]

M = molarity of the titration solution [\ mumol / ml]

VF = dilution factor

VE = volume of enzyme solution [ml]

t = reaction duration [min].

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5.- Determination of the activity of the cellulase-C1 with Avicel

(Sugar measurement reducer)

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Trial Principle

The cellulase unfolds the link β-1,4 between glucose units within a starch molecule. The glucose that is released with it is detected photometrically with 3,5-dinitrosalicylic acid (DNSS)

During acid heating 3,5-Dinitrosalicylic (DNSS) with reducing sugar in alkaline medium acid is formed 3-amino-5-nitrosalicylic (Equation 1). In this case a nitro group is reduced, obtaining a amino group, while the aldehyde group of the monosaccharide is oxidized in the form of a carboxyl group (Kakác and Vejdelek, 1974).

2

Different sugars result in various colorations, whose maximum absorption is between 500 and 550 nm.  In this way, by this test, they can also be detected mono-, di-, oligo- and polysaccharides, as well as methylpentoses and O-methylsaccharides.

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Reagents

DNSS-phenol reagent:

- Solution to:

Enter by weighing 38.55 g of tartrate K-Na in a 200 ml glass cup, and dissolve in 125 ml distilled water. Then 2,425 g of NaOH are dissolved (cookie).

- Solution B:

Dissolve 1,325 g of acid 3,5-dinitrosalicylic (C 7 H 4 N 2 O 7; acid 2-hydroxy-3,5-dinitrobenzoic acid) in a screw bottle, brown, in 125 ml of distilled water.

- Solution C:

Dissolve 1.05 phenol in 12.5 ml of water distilled Add 0.25 g of NaOH (biscuit) and 1.05 g consecutively of Na 2 SO 4, and dissolve under stirring.

- Usage Solution:

Solution A and solution C are poured into the solution B, without subsequent rinsing, and homogenize for 10 min. Leave the solution at rest before use for at least one night, and always keep in the dark.

Substrate: 2.0 g of Avicel in 90 ml of tampon

Buffer: Na citrate buffer, 0.1 M, pH 4.5

Standard solution: 2.0 g / l glucose in water distilled

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Realization

Submit to incubation (remove under agitation!) 1.5 ml of substrate solution for 10 min at 50 ° C, then add 0.5 ml of liquid enzyme preparation (for the value per 0.5 M buffer defect). After exactly 20 min of time The reaction is cooled in an ice bath, and centrifuged. for 20 min at 4,200 rpm. 1.0 ml of material is mixed supernatant with 2.0 ml of DNSS-phenol reagent, and boil for 5 min in the boiling water bath. Cool the mix for about 5 min in the ice bath, and measure the extinction at room temperature and under 546 nm.

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Calibration

Instead of enzyme substrate solution, in The test uses 2 ml of standard solution. Dilutions of the Standard solution for drawing calibration lines:

C (glucose) in g / l Standard solution in \ mul distilled water in \ mul 0.1 100 1,900 0.125 120 1,880 0.16 160 1,840 0.22 220 1,780 0.24 240 1,760 0.28 280 1,720 0.3 300 1,700

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Evaluation

Activity of Cellulase-C1 [U / ml] = \ frac {c (Glucose)} {M (Glucose)} \ cdot \ frac {V_ {solution \ reactive}} {V_ {enzyme} \ cdot t_ {reaction}} \ Diluc \ cdot

M (Glucose) = 180 g / mol

T_ {reaction} = 20 min

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6.- Determination of the activity of the laminarinase - (β-1,3-glucanase)

(Sugar measurement reducer)

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Trial Principle

Laminarinase unfolds the bond β-1,3 between glucose units within the laminarin The glucose that is released with it is detected photometrically with 3,5-dinitrosalicylic acid (DNSS)

During acid heating 3,5-Dinitrosalicylic (DNSS) with reducing sugars in alkaline medium, acid is formed 3-amino-5-nitrosalicylic (Equation 1). In this case, a nitro group is reduced, obtaining an amino group, while the aldehyde group of the monosaccharide is oxidized in the form of a carboxyl group (Kakác and Vejdelek, 1974).

