SK500332008A3 - The method for producing D-galactose - Google Patents

Abstract

A method is described in which immobilized yeast is immobilized of the genus Saccharomyces and immobilized enzyme β-galactosidation cultures submerged at a temperature of 10 to 40 ° C, at pH 3.0 to 7.0 in the presence of 5 to 25% lactose medium, wherein D-galactose can be prepared from fermented media by crystallization.

Description

Process for the preparation of D-galactose.

Technical field

The invention relates to a process for the preparation of D-galactose

BACKGROUND OF THE INVENTION

Anhydrous D (+) galactose is hexose (C 6 H 12 O 6) with a molecular weight of 180.1 g / mol. It is a component of oligosaccharides such as lactose, melibiosis arafinose, as well as glycoproteins and glycolipids. It is mainly found in hydrolysed milk, whey and other dairy products, while a very small amount is also present in breast milk.

It is used in biochemistry, microbiology, cosmetics, and also as a starting material for the production of other rare carbohydrates such as arabinose, pentitol, arabinitol, globotriose, arabitol, galactitol, xylitol or tagatose. It is used as a stabilizer in injectable medical solutions, especially ferric and related compounds. It is also used as a substitute for phenols in resins, in the manufacture of contrast agents, or as a sweetener for the prevention of dental diseases.

The process for the preparation of D-galactose consists of three main steps: 1. hydrolysis of lactose, 2. separation from a mixture of D-galactose and D-glucose, 3. purification of D-galactose. One of the most common methods of production is hydrolysis, chemical or enzymatic, by the enzyme βgalactosidase, milk sugar (lactose) present in milk or whey. The chemical route requires the use of strong acids at high temperatures, causing the formation of a large number of substances that are formed after the hydrolysis of macromolecules and the thermal decomposition of organic substances, resulting in problems in isolation of the pure product.

β-galactosidase is found in a number of microorganisms, plants and animal tissues. It catalyzes the hydrolytic decomposition of lactose into D-glucose and D-galactose. Potassium ions (K ⁺ ) and magnesium ions (Mg ²⁺ ) are required for optimal activity.

The ability of β-galactosidase to hydrolyze lactose is applied in the food industry, mainly in the field of dairy products due to nutritional (lactose intolerance), technological (crystallization) and environmental (contamination) problems associated with the use of lactose. The added value obtained by the hydrolysis of lactose consists in increasing the usefulness of hydrolysed lactose as a food hydrocarbon. Lactose itself has limited use because of its low sweetness, solubility and digestibility. Its hydrolysis products have these properties much better. Increased sweetness and solubility increase the technical utility of whey products.

Various substrates can be used for the preparation of D-galactose, in particular its preparation from milk or milk serum is described (Pat US2007 / 0134373 A1). EP 499165 describes a process for producing syrups containing galactose, fructose and glucose from pre-treated whey. Another method for preparing the desired monosaccharide is by isolation from plant materials, either from homologous galactose polymers (e.g. galactan) or non-homologous polysaccharides (melibiosis, maninotriose, raffinose, stachyosis, etc.) (Pat. WO 0063445, Pat. US 2004/0198965 A1) ). Various non-traditional substrates for the preparation of D-galactose have also been described, such as larch extract (Pat. US 1718837) or cashew nut shells (Ingle: Research and Industry, 21 (4), 243-246, 1976.).

Several methods for the preparation of D-galactose are known. In US925380, an aqueous lactose solution is hydrolyzed with sulfuric acid at 100 ° C. The crude D-galactose preparation is then dissolved in ethanol or methanol to remove insoluble impurities and Dgalactose is obtained by crystallization. US4156076 discloses the process of converting lactose to monosaccharides and their derivatives using oxidative hydrolysis of a lactose solution. The obtained D-galactose and gluconic acid are then separated.

EP499465 discloses a process for producing a syrup comprising D-galactose, D-fructose and D-glucose. This syrup prepared from whey ultrafiltrate undergoes purification, desalinization, lactose concentration and hydrolysis, glucose isomerization to fructose, and product purification and concentration.

Hydrolysis of whey lactose to D-glucose and D-galactose is described in US4067748. Lactose comes into contact with a solid, insoluble, strongly acidic ion exchange resin formed by selected interconnected polystyrenes or selected hydrocarbons in water. EP168127 discloses a process and apparatus for the continuous hydrolysis of lactose to D-glucose and D-galactose using a flow reactor in which the lactase system is immobilized.

US3981773 describes a process for preparing D-galactose and beverages based on its content from solutions containing lactose. The process involves culturing mutant, non-pathogenic prototrophic yeasts or bacteria having β-galactoside activity and non-neutralizing Dgalactose, followed by separation of D-galactose. This patent also points out the need to remove proteins before fermentation. In SK281153, a glucose-galactose syrup is prepared by acidic or enzymatic hydrolysis of a lactose solution which is fermented with yeast of 5. cerevisiae or S. oviformis.

One of the main advantages of technologies with immobilized biocatalysts is a significant reduction in the cost of the process coupled