RU2441024C1 - Method of producing low-molecular pectin - Google Patents

Abstract

FIELD: chemistry. ^ SUBSTANCE: invention relates to methods of producing low-molecular pectin. The method involves hydrolysis of pectin in aqueous solution of mineral acid while heating. Further, the liquid phase is separated from the insoluble pectin residue and neutralised to pH lower than 4.0. Low-molecular pectin hydrolysis products are extracted through precipitation thereof using a water-miscible organic solvent. After precipitation, the obtained low-molecular pectin hydrolysis products are dried. The starting material used for hydrolysis is low-etherified pectin with etherification degree of not more than 30%. Hydrolysis is carried out in cycles. The duration of one cycle is calculated using a given formula. After separation from the pectin residue, the liquid phase undergoes further hydrolysis. The duration of one cycle of additional hydrolysis is calculated using a formula. ^ EFFECT: invention enables to obtain low-molecular pectin without process loss of material and also significantly ensures destruction of oligogalacturonides, thus increasing ultimate output of the product. ^ 2 cl, 6 ex

Description

The invention relates to methods for producing low molecular weight pectin and can be used in various industries and medicine, in particular in the pharmaceutical industry to create new therapeutic and prophylactic agents and low molecular weight sorbents.

Pectins are high molecular weight natural polymers. They are present in all higher flowering plants, are part of the primary cell walls and median plates. The basis of pectin molecules is poly-D-galacturonic acid. Part of the residues of D-galacturonic acid in pectin is in the form of methyl esters. If the proportion of such residues is less than 50%, then such pectin is considered to be low esterified. Fully deesterified, that is, containing no methyl esters or containing a small amount of them, pectin is also called pectic acid.

In the course of studies of the biological activity of pectins, a number of pharmacological effects were found in low molecular weight derivatives of pectins - oligogalacturonides. In this regard, oligogalacturonides with a degree of polymerization from 6 to 25-30 monosaccharide residues are of particular interest, which corresponds to their molecular weight from 1 to 4.5-5 kDa. In this group of oligogalacturonides, immunomodulating, interferon-inducing, macrophage-activating and antitumor activity was found (Kumazawa Y., Mizunoe K., Otsuka Y. Immunostimulating polysaccharide separated fromhot water extract of Angelic acutiloba Kitagawa (Yamatohoki). .47. P.75-83). In addition, these oligosaccharides stimulated apoptosis of intestinal adenocarcinoma cells in humans (Olano-Martin E., Rimbah GH, Gibson GR, Rastall RA Pectin and pectic-oligosaccharides induce apoptosis in in vitro human colonicadenocarcinoma cell // Anticancer Res. 2003. Vol.23 P.341-346).

It was found that oligogalacturonides with a degree of polymerization of at least 6–9 are capable of forming strong complexes with ions of many heavy metals, such as lead, mercury, cadmium, and some others (Kohn R. Binding of divalent cations to oligomeric fragments of pectin // Carbohydr. Res . 1987. Vol. 160. P.343-353). This opens up the prospect of creating, on the basis of oligogalacturonides, effective means for removing the deposited forms of these toxic elements from the human and animal body, because due to its small molecular weight, oligogalacturonides can be easily absorbed in the gastrointestinal tract, and then excreted through the kidneys.

Obtaining oligogalacturonides is usually carried out by depolymerization of natural high molecular weight pectins. A number of methods can be used for this. The most famous is the method of enzymatic hydrolysis of pectins using pectinases. So, back in 1944, a method was proposed for the production of D-galacturonic acid by enzymatic hydrolysis of pectin (US Pat. USA No. 2,338,534, publ. 04.01.1944).

A known method of obtaining a composition of oligogalacturonides with antibacterial effect, also by enzymatic hydrolysis of pectin (JP 10226701 A, C08B 37/00, publ. 08/25/2010). According to this method, pectin is first subjected to preliminary hydrolysis in an aqueous solution at 80-100 ° C, and then the final hydrolysis of the mixture is carried out using pectinase. The resulting product contains a mixture of saturated and unsaturated (having double carbon-carbon bonds in the molecules as a result of oxidative degradation) oligogalacturonides with a degree of polymerization from 2 to 15 (0.4-2.7 kDa).

The disadvantage of this method is that for the successful implementation of enzymatic hydrolysis requires, as a rule, a certain preliminary processing of pectin, for example, as in this case, partial depolymerization and deesterification during preliminary hydrolysis. Another significant drawback of enzymatic hydrolysis is that, due to the specificity of the action of pectinolytic enzymes, mono-, di- and trisaccharides always prevail in the obtained hydrolysates, and the fraction of molecules with a mass of 1 kDa (which corresponds to polymerization degree 6) is even more insignificant.

Another known method is the method of alkaline depolymerization of pectin (EP 0487340 A1, A23P 1/08, publ. 05.27.92). According to this method, a suspension of pectin in 60% vol. I treat isopropanol with three times (according to the chemical equivalent of galacturonic acid) amount of sodium hydroxide for 5 days at 25 ° C or 2 days at 40 ° C, followed by separation of the product on the filter, neutralization and drying. The resulting product contains pectins with a molecular weight of 2 to 20 kDa and is intended as an auxiliary substance in cooking.