3

Different sugars result in different colorations, whose maximum absorption is between 500 and 550 nm. In this way, by this test, they can also be detected mono-, di-, oligo- and polysaccharides, as well as methylpentoses and O-methylsaccharides.

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Reagents

DNSS-phenol reagent:

Solution to:

Enter by weighing 38.55 g of tartrate K-Na in a 200 ml glass cup, and dissolve in 125 ml distilled water. Then 2,425 g of NaOH are dissolved (cookie).

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Solution B:

Dissolve 1,325 g of acid 3,5-dinitrosalicylic (C 7 H 4 N 2 O 7; acid 2-hydroxy-3,5-dinitrobenzoic acid) in a brown bottle of thread, in 125 ml of distilled water.

Solution C:

Dissolve 1.05 phenol in 12.5 ml of water distilled Add 0.25 g of NaOH (biscuit) and 1.05 g consecutively of Na 2 SO 4, and dissolve under stirring.

Usage Solution:

Solution A and solution C are poured into the solution B, without subsequent rinsing, and homogenized for 10 min. Leave the solution at rest before use for at least one night, and always keep in the dark.

Substrate: 0.5% laminarin in citrate buffer Sodium 0.1 M, pH 4.5: heat the buffer to approximately 40 ° C, add laminarin under stirring.

Buffer: Na citrate buffer, 0.1 M, pH 4.5

Standard solution: 2.0 g / l glucose in water distilled

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Realization

Probes:

Mix 0.8 ml of substrate solution with 0.1 ml of enzyme solution; incubate at 40 ° C for 15 min, then immediately place in ice bath. Add to it 2.0 ml of DNSS-phenol reagent and boil for 5 min in the boiling water bath. Cool the mixture for approximately 5 min in an ice bath, and measure the extinction at ambient temperature and low 546 nm.

Default values:

Mix 0.9 ml of standard solution with 0.1 ml of water. To this add 2.0 ml of DNSS reagent - phenol, and boil for 5 min in the boiling water bath. Cool the mixture for about 5 min in the ice bath, and measure the extinction at room temperature and at 546 nm.

Standard Solution:

Mix 1.0 ml of diluted standard solution with 2.0 ml of DNSS reagent - phenol, and boil for 5 min. Cool the mix for about 5 min in the ice bath, and measure the extinction at room temperature and at 546 nm.

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Calibration

Dilutions of the standard solution for the plotting the calibration line:

C (glucose) in g / l Standard solution in \ mul distilled water in \ mul 0.092 fifty 950 0,110 60 940 0.147 80 920 0.221 120 880 0.258 140 860 0.294 160 840

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Evaluation

Activity of laminarinase [U / ml] = \ frac {c (Glucose)} {M (Glucose)} \ cdot \ frac {V_ {solution \ reactive}} {V_ {enzyme} \ cdot t_ {reaction}} \ cdot Diluc \ cdot

M (Glucose) = 180 g / mol

T_ {reaction} = 20 min

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7.- Determination of xylanase activity

(Sugar measurement reducer)

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Trial Principle

Xylanase unfolds the link β-1,4 between the xylose units within a Xylan molecule. The xylose that is released with it is detected photometrically with 3,5-dinitrosalicylic acid (DNSS)

During acid heating 3,5-Dinitrosalicylic (DNSS) with reducing sugar in alkaline medium acid is formed 3-amino-5-nitrosalicylic (Equation 1). In this case a nitro group is reduced, obtaining a amino group, while the aldehyde group of the monosaccharide is oxidized in the form of a carboxyl group (Kakác and Vejdelek, 1974).

4

Different sugars result in various colorations, whose maximum absorption is between 500 and 550 nm.  In this way, by this test, they can also be detected mono-, di-, oligo- and polysaccharides, as well as methylpentoses and O-methylsaccharides.

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Reagents

DNSS-phenol reagent:

Solution to:

Enter by weighing 38.55 g of tartrate K-Na in a 200 ml glass cup, and dissolve in 125 ml distilled water. Then 2,425 g of NaOH are dissolved (cookie) in the solution.

Solution B:

Dissolve 1,325 g of acid 3,5-dinitrosalicylic (C 7 H 4 N 2 O 7; acid 2-hydroxy-3,5-dinitrobenzoic acid) in a brown bottle of thread, in 125 ml of distilled water.