The disadvantage of this method is the long duration of the process and the large dispersion of the molecular masses of pectins in the product. And the fact that galacturonic acid is unstable in an alkaline environment and easily decomposes with the formation of unsaturated, highly colored and strongly odorous compounds (Kertesz Z. I. The pectic substances. Interscience Publishers, Inc., N.Y., L., 1951, 576 p.). There is also known a method of hydrolysis of pectin at high temperature (WO 2009/004153, C08B 37/06, publ. 08.01.2009). According to this method, a 1% pectin solution with a pH of 4-5 is heated under pressure at 110 ° C for 30-60 minutes, then the solution is cooled, oligogalacturonides are precipitated with isopropanol and dried. The resulting product consists of 95% oligogalacturonides with a degree of polymerization of 4-11 (0.7-2 kDa). The disadvantage of this method is that at temperatures above 100 ° C, the processes of thermal decomposition of oligogalacturonides — breaks in the pyranose cycle, oxidation, decarboxylation, formation of furfural and its derivatives are sharply accelerated. This is accompanied by a decrease in the yield of oligogalacturonides and contamination of the hydrolyzate with products of their decomposition (Kertesz Z. I. The pectic substances. Interscience Publishers, Inc., N.Y., L., 1951, 576 p.). From a chemical point of view, oligogalacturonides containing the products of thermal degradation of galacturonic acid in the molecules cannot be fully considered oligogalacturonides. A known method of depolymerization of polyuronides - alginates and pectins using an oxidizing agent - hydrogen peroxide (US Pat. US US 2002/0016453 A1, C08B 37/06, publ. 07.02.2002). According to this method, pectic acid in the form of lithium pectate is oxidized with hydrogen peroxide at a concentration of about 2% in the presence of ferrous sulfate for 4 hours. Then oligogalacturonides precipitated with acetic acid, washed with propanol and dried. The average degree of polymerization of oligogalacturonides is 16 (2.8 kDa). The product yield is 38%. The disadvantage of this method is, firstly, the low yield of the product. Secondly, hydrogen peroxide simultaneously with the depolymerization of pectin causes a significant destruction of galacturonic acid residues - decarboxylation, breaks of the pyranose cycle, oxidation of alcohol groups and, as noted in the previous example, the products obtained cannot be fully considered oligogalacturonides (Kertesz ZI The pectic substances. Interscience Publishers, Inc., NY, L., 1951, 576 p.).

The closest in technical essence to the claimed method is a method for producing oligogalacturonides from flax pectins by acid hydrolysis (Bedouet L., Courtois B., Courtois J. Methods for obtaining neutral and acid oligosaccharides from flax pectins // Biotechnol. Lett. 2005. Vol. 27. P.33-40). The method is as follows. An aqueous pectin concentration of 5 mg / ml (0.5%) is prepared from a 50 mg pectin sample containing 22.9 mg (45.8%) of uronic (galacturonic) acid. Then add a 12 M solution of hydrochloric acid to a concentration of 100 mm (0.1 g-equiv / l) and carry out hydrolysis at 80 ° C for 24 hours. The resulting hydrolyzate is cooled to room temperature. The liquid phase containing low molecular weight hydrolysis products is separated from high molecular weight polygalacturonic acid by centrifugation at 4 ° C and a relative centrifugal acceleration of 20,000 g for 30 minutes. Then low molecular weight hydrolysis products - oligogalacturonides are precipitated from the liquid phase with isopropanol and separated by centrifugation. The yield of oligogalacturonides is 33.9% of the initial content of galacturonic acid.

A significant disadvantage of this method is the low initial concentration of pectin in the solution. The result of this is, firstly, a low rate of hydrolysis, since at a constant hydrolysis constant it is proportional to the concentration of pectin in the solution. Secondly, a low initial concentration of pectin leads to a corresponding low concentration of the final product in the finished hydrolyzate - the content of oligogalacturonides in the finished hydrolyzate obtained by the above method is only 0.78 mg / ml for 24 hours of hydrolysis (product yield is calculated by multiplying the initial concentration of pectin in the solution on the relative content of galacturonic acid in pectin and on the relative yield of galacturonides is 5 mg / ml × 0.458 × 0.339 = 0.78 mg / ml, respectively). And this, in turn, leads to higher unit costs for the production of a unit mass of the product. In this case, this is, for example, the costs associated with thermostating the entire volume of the hydrolyzate at 80 ° C and the acid consumption for hydrolysis.

An obstacle to a significant increase in the concentration of pectin in the solution is the fact that high molecular weight pectins even at a concentration of about 1% form fairly viscous solutions and the use of more concentrated pectin solutions can create certain technological difficulties. Highly viscous solutions at low concentrations form almost all commercial pectins, since this ability is associated with one of the main directions of their use - as thickeners and structuring agents in the food industry. For example, the viscosity of 1.5% solutions of the most common commercial pectins - apple and citrus (manufacturer - Herbstreith & Fox KG, Germany) is 25-30 times higher than the viscosity of water.

Another disadvantage is the low technological efficiency of the method, which leads to a low yield of the product. The yield of the product - oligogalacturonides is about 34%, 33% is the remainder of high molecular weight polygalacturonic acid and 33% is the loss of oligogalacturonides due to their structural degradation. Thus, the loss of the product is almost the same as its yield. The technological drawback of the method is that the separation of the liquid phase containing the finished product requires too harsh conditions: the relative centrifugal acceleration of 20,000 g for most industrial centrifuges and separators is either unattainable or close to their maximum operating mode, the need to cool the system to 4 ° C also creates additional technical problems and leads to an increase in the cost of the process.

The task of the invention is to obtain low molecular weight pectin enriched in a fraction of oligogalacturonides with a molecular weight of 1-5 kDa, increasing the yield of the target product, simplifying and reducing the cost of the method.

This goal is achieved by the fact that in the known method for producing low molecular weight pectin (oligogalacturonides), which includes hydrolysis of pectin in an aqueous solution of a mineral acid by heating, followed by separation of the liquid phase from the insoluble pectin residue, isolation of low molecular weight products of pectin hydrolysis by precipitation with an organic solvent with water, according to the invention, low esterified pectin with a degree of esterification is used as a feedstock for hydrolysis not more than 30%, the hydrolysis is carried out cyclically, with the time of one cycle is calculated by the formula:

where T is the time of one cycle, h;

C - acid concentration: 0.1-2.0 g-mol / l;

t - process temperature: 70-100 ° C;

K is the coefficient of proportionality, the values of which are in the range of 10-15,

the liquid phase after separation from the pectin residue is subjected to additional hydrolysis, the time of which is calculated by the formula:

where T ₁ is the time of additional hydrolysis of the liquid phase, h;

C - acid concentration: 0.1-2.0 g-mol / l;

t - process temperature: 70-100 ° C;

To ₁ - the coefficient of proportionality for additional hydrolysis of the liquid phase, the values of which are in the range of 1.7-2.5;

in this case, before the precipitation of low molecular weight products of pectin hydrolysis with an organic solvent, the liquid phase is neutralized to a pH of at least 4.0. After precipitation, the resulting low molecular weight pectin hydrolysis products are dried.

The use of low-esterified pectin with a degree of esterification of not more than 30% as a raw material for hydrolysis, with limited swelling in an acidic medium, allows acid hydrolysis in a heterophasic system — a suspension: a low-esterified pectin gel (solid phase) —a mineral acid solution (liquid phase) , which, firstly, allows in practice to increase the concentration of pectin in suspension to 5-15% (in terms of the dry matter of pectin). The use of such high, in comparison with the prototype, concentrations of the feedstock, ceteris paribus (with the same degree of pectin hydrolysis) allows to obtain hydrolysates with a correspondingly high percentage of the final product - oligogalacturonides, which significantly reduces the unit cost of producing a unit mass of the product and, as a result leads to the simplification and cost reduction of the process of obtaining the target product.

Secondly, the hydrolysis of pectin in a heterophase system allows the process to be carried out in cycles, due to which it is possible to fully use the feedstock and prevent the degradation of oligogalacturonides in the liquid phase, thereby increasing the yield of the target product and lowering the specific costs for its production.

With an increase in the degree of esterification of more than 30%, there is a sharp increase in the volume of the swollen pectin gel and a significant decrease in its mechanical strength, which greatly complicates the subsequent separation of pectin gel from the liquid phase, and partial transition of high molecular weight pectin to the liquid phase is also possible.

Almost any commercial pectin can be used to carry out the claimed method. For this, a reduction in its degree of esterification to the required value in the range of 0-30% is preliminarily carried out using any of the known methods of deesterification: alkaline, acidic or enzymatic. The most convenient and economical, according to the authors, can be considered the method of alkaline deesterification of pectin in an organic solvent, for example ethanol or isopropanol. This method is given below in the description of the implementation of the proposed method.

The use of formulas (I) and (II) developed by the authors allows in the indicated temperature range (70-100 ° С) and acid concentration (0.1-2.0 g-mol / l) using the proportionality coefficient K = 10- 15, with satisfactory accuracy, calculate the time of one cycle of pectin hydrolysis, and using the proportionality coefficient K ₁ = 1.7-2.5, with satisfactory accuracy, calculate the time of additional hydrolysis of the liquid phase necessary to obtain a product with a maximum relative content of oligogalacturonides fraction of 1-5 kDa .

Carrying out additional hydrolysis of the liquid phase allows to increase the content of the target fraction of oligogalacturonides 1-5 kDa in the final product due to the hydrolysis of the high molecular weight fraction of oligogalacturonides (more than 5 kDa). Moreover, the hydrolysis time calculated by formula (II) with a proportionality coefficient of K ₁ = 1.7-2.5 allows for additional hydrolysis of the liquid phase in such a way that the increase in the target fraction of oligogalacturonides due to the hydrolysis of the high molecular weight fraction of oligogalacturonides significantly exceeds the decrease in this target fraction resulting from its partial hydrolysis and transition to a low molecular weight fraction of oligogalacturonides (less than 1 kDa).

In the course of a study by the authors of heterophasic acid hydrolysis of low esterified pectin, it was found that oligogalacturonides with a molecular weight of 1-5 kDa formed during the hydrolysis of pectin almost completely pass into solution - the liquid phase, while the fraction of such molecules remaining in the pectin gel is solid phase does not exceed 1-2% of their total amount. Thus, an important advantage of the proposed method of heterophasic acid hydrolysis of low esterified pectin is the presence of effective and constant molecular separation that occurs naturally in the process of hydrolysis itself.

The combination in this method of the effect of interfacial molecular separation and the technological simplicity of phase separation allows you to stop the hydrolysis process at the right time, quickly separate the liquid phase containing the finished product, and then carry out the next hydrolysis cycle of the remaining high molecular weight pectin.

The indicated limitations of temperature and acid concentration are associated primarily with economic and technological reasons. So, at a medium temperature below 70 ° C and an acid concentration below 0.1 g-mol / L, a significant decrease in the rate of pectin hydrolysis and a significant increase in the process time, this leads to an increase in the cost of production of the target product.