Solution C:

Dissolve 1.05 phenol in 12.5 ml of water distilled Add 0.25 g of NaOH (biscuit) and 1.05 g consecutively of Na 2 SO 4, and dissolve under stirring.

Usage Solution:

Solution A and solution C are poured into the solution B, without subsequent rinsing, and homogenize for 10 min. Leave the solution at rest before use for at least one night, and always keep in the dark.

Substrate: 2.0 g of xylan in 90 ml of tampon

Buffer: Na citrate buffer, 0.1 M, pH 4.5

Standard solution: 2.0 g / l of xylose in water distilled

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Realization

Submit to incubation (remove under agitation!) 1.5 ml of substrate solution for 10 min at 50 ° C, then add 0.5 ml of liquid enzyme preparation (for the value per default buffer 0.5 ml). After exactly 20 min of time reaction the mixture is cooled in the ice bath, and centrifuged for 20 min at 4,200 rpm. 1.0 ml of material is mixed supernatant with 2.0 ml of DNSS-phenol reagent, and boil for 5 min in the boiling water bath. Cool the mix for about 5 min in the ice bath, and measure the extinction at room temperature and under 546 nm.

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Calibration

Instead of substrate and enzyme solution, in The test uses 2 ml of standard solution. Dilutions of the Standard solution for drawing the calibration line:

C (glucose) in g / l Standard solution in \ mul distilled water in \ mul 0.1 100 1,900 0.125 120 1,880 0.16 160 1,840 0.22 220 1,780 0.24 240 1,760 0.28 280 1,720 0.3 300 1,700

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Evaluation

Activity of xylanase [U / ml] = \ frac {c (xylose)} {M (xylose)} \ cdot \ frac {V_ {solution \ reactive}} {V_ {enzyme} \ cdot t_ {reaction}} \ cdot Diluc \ cdot

M (Xylosa) = 150 g / mol

T_ {reaction} = 20 min

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8.- Determination of lipase activity and phospholipase

Lipase activity is determined by the pH-Stat method. As a substrate it is used triolein A consistent substrate solution (30 ml) is prepared in 2% weight / volume of gum arabic, 5% volume / volume of substrate in water, and emulsified with a homogenizer. To this is added 1 ml of enzyme solution. The acid released in the reaction is reticulate with 0.01 M soda bleach, in order to maintain the pH constant. The amount of acid released is calculated from the amount of NaOH, and then enzyme activity. For the determination of autohydrolysis replaces the solution of enzymes with distilled water. The measurements are carried out at 37 ° C and at pH 7.5 in each case, three times. Activity values obtained in U / ml are converted mathematically for the composition total olives or components of olives and mixture of enzymes (that is, the activity in the enzyme mixture already used for oil production).

The activity of phospholipase-C is carried out with p-nitrophenylphosphorylcholine as test substance At 50 µl of a solution of N-nitrophenylphosphorylcholine (100 mM in 50 mM borax-HCl, pH 7.5), 50 µl of enzyme solution The p-nitrophenol formed by the hydrolysis of p-nitrophenylphosphorylcholine, is measured at 410 nm, by photometer. As a negative control, the Enzyme solution with distilled water. For calibration it is apply a fresh solution of p-nitrophenol (100 mM in 50 mM Borax-HCl, pH = 7.5). By means of the preparation of appropriate dilutions and measurement of these solutions at 410 nm in the photometer, it is possible to prepare a curve of calibration. In each case, each of the measurements is taken to out three times. The activity values obtained, expressed in U / ml, are converted by calculation into the equivalent of the mixture of enzymes On the basis that the p-nitrophenyl phosphorocoline does not unfolds by enzyme solutions, there is no activity of phospholipase-C present.