At temperatures above 100 ° C, the processes of thermal decomposition of oligogalacturonides are sharply accelerated - breaks in the pyranose cycle, oxidation, decarboxylation, formation of furfural and its derivatives. This is accompanied by a decrease in the yield of oligogalacturonides and contamination of the hydrolyzate with their decomposition products. An increase in the acid concentration of more than 2.0 g mol / L does not give a significant increase in the rate of hydrolysis, but leads to a significant cost overrun of the acid and additional costs for its subsequent neutralization and release of the product from the formed salts, which, in the final result, complicates the process of obtaining target product.

The neutralization of the liquid medium to a pH of not less than 4.0 before the precipitation of low molecular weight products of pectin hydrolysis with an organic solvent provides a precipitate of oligogalacturonide pectin with an optimal consistency - dense, well separated from the liquid phase. At pH below 4.0, the specific volume of the precipitate increases and its structure weakens, and, as a result, separation of the precipitate from the liquid phase becomes difficult.

As a mineral acid, in particular, hydrochloric, sulfuric, and nitric acids can be used.

As the organic solvent, known organic solvents miscible with water, in particular acetone, propanol, isopropanol, ethanol, methanol, can be used.

The separation of the liquid phase from the insoluble precipitate can be carried out either by filtration or by centrifugation at relatively small values of centrifugal acceleration of 500-1000 g. This significantly reduces costs and simplifies the process of obtaining the target product in comparison with the prototype, since in the prototype centrifugation is carried out at an acceleration of 20,000 g and a temperature of 4 ° C.

Another advantage of the proposed method is the ability to maintain a constant amount of low esterified pectin in the reaction medium by adding new portions of the feed to the pectin residue after separation of the liquid phase to compensate for its consumption during hydrolysis. This allows you to consecutively carry out any number of the same type of hydrolysis cycles and get any desired amount of the target product with the desired molecular weight, 1-5 kDa.

Raw materials can be added to the pectin residue after each or after several hydrolysis cycles, and its amount can be calculated either by changing the volume of the pectin gel or by the amount of pectin remaining after separation of the liquid phase.

In the first case, to determine the amount of raw material added by changing the volume of the pectin gel before hydrolysis, the suspension of pectin in mineral acid is centrifuged at a relative centrifugal acceleration of 500-1000 g for 5-15 minutes and the initial volume of the gel is pectin, V ₁ , ml. Before subsequent cycles of hydrolysis, the pectin residue after hydrolysis is centrifuged under the same conditions - at the same relative centrifugal acceleration and time, fixing V ₂ is the volume of the pectin residue after hydrolysis, ml.

To determine the amount of added raw materials by volume of the pectin residue after centrifugation, it is calculated by the formula:

where m is the mass of added raw materials, g;

m ₁ - mass of feedstock, g;

V ₁ - the initial volume of the swollen raw material is a gel of low esterified pectin, ml;

V ₂ - the volume of the pectin residue after hydrolysis, ml

Using for calculation of formula (III), proposed by the authors, it is possible to determine with sufficient accuracy the mass of added raw materials, ensuring the restoration of the pectin content to the initial concentration. In the final result, this ensures the most complete use of the pectin residue, increasing the yield of the target product and the possibility of obtaining it in the required amount.

In the second case, to determine the amount of added raw materials by the amount of pectin remaining after separation of the liquid phase, before starting the hydrolysis process, the mass of the feedstock, m ₁ , is fixed, the content of galacturonic acid, C ₁ , is also determined in it. At the end of the hydrolysis time calculated by the formula (I), the pectin residue is separated from the liquid phase, its mass m _{2 is} fixed and its content of galacturonic acid C _{2 is} determined.

The amount of added raw materials is calculated by the formula:

where m is the mass of added raw materials, g;

m ₁ - mass of feedstock, g;

m ₂ - mass of pectin residue after separation of the liquid phase, g;

With ₁ - the content of galacturonic acid in the feedstock,%;

C ₂ - the content of galacturonic acid in the pectin residue,%.

Formula (IV), proposed by the authors, as well as formula (III), allows to determine the amount of added raw materials with the necessary accuracy.

To determine the content of galacturonic acid in the feedstock and the pectin residue, any of the known methods can be used, for example, titrimetric (Afanasyev S.P., Chirva V.Yu., Katseva G.N. et al. Modification of titrimetric methods for the analysis of pectin substances // Chemistry of Natural Compounds. 1984. No. 4. P. 428-431) or photometric (Blumenkrantz N., Asboe-Hansen G. New method for quantitative determination of uronic acids // Anal. Biochem. 1973. Vol.54. P. 484-489). Considering that approximately the same amount of raw materials is consumed during each hydrolysis cycle, there is no need to calculate the amount of added raw materials after each hydrolysis cycle - it is enough to carry them out every 5-10 cycles.

The method is as follows.

Example 1

Take 30.0 g of pectic acid (Serva, No. 31680) with a galacturonic acid content of 82.2% and an esterification degree of less than 1% and mixed with 200 ml of a hydrochloric acid concentration of 0.1 g mol / L (C). The resulting 15% suspension is heated to 100 ° C (t) in a boiling water bath and kept at this temperature with constant stirring for a time (T) calculated by formula (I) with a proportionality coefficient of K = 15:

Then the suspension is cooled and filtered; a PL-30 polypropylene filter-cloth (LOSMA, Italy) is used to filter the suspension. The remaining pectin on the filter is washed with 200 ml of hydrochloric acid at a concentration of 0.1 g mol / L (C) to more fully separate oligogalacturonides. A solution of hydrochloric acid with a concentration of 0.1 g mol / L is added to the pectin residue until the initial volume of the suspension is restored. The pectin residue is resuspended in the liquid phase and the next hydrolysis cycle is carried out under the previous conditions — T = 3.4 hours at t = 100 ° C. A total of 5 cycles of hydrolysis are carried out, each time separating and washing the remainder of the pectin, as described above. The total volume of the obtained filtrate is 1440 ml, the content of oligogalacturonides is 7.02 mg / ml.

The resulting liquid phase is subjected to additional hydrolysis at 100 ° C (t) for a time (T ₁ ) calculated by the formula (II) with a proportionality coefficient K ₁ = 2.5:

Then the liquid phase is cooled to room temperature, neutralized to pH 4.0 with a 5.0 M sodium hydroxide solution, and oligogalacturonides are precipitated by the addition of 4.3 l of ethanol. The mixture is stirred vigorously for one minute, then after 12 hours the precipitated precipitate of oligogalacturonides is separated by centrifugation at a BCC of 1000-1500 g for 15 minutes and dried at 80 ° C. The yield of the product is a dry powder of sodium oligogalacturonate - 11.41 g (the content of pure galacturonic acid in sodium oligogalacturonate is 88.5%). To determine the relative (by raw) yield of the product, in terms of galacturonic acid, the consumption of raw materials is determined by the difference between the total amount of pectic acid used for hydrolysis (30.0 g) and the amount of pectin remaining after hydrolysis. For this, the amount of pectic acid is converted to the content of galacturonic acid in it, and the total content of galacturonic acid in the pectin residue is determined by the photometric method. The established consumption of raw materials for galacturonic acid is 11.28 g. The relative yield of product for galacturonic acid is 89.5%, respectively. According to size exclusion chromatography of the obtained product in 0.05 M acetate buffer with a pH of 3.0 on a Sephadex G-50 superfine column, calibrated using a standard set of pullulans, the relative content of the 1-5 kDa oligogalacturonide fraction in the resulting low molecular weight pectin is 72.5 %

Example 2

Take 10.0 g of pectic acid (Serva, No. 31680) (m ₁ ) with a galacturonic acid content of 82.2% and an degree of esterification of less than 1% and mixed with 200 ml of a hydrochloric acid concentration of 0.1 g mol / L (C ), the volume of the obtained 5% suspension is recorded - it is 210 ml, then the suspension is centrifuged for 15 minutes at a relative centrifugal acceleration (BCC) of 500 g and the initial volume of pectin gel is fixed - it is 48 ml (V ₁ ). Then the precipitate of the pectin gel is resuspended in the liquid phase, heated to 100 ° C (t) in a boiling water bath and kept at this temperature with constant stirring for a time (T) calculated by formula (I) with a proportionality coefficient of K = 15:

Then the suspension is cooled to room temperature and centrifuged for 15 minutes at a BCC of 500 g. The liquid phase is separated from the precipitate of pectin gel, the volume of the precipitate is measured - it is 41.2 ml (V2). Substituting the obtained data on the initial volume of the low esterified pectin gel (V ₁ ) and the volume of the pectin gel residue after hydrolysis (V ₂ ) in formula (III), the required raw material addition (m) is calculated to restore the initial amount of pectic acid:

The precipitate was added the indicated amount of pectic acid and a solution of hydrochloric acid with a concentration of 0.1 g mol / L (C) until the initial volume of the suspension was restored. The precipitate was resuspended in the liquid phase and the next hydrolysis cycle was carried out under the previous conditions — 3.4 hours at 100 ° C. A total of 5 cycles of hydrolysis are carried out. In this case, each time after separation of the liquid phase, the volume of pectin precipitate is measured: if it remains constant (equal to 41.2 ml (V ₂ )), then the amount of raw material added to the pectin residue also remains constant - 1.42 g (m), if the sediment volume changes, then the necessary raw material addition is recalculated by the formula (II). The volume of the obtained centrate — the liquid phase — is 810 ml, the content of oligogalacturonides is 6.10 mg / ml. The resulting liquid phase is subjected to additional hydrolysis at 100 ° C (t) for a time (T ₁ ) calculated by the formula (II) with a proportionality coefficient K ₁ = 2.5:

Then the liquid phase is cooled to room temperature, neutralized to pH 4.0 with a 5.0 M sodium hydroxide solution, and oligogalacturonides are precipitated by the addition of 2.4 L of acetone. The mixture is stirred vigorously for one minute, then after 12 hours the precipitated precipitate of oligogalacturonides is separated by centrifugation at a BCC of 1000-1500 g for 15 minutes and dried at 80 ° C. The yield of the product — dry powder of sodium oligogalacturonate — 5.55 g. The relative yield of product according to galacturonic acid, determined as described in the previous example, is 89.1%, respectively. The relative content of the fraction of oligogalacturonides 1-5 kDa in the resulting low molecular weight pectin is 72.0%.

Example 3

A low esterified pectin with an esterification degree of 30% is obtained by controlled alkaline deesterification of a highly esterified pectin.