The invention will be explained in greater detail below. detail with the help of the following examples, which are not limiting

Example 1 Laboratory test, for explanation

Approximately 900 g of previously desalted olive mass ( Olea European Hojiblanca cv., Second stage of maturity) were introduced by weighing, mixed with 50 ml of water or aqueous enzyme solution, and incubated for 60 min at 40 ° C in a 2,000 ml glass vessel under stirring with a spiral stirrer (150 rpm). A solid-liquid separation was then carried out by centrifugation in a 500 ml stainless steel cup in a Heräus centrifuge type KS IV (2,000 g, 20 ° C). The liquid / solids separation took place in a separating funnel at room temperature overnight. The aqueous phase was drained, the drained material was dried by rinsing, and the amount of remaining oil was determined gravimetrically. The relative yield in oil by exclusive addition of water without enzyme mixture (comparison sample) was 0.41% (weight / weight), in the case of a 10 µL addition of an enzyme mixture according to the invention (sources and sources of enzymes, as indicated above):

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Exo -PGasa: 900 U / ml Pectin liasa: 61,400 U / ml Endo -PGasa: 5,500 U / ml (viscosity test; substrate: pectin) Endo -PGasa: 2,060 U / ml (viscosity test; substrate: PGA) Pectin esterase: 340 U / ml Cellulase-C1 (Avicelasa): 2.8 U / mL Laminarinase: 9.3 U / ml Xylanase: 1.820 U / ml

the yield was 1.11% (weight / weight), or of 270% of the comparison sample; for an addition of 50 µl of the enzyme mixture according to the invention, the yield was 1.14% (weight / weight), or 278% of the comparison sample; for one adding 100 µl of the enzyme mixture according to the invention, the yield was 1.26% (weight / weight), or 307% of the comparison sample, and for an addition of 200 µl of the enzyme mixture according to the invention, the yield was 1.45% (weight / weight), or 354% of the comparison sample.

Example 2 Laboratory test, for explanation

Approximately 600 g of previously desalted olive mass ( Olea European Hojiblanca cv., Third stage of maturity) were introduced by heavy weighing and were incubated without or with aqueous enzyme solution for 60 min at 35 ° C in a low stainless steel container stirring with a spiral stirrer (150 rpm). The solids-liquid separation took place by centrifugation at 3,500 g for 60 seconds. The oil yield was determined volumetrically in a test tube with a foot after a phase separation of one hour.

The relative yield in oil without adding Enzymes (comparison sample) was 2.5 ± 0.1% (weight / weight); for an addition of 150 µl of the enzyme mixture according to the invention (cf. above), the relative yield was 4.0 ± 0.2% (weight / weight) or 163.6 ± 4.2% of the sample of comparison; for an addition of 250 µl of the enzyme mixture According to the invention, the relative yield was 4.3 ± 0.2% (weight / weight), or 177.3 ± 3.9% (weight / weight) of the sample of comparison; for an addition of 350 µl of the mixture of enzymes according to the invention, the relative yield was 4.8 ± 0.1% (weight / weight), or 195.5 ± 2.0% of the sample of comparison; and for an addition of 450 µl of the mixture of enzymes according to the invention, the relative yield was 4.9 ± 0.1% (weight / weight) or 200.0 ± 2.0% (weight / weight) of the comparison sample.

Example 3 Industrial test, Polytechnic Institute, for explanation

1,500 kg of olives of the species were weighed Olea European Hojiblanca CV, second stage of maturity, is the crushed (hammer mill), and incubated with 1.5% (weight / weight) of talc (22.2 kg) for 68 min at 28-30ºC in a horizontal shaking bath (Pieralsi). The mass flow rate was Q = 1,100 kg / h. The oil by separating solids-liquids in a horizontal two phase decanter (Model M-2 of Pieralsi) and separation of liquids / liquids in a centrifuge (in each case, continuously). The content of residual oil and moisture of the dehydrated olive mass (Trials 2F0498 and 3F0502). Then weighed 1,250 kg of the mass of olives unleashed from the first step of the process. Be subjected the dough to incubation without adding olives (sample of reference) or with an addition of 150 ml of the enzyme mixture according to the invention (cfs with the foregoing) per tonne, but in both cases without further addition of talc, for 50 min at 37-40 ° C (reference sample), or at 39-40 ° C (sample treated with enzymes) in the bath horizontal agitation The oil was isolated by separation of solid-liquids in a horizontal decanter of two phases (Pieralisi M-2 Model) and separation of liquids / liquid in a centrifuge (in each case, so keep going). Again, the mass flow rate was Q = 1,100 kg / h. The residual oil content and moisture in the reference sample (Test 3F0500) and in the treated sample (3F0504). For double phases, see description.

Content determination was undertaken residual in oil by extraction of Soxhlet (Spanish Standard Unified, UNE 55030), and moisture content determination It was carried out by drying at 110 ° C until constant weight.

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Test 3F0498 (olive pulp after the first pressing of the sample of reference)

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MS = dry mass

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Test 3F0500 (olive pulp after the second pressing of the sample of reference)

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Test 3F0502 (olive pulp after the first pressing of the sample treated)

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Test 3F054 (olive pulp after the second pressing of the sample treated)

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The relative oil yield of the second pressing, results from the calculation of the difference between the content residual oil in the gross mass after the first step and the residual equivalent oil content after the second step, divided by the residual oil content of the first step.

For reference sample 3F0500, this means that in relation to sample 3F0498 a relative yield in oil of 1.50 ± 0.40%: 3.62 ± 0.03%, corresponding to 41.44% ± 1.42% based on mass gross Referred to the dry mass, it was a relative yield in oil, 2.73% ± 0.08%; 7.75% ± 0.04%, corresponding to 35.22 ± 1.21%.

For sample 3F0504 treated with the mixture of enzymes according to the invention, this means that in relation to Sample 2F0502 achieved a relative yield in oil of 1.70% ± 0.04%: 3.64% ± 0.04%; corresponding to 46.70% ± 1.21%, based on the gross mass. Referring to the dry mass, a relative oil yield of 3.26% ± 0.09% resulted: 7.88% ± 0.09%, corresponding to 41.37% ± 1.62%.

In this test, for an addition of 150 ml of the mixture of enzymes according to the invention, and per ton of the mass of olives previously unleashed, could achieve a average additional yield of 17.5%, based on dry mass.

Example 4 Industrial test, industrial installation, "stored", for explanation purposes

A total of 31,000 kg of mass of pre-deaceted olives, obtained from the Olea European picual cv species, which had been stored under refrigeration for 24 days under cover of air, was used as the starting material for the test described below. In a semi-parallel preparation, 15,500 kg of this olive mass were first incubated in each case, without or with the addition of the enzyme mixture according to the invention (in total 250 g / t, incorporated in 1.5 l of water) for 60 min. at 33-35ºC in horizontal shaking baths with two planes (Pieralisi). The oil was isolated by separation of solids / liquids in a two-phase horizontal decanter (Model SPI7 of Pieralisi, flow 2,500-2,725 kg / h) and separation of liquids / liquids in a centrifuge (Model P2000 of Pieralisi) under the addition of 300 l / h of water at a temperature of 38 ° C. The isolated oil was then stored, and the yield was determined by reading a level indicator in the storage containers, in which a level difference of 1 cm corresponded to an oil quantity of 95.26 kg.

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specs

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In this test, for an addition of 250 grams of enzyme mixture according to the invention per tonne of pre-oiled olives under the given conditions, could be achieved a yield of 63%, based on the determined dry mass (MS), compared to the untreated reference sample. The content residual, minimum, of oil in the mass of treated olives, makes that further processing in a procedure is superfluous chemical-extractive.

Example 5 Verification of ideal relationships between enzymes (for explanation purposes)

In the investigations the product was involved Olivex® (designated below, as KP1) from Nozymes A / S, DK, obtainable in commerce. For the experiments described to then all enzyme activities were determined according to the procedure indicated in the preceding chapter referred to the methods. In this case, the individual activities indicated in the following table:

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(*) Determined according to the chapter of methods, with PGA (pectinic acid) as a substrate

For comparative measurements they were weighed in each case 600 g of dehydrated olive mass (European Olea hojiblanca, cv., third stage of maturity) and submitted it to incubation without or with aqueous enzyme solution (150 µl of cooperative product, eventually more pectin addition esterase) for 60 min. at 35 ° C in a steel container stainless under stirring with a spiral stirrer 150 rpm). The separation of solids and liquids took place by centrifugation at 3,500 g for 60 seconds. The oil yield is determined volumetrically after phase separation for an hour in a test tube with foot.