Take 50 g of highly esterified citrus pectin (Herbstreith & Fox KG, Germany), suspended in 0.5 l of 60% vol. ethanol and a 5.0 M sodium hydroxide solution is gradually added in portions, controlling the degree of pectin esterification in samples taken after adding each portion of sodium hydroxide. After reaching an esterification degree of 30%, 20 ml of concentrated hydrochloric acid (density d = 1.19 g / cm ³ ) was added to the suspension, the pectin was filtered off, washed with 150 ml of 50% ethanol and dried at 80 ° C. The yield of low esterified pectin is 47 g, the content of galacturonic acid is 74.8%, the degree of esterification is 30.0%.

Take 10.0 g of the obtained low esterified pectin (m ₁ ), mix with 200 ml of hydrochloric acid at a concentration of 0.5 g mol / L (C) and fix the volume of the obtained 5% suspension - it is 210 ml, then the suspension is centrifuged for 5 minutes with BCC of 1000 g and the initial volume of pectin gel is fixed - it is 112 ml (V ₁ ). Then the precipitate of the pectin gel is resuspended in the liquid phase and thermostated at 80 ° C (t) and with constant stirring for a time (T) calculated by formula (I) with a proportionality coefficient of K = 10:

Then the suspension is cooled to room temperature and centrifuged for 5 minutes at a BCC of 1000 g. The liquid phase is separated from the precipitate of pectin gel, and the precipitate is washed twice with an acid solution for more complete separation of oligogalacturonides: the precipitate is suspended in 100 ml of hydrochloric acid at a concentration of 0.5 g mol / L (C), then centrifuged for 5 minutes at 1000 g of OTC, liquid the phase is drained and the washing of the precipitate is repeated. After washing, the volume of pectin precipitate after hydrolysis is measured - it is 98.5 ml (V ₂ ). Substituting the obtained values of V ₁ , V ₂ and m ₁ in the formula (III), calculate the necessary addition of raw materials (m) to restore the original amount of pectin:

The precipitate was added the indicated amount of low esterified pectin (1.43 g) and a solution of hydrochloric acid with a concentration of 0.5 g mol / L (C) until the initial volume of the suspension was restored. The precipitate was resuspended in the liquid phase and the next hydrolysis cycle was carried out under the previous conditions — T = 5.8 hours at t = 80 ° C. A total of 5 cycles of hydrolysis are carried out. Moreover, each time after separation of the liquid phase, the volume of the precipitate is measured: if it remains constant (equal to 98.5 ml), the amount of raw material added to the pectin residue also remains constant - 1.21 g, if the volume of the precipitate changes, then according to the formula (II) re-calculate the necessary addition of raw materials. The volume of the obtained centrate — the liquid phase — is 1450 ml, the oligogalacturonide content is 2.94 mg / ml. The resulting liquid phase is subjected to additional hydrolysis at 80 ° C (t) for a time (T ₁ ) calculated by the formula (II) with a proportionality coefficient K ₁ = 1.7:

Then the liquid phase is cooled to room temperature and neutralized: at the beginning with dry sodium hydroxide to pH 1-2, then with a 5.0 M sodium hydroxide solution to pH 6.0. Oligogalacturonides are precipitated by the addition of 4.5 L of ethanol. The precipitated precipitate of oligogalacturonides is separated by centrifugation for 15 minutes at a BCC of 1000-1500 g, then purified from salts: resuspended in 0.6 l of 70% ethanol and centrifuged again. The wet cake is dried at 80 ° C. The yield of the product is a dry powder of sodium oligogalacturonate - 4.81 g. The relative yield of the product from galacturonic acid, determined as described in the previous example, is 94.5%. The relative content of the fraction of oligogalacturonides 1-5 kDa in the obtained low molecular weight pectin is 78.0%.

Example 4

Take 30 g of pectic acid (see example 1) (m ₁ ) with a galacturonic acid content of 82.2% (C ₁ ), mix with 200 ml of a sulfuric acid solution at a concentration of 2.0 mol / L (C) and fix the volume of the obtained 15 % suspension - it is 230 ml. The suspension is thermostated at 70 ° C (t) and with constant stirring over time (T) calculated according to formula (I) with a coefficient of K = 10:

Then the hot suspension is filtered, to filter the suspension using a polypropylene filter cloth PL-30 (LOSMA, Italy). The remaining pectin on the filter is washed with 200 ml of sulfuric acid at a concentration of 2.0 mol / L (C) to more fully separate oligogalacturonides. The resulting filtrate was cooled to room temperature. The pectin residue is weighed and the percentage of galacturonic acid is determined in it by the titrimetric method. According to the obtained values of the mass of the pectic acid residue - 127.5 g (m ₂ ), the content of galacturonic acid in it - 16.4% (C ₂ ) and the content of galacturonic acid in the feedstock (pectic acid) - 82.2% (C ₁ ) calculated by the formula (IV) the necessary addition of raw materials (m) to restore the original amount of pectic acid:

The calculated amount of pectic acid (m) and a solution of sulfuric acid with a concentration of 2.0 mol / L (C) are added to the pectin residue until the initial volume of the suspension is restored. The pectin residue is resuspended in the liquid phase and the next hydrolysis cycle is carried out under the previous conditions - T = 6.25 hours at t = 70 ° C. A total of 5 cycles of hydrolysis are carried out. Moreover, each time after separation of the liquid phase, the pectin residue is again weighed, the percentage of galacturonic acid is determined in it, and the necessary raw material addition is calculated by formula (IV). The total volume of the obtained filtrate is 1285 ml, the content of oligogalacturonides is 14.0 mg / ml. The resulting liquid phase is subjected to additional hydrolysis at 70 ° C (t) for a time (T ₁ ) calculated by the formula (II) with a proportionality coefficient K ₁ = 1.7:

Then the liquid phase is cooled to room temperature and neutralized: at the beginning with dry sodium hydroxide to pH 1-2, then with a 5.0 M sodium hydroxide solution to pH 8. Oligogalacturonides are precipitated by adding 4.0 L of isopropanol. The precipitated precipitate of oligogalacturonides is separated by centrifugation for 15 minutes at a BCC of 1000-1500 g, then purified from salts: resuspended in 2.0 L of 70% isopropanol and centrifuged again. The wet cake is dried at 80-100 ° C. The yield of the product is a dry powder of sodium oligogalacturonate - 20.26 g. The relative yield of product by galacturonic acid is 96.0%. The relative content of the fraction of oligogalacturonides 1-5 kDa in the obtained low molecular weight pectin is 81%.