Various relationships between pectin were adjusted esterase and the other enzymes in the assays by adding defined amounts of an orange peel esterase pectin (Sigma / Aldrich, Catalog No. P5400). For this the enzyme was collected lyophilized in a buffer ((NH4) 2 SO4 3.2M and 0.1 M NaCl, pH = 7.0), and adjusted to an activity of 75 U / ml. The addition was carried out in increments of in each case 7.5 U; be made a double determination. The results have been summarized experimental in the following table. The oil yield without No type of enzyme addition, was 14.7% ± 0.6 ml, the yield in oil under the addition of 150 µl of the mixture of Enzymes according to the invention, was 24.5 ± 0.4 ml.

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An evaluation can be found in Figure 1 graph of both analyzes. It is evident that the modification of relationships between enzyme activities by adding from pectin esterase to product KP1 leads to a manifest improvement of oil yield.

Modifications between the relationships of activities, resulting from the addition of pectin esterase, have been represented in the following tables. In this case I don't know took volume effects into account. From the tables it follows that adjusting the relationships between enzyme activities in the intervals according to the intervention is correlated with a strong performance rise in oil in case of KP1.

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Adjustment of the activities of enzyme KP1

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The following Examples 6 and 7 were carried out for the verification of the biotechnological optimization of the process in the production of oil under use of the enzyme mixture according to the invention.

Example 6 Reduction of the oil content in the aqueous discharge of the decanter depending on the process temperature; increase of oil yield

From a reserve pool, the pulp of olive, pre-oiled, was continuously supplied to a mixing vessel with vertical stirring rods. The yield of this mixing vessel was 25 tm / h, with an average residence time of 60 min. In this case, the pulp was heated to a temperature of 45 ° C. From this mixing vessel, the olive pulp was transported to a mixing vessel with horizontal stirring rods (Pieralisi Kneading 900 4 piles). With an average residence time of 40 min, the performance of this processing unit was 11 t / h. The olive pulp was transported from this horizontal mixing bath to two three-phase decanters of Type Pieralisi Giant 2. These decanters had a nominal yield of approximately 7 tm of oil pulp per hour, but had been regulated to the pre-established mass flow by the process The results described below come from the analysis of samples that were extracted from the aqueous drain ( porridge ) of both decanters.

For investigation of the effect of the enzyme mixture according to the invention according to Example 1, they were added to the olive pulp led to the horizontal mixing vessel, directly in the inlet opening and over a time interval of 12 hours, 80, 160 or 240 ml / tm of the enzyme mixture according to the invention according to Example 1, by means of a pump. The bath temperature was 45 ° C, 37 ° C and 41 ° C, respectively. The corresponding samples for the analysis of the functioning of the enzyme composition according to the invention were extracted at 6 h after the start of the addition of enzymes, from the porridge in the decanters. At 6 hours after the termination of the enzyme addition, comparison samples were taken at the same extraction sites. In this way, a direct comparison between the results of the process was possible, with the addition of the enzyme mixture according to the invention, and without such addition.

During the analysis of the samples They determined the results for the following parameters: water content in the porridge, and oil content in the pap. The results for the addition of 80, 160 and 160 ml / tm, of the enzyme mixture according to the invention have been summarized in The following Tables 1, 2 and 3.

TABLE 1 Experimental results in case of adding 80 ml / tm of the enzyme mixture according to the invention (T = 45 ° C)

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TABLE 2 Experimental results in case of adding 160 ml / tm of the enzyme mixture according to the invention (T = 37 ° C)

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The additional evaluation of this data from analysis yielded the following results:

1.- The addition of 80 ml of the enzyme mixture according to the invention per tonne of olive pulp under the given process conditions (T = 45 ° C) leads, compared to the pulp of untreated olives, to an elevation of the content of water in the porridge, up to 2.61%, and a reduction in content of oil in the porridge, up to 28.44%. In this step of the process, The oil yield rose to 38.26%.

2.- The addition of 160 ml of the enzyme mixture according to the invention per tonne of olive pulp under the given process conditions (T = 37 ° C) leads, compared to the pulp of untreated olives, to an elevation of the content of water in the porridge, up to 2.78%, and a reduction in content of oil in the porridge, up to 38.30%. In this step of the process, the oil yield rose to 29.03%.

Under the given conditions, the addition of 160 ml of the enzyme mixture according to the invention per tonne of olives, showed an especially advantageous effect in terms of water and oil content in the porridge, as well as in the oil yield A positive effect was also achieved, referred to the mentioned parameters, by adding 80 ml of the enzyme mixture according to the invention, although in this case it is carried out a temperature rise of 8 ° C, in comparison with the test with 160 ml / t.

It can also be verified that the addition of 160 ml of the enzyme mixture according to the invention per tonne of olive pulp at a temperature of approximately 37 ° C has an especially advantageous effect on the separation capacity, as well as the oil yield in the process considered, but from the analysis of the other results it is also concluded that it is possible to observe other advantageous effects with other temperatures and with other enzyme concentrations.

Example 7 Economy of the Global Process

From a reserve pool it was contributed in a manner continuous pulp of olives, unleashed beforehand, to a mixing vessel provided with vertical stir bars. The yield of this mixing vessel was 25 tm / h with a average residence time of 60 min. From this vessel of mixed the oil pulp was transported directly, it is say without interposition of a mixing container with bar horizontal stirring, towards three phase decanters (Pieralisi, Type Giant 2). These decanters had a capacity nominal of approximately 7 tm of olive pulp per hour, but they had been regulated to the mass flow preset by the process. The results described below come from the analysis of samples that were extracted directly from a Decanter chosen arbitrarily. The pulp temperature of Olive in the process was 32 ° C.

For investigation of the action of the enzyme mixture according to the invention according to Example 1, it was added to the olive pulp introduced into the vertical mixing vessel, directly into the inlet opening over a period of time 12 hours, 160 or 240 ml / tm, respectively, of the enzyme mixture according to the invention, by means of a pump. The corresponding samples for the analysis of the functioning of the enzyme composition according to the invention from the process streams of the decanters (olive pulp, processed ( Alperujo ), aqueous phase ( porridge )) were extracted 6 hours after the start of the addition of enzymes. The amount of oil produced was determined continuously by the measurement technique available on the side of the installation. After 12 h the addition of enzymes was stopped. At 6 hours after the addition of enzymes, comparison samples were extracted from the same extraction sites. In this way, a direct comparison between the results of the process, with or without addition, of the enzyme mixture according to the invention was possible.

During the analysis the results for the following parameters: water content in the alperujo, oil content in the alperujo, water content in the porridge, and oil content in the porridge. Also I know determined the efficiency of the process, referring to the performance in oil, at the time of the corresponding sample extraction. The following Tables 3 and 4 have summarized the results for the addition of 160 or 240 ml / tm of the enzyme mixture according to the invention.

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TABLE 3 Experimental results in case of adding 160 ml / tm of the enzyme mixture according to the invention

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TABLE 4 Experimental results in case of adding 240 ml / tm of the enzyme mixture according to the invention

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The further evaluation of these data from analysis provided the following results:

1.- The addition of 240 ml of the enzyme mixture according to the invention per tonne of olive pulp under the given process conditions, drive, compared to the pulp of untreated olives, to an elevation of the water content in the alperujo, of 4.24%, to a reduction of the oil content in the alperujo of 12.42%, at an elevation of the water content in the porridge, 1.07%, and a reduction in oil content in the porridge, 8.73%. In this step of the process, the performance in Oil fell by 2.22%.

2.- The addition of 160 ml of the enzyme mixture according to the invention per tonne of olive pulp under the given process conditions, drive, compared to the pulp of untreated olives, to a reduction of the water content in the alperujo of 13.85%, to a reduction of the oil content in the alperujo of 13.25%, at an elevation of the water content in the 0.13% porridge, and a reduction in oil content in the 3.94% porridge. In this process step, the oil yield increased fell by 4.94%.

Under the given conditions, the addition of 160 ml of the enzyme mixture according to the invention per tonne of olives, shows an especially advantageous action with respect to The economy of the whole process. A positive action, referred to the global economy of the process, it is also achieved through the addition of 240 ml of the enzyme mixture according to the invention, although not as pronounced, as in the case of the addition of 160 ml.

It is especially important that these experiments were carried out at a process temperature of 32 ° C. Also in the case of this comparatively low temperature, the use of the enzyme mixture according to the invention had a strong positive effect on the economy of the process.