Example 5

Get low esterified pectin. To do this, 20.0 g of sodium hydroxide is dissolved in 0.5 l of 50% ethanol, then 100.0 g of highly esterified citrus pectin is added with stirring (Herbstreith & Fox KG, Germany). The mixture is stirred for 50 minutes, then 40.0 ml of concentrated hydrochloric acid are added in small portions, the pectin is filtered off, washed with 300 ml of 50% ethanol and dried at 80 ° C. The yield of low esterified pectin is 93.3 g, the content of galacturonic acid is 76.5% (C ₁ ), the degree of esterification is 0%.

Take 20.0 g (m ₁ ) of the obtained low esterified pectin, mix with 200 ml of a solution of nitric acid with a concentration of 0.5 g mol / L (C) and fix the volume of the obtained 10% suspension - it is 220 ml. The suspension is thermostated at t = 90 ° C and with constant stirring over time (T) calculated according to formula (I) with a coefficient of K = 12:

At the end of the hydrolysis, the suspension is cooled and filtered; a PL-30 polypropylene filter cloth is used to filter the suspension. The remaining pectin on the filter is washed with 150 ml of nitric acid with a concentration of 0.5 g mol / L (C) to more fully separate oligogalacturonides. A solution of nitric acid with a concentration of 0.5 g mol / L (C) is added to the pectin residue until the initial volume of the suspension is restored. The pectin residue is resuspended in the liquid phase and the next hydrolysis cycle is carried out under the previous conditions - 3.0 hours (T) at 90 ° C (t). In total, 3 hydrolysis cycles are carried out. The washed pectin residue after the last hydrolysis cycle is weighed and the percentage of galacturonic acid is determined in it by the titrimetric method. According to the obtained values of the mass of the pectin residue - 59.2 g (m ₂ ) and the content of galacturonic acid in it - 17.1% (C2), the necessary raw material addition (m) is calculated using formula (IV) to restore the initial amount of pectin:

The indicated amount of low esterified pectin is added to the pectin residue and the following 3 cycles of hydrolysis are carried out under the same conditions. The total volume of the obtained filtrate is 1660 ml, the content of oligogalacturonides is 5.79 mg / ml. The resulting liquid phase is subjected to additional hydrolysis at 80 ° C (t) for a time (T ₁ ) calculated by the formula (II) with a proportionality coefficient K ₁ = 2.0:

Then the liquid phase is cooled to room temperature and neutralized: at the beginning with dry sodium hydroxide to pH 1-2, then with a 5.0 M sodium hydroxide solution to pH 5. Oligogalacturonides are precipitated by adding 5.0 L of ethanol. The precipitated precipitate of oligogalacturonides is separated by centrifugation for 15 minutes at a BCC of 1000-1500 g, then purified from salts: resuspended in 1.3 l of 70% vol. ethanol and re-centrifuged. The wet cake is dried at 80 ° C. The yield of the product is a dry powder of sodium oligogalacturonate - 10.85 g. The relative yield of the product for galacturonic acid is 93.7%. The relative content of the fraction of oligogalacturonides 1-5 to Yes in the obtained low molecular weight pectin is 76.5%.

Example 6

Get low esterified pectin.

In 0.4 l, 50% vol. 20 g of sodium hydroxide is dissolved in isopropanol, then 100 g of highly esterified apple pectin is added with stirring (Herbstreith & Fox KG, Germany). The mixture is stirred for 10 minutes, then gradually 40 ml of concentrated hydrochloric acid are added in small portions, the pectin is filtered off, washed with 300 ml of 50% isopropanol and dried at 80-100 ° C. The yield of low esterified pectin is 94.5 g, the content of galacturonic acid is 78.4% (C ₁ ), the degree of esterification is 13.8%.

Take 20 g of the obtained low esterified pectin (m ₁ ), mix with 200 ml of a solution of sulfuric acid at a concentration of 1.0 mol / L (C) and fix the volume of the obtained 10% suspension - it is 220 ml. The suspension is thermostated at t = 90 ° C and with constant stirring over time (T) calculated according to formula (I) with a coefficient of K = 13:

Then the suspension is cooled and filtered, to filter the suspension using a polypropylene filter cloth PL-30. The remaining pectin on the filter is washed with 150 ml of sulfuric acid at a concentration of 1.0 mol / L (C) for a more complete separation of oligogalacturonides. The pectin residue is weighed and the percentage of galacturonic acid is determined in it by the titrimetric method. According to the obtained values of the mass of the pectin residue - 79.2 g (m ₂ ) and the content of galacturonic acid in it - 16.2% (C ₂ ), the necessary raw material addition (m) is calculated using formula (IV) to restore the initial amount of pectin:

The indicated amount of low esterified pectin and a solution of sulfuric acid with a concentration of 1.0 mol / L (C) are added to the pectin residue until the initial volume of the suspension is restored. The pectin residue is resuspended in the liquid phase and the next hydrolysis cycle is carried out under the previous conditions T = 2.0 hours at t = 90 ° C. A total of 5 cycles of hydrolysis are carried out. Moreover, each time after separation of the liquid phase, 3.63 g of low esterified pectin (m) is added to the pectin residue. Since, during the hydrolysis of pectin, a certain acceptable discrepancy may occur between the amount of added raw materials and the actual value of the consumption of raw materials, in order to eliminate this possible discrepancy, after 5 cycles of hydrolysis, the necessary raw material addition is again calculated. For this, the washed pectin residue after the last hydrolysis cycle is again weighed and the percentage of galacturonic acid is determined in it by the titrimetric method. According to the obtained values of the mass of the pectin residue - 85.7 g (m ₂ ) and the content of galacturonic acid in it - 15.5% (C ₂ ), the necessary one-time corrective additive of the raw material (m) is calculated using formula (IV) to restore the initial amount of pectin:

The indicated amount of low esterified pectin is added to the pectin residue. Then carry out the following 5 cycles of hydrolysis under the same conditions. Moreover, each time after separation of the liquid phase, 3.63 g of low esterified pectin is still added to the pectin residue. The total volume of the obtained filtrate is 2810 ml, the content of oligogalacturonides is 9.10 mg / ml. The resulting liquid phase is subjected to additional hydrolysis at 90 ° C (t) for a time (T ₁ ) calculated by the formula (II) with a proportionality coefficient K ₁ = 2.2:

Then the liquid phase is cooled to room temperature and neutralized: at the beginning with dry sodium hydroxide to pH 1-2, then with a 5.0 M sodium hydroxide solution to pH 5. Oligogalacturonides are precipitated by adding 8.0 L of isopropanol. The precipitated precipitate of oligogalacturonides is separated by centrifugation for 15 minutes at a BCC of 1000-1500 g, then purified from salts: resuspended in 2.6 l of 70% vol. isopropanol and centrifuged again. The wet cake is dried at 80-100 ° C. The yield of the product is a dry powder of sodium oligogalacturonate - 28.81 g. The relative yield of the product for galacturonic acid is 91.1%. The relative content of the fraction of oligogalacturonides 1-5 kDa in the obtained low molecular weight pectin is 73.8%.

As can be seen from the above examples, the inventive method of heterophasic hydrolysis allows, due to the insolubility of the starting pectin in acid, to carry out hydrolysis purposefully to obtain a product with the desired molecular weight and at the same time to use the starting pectin almost completely avoids any significant technological losses of raw materials and increase the final yield of the product (galacturonic acid) compared with the prototype in 2.6-2.8 times to 89-96% of the feedstock.

In addition, in the inventive method, in contrast to the known method, the hydrolysis of raw materials is carried out in separate, relatively short cycles, which can significantly limit the destruction of oligogalacturonides during hydrolysis and, as a result, further increase the yield of the target product.

Moreover, the technical solution proposed by the authors provides a higher process speed compared to the prototype and, accordingly, a reduction in the specific production costs and time to produce one kilogram of the product with significant raw material savings, also eliminates the use of complex and expensive technological equipment for separating the liquid phase from the residue pectin.

Claims (2)

1. The method of obtaining low molecular weight pectin, comprising hydrolysis of pectin in an aqueous solution of mineral acid by heating, followed by separation of the liquid phase from the insoluble pectin residue, separation of low molecular weight products of pectin hydrolysis from them by precipitation with an organic solvent miscible with water, characterized in that as feedstocks for hydrolysis use low esterified pectin with an esterification degree of not more than 30%, hydrolysis is carried out cyclically, while the time of one qi la is calculated using the formula:

where T is the time of one cycle, h;
C is an acid concentration of 0.1-2.0 g mol / L;
t is the process temperature of 70-100 ° C;
K is the coefficient of proportionality, the values of which are in the range of 10-15;
the liquid phase after separation from the pectin residue is subjected to additional hydrolysis, the time of which is calculated by the formula:

where T ₁ is the time of additional hydrolysis of the liquid phase, h;
C is an acid concentration of 0.1-2.0 g mol / L;
t is the process temperature of 70-100 ° C;
To ₁ - the coefficient of proportionality for additional hydrolysis of the liquid phase, the values of which are in the range of 1.7-2.5;
before precipitation of low molecular weight products of pectin hydrolysis with an organic solvent, the liquid phase is neutralized to a pH of not less than 4.0, and after precipitation, low molecular weight products of pectin hydrolysis are dried. 2. The method according to claim 1, characterized in that after each or several hydrolysis cycles, a new portion of the feed is added to the pectin residue, the amount of which is calculated by the formula:

where m is the mass of added raw materials, g;
m ₁ - mass of feedstock, g;
V ₁ - the volume of the pectin gel before hydrolysis, ml;
V ₂ - the amount of pectin gel residue after hydrolysis, ml,
wherein V ₁ and V _{2 are} determined after centrifugation of the pectin gel, or by the formula:

where m is the mass of added raw materials, g;
m ₁ - mass of feedstock, g;
m ₂ - mass of pectin residue after separation of the liquid phase, g;
C ₁ - the content of galacturonic acid in the feedstock,%;
C ₂ - the content of galacturonic acid in the pectin residue,%.