Claims (20)

1. Procedure for the production of oil a from masses of olives and / or components of olives, which covers the following steps: a.- making available a mass of olives and / or components of olives, without the separation of bones of olives; b.- adding an enzyme or a mixture of Enzymes containing at least one pectin esterase, in form liquid or solid; c.- mixed mixture of dough and mixture of enzymes; d.- separation of the oil phase from the dough, with the help of a three phase decanter. 2. Method according to claim 1, which also covers the following step: incubation of the mixture of the claim 1c, especially at a temperature between 20 and 60 ° C, and especially for 5 to 120 min. 3. Procedure according to one of the preceding claims, wherein as a mass of olives and / or of components of olives a mass is used previously unwanted 4. Procedure according to one of the preceding claims, in which at least one step is contributes in a decanter of three phases, pulp of olives unwanted in advance without interposition of a container of mixed with horizontal stirring bars (kneader), nor of a mixing bowl with vertical stir bars. 5. Method according to one of the preceding claims, wherein the enzyme mixture contains at least one pectin esterase, at least one endo- polygalacturonase and at least one exo- polygalacturonase, and in which the ratio between pectin esterase activity and endo- polygalacturonase activity is preferably at least 0.13, and in which the ratio between pectin esterase activity and exo- polygalacturonase activity is preferably 0.3 . Method according to one of the preceding claims, characterized in that the relationship between the activity of pectin esterase and the activity of endo- polygalacturonase is at least 0.15, preferably at least 0.16. Method according to one of the preceding claims, characterized in that the relationship between the activity of pectin esterase and the activity of exo- polygalacturonase is at least 0.33. Method according to one of the preceding claims, characterized in that at least one laminarinase is contained in the enzyme mixture according to the invention, and because the relationship between the activity of pectin esterase and the activity of laminarinase is of at least 20, preferably at least 30. Method according to one of the preceding claims, characterized in that at least one C1 cellulase is contained in the enzyme mixture according to the invention and because the relationship between pectin esterase activity and cellulase activity. C1 is at least 75, preferably at least 100. Method according to one of the preceding claims, characterized in that the following enzyme activities are contained in the enzyme mixture according to the invention:

to.-

Pectin esterase: more than 300 U / ml, in special, more than 320 U / ml;

b.-

exo- polygalacturonase: less than 1,000 U / ml:

C.-

endo- polygalacturonase: between 1,500 and 2,500 U / ml,

d.-

laminarinase: less than 15 U / ml, in special less than 12 U / ml;

and.-

cellulase-C1: less than 3.3 U / ml;

F.-

Pectin lyase: between approximately 25,000 and 150,000 U / ml.

Method according to one of the preceding claims, characterized in that therein are contained per tonne of dough, especially between 50 and 400 ml, preferably between 100 and 300 ml, especially preferably between 125 and 250 ml of The enzyme mixture.

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12. Method according to one of the preceding claims, characterized in that in the enzyme mixture the protein concentration is between 2 and 20 mg / ml, preferably between 2 and 12 mg / ml, especially between 3 and 10 mg / ml. . 13. Method according to one of the preceding claims, characterized in that it is a process for the production of oil from previously unaltered masses of olives and / or components of olives. 14. Method according to one of the preceding claims, characterized in that it is a physical or mechanical-physical process for the production of oil. 15. Use of a mixture of enzymes according to a of the preceding claims, defined in a process according to one of the preceding claims for the improvement of the separation between solids and liquids for oil production, especially in the de-oiling of a previously de-oiled mass of olives or olives components. 16. Use according to claim 15, characterized in that the separation of solids / liquids takes place exclusively with the aid of at least one three phase decanter. 17. Use according to one of claims 15 or 16, characterized in that the oil yield in the solids / liquids separation is increased. 18. Use according to one of claims 15 to 17, characterized in that the oil content in the aqueous discharge of the three phase decanter is reduced and / or because the amount of water separated in the aqueous discharge is raised. 19. Use according to one of claims 15 to 18, characterized in that the oil and / or water content of the solid phase obtained with the aid of the three phase decanter is reduced. 20. Use according to one of the claims 15 to 19, to be able to reduce the process temperature while maintaining at least constant oil yield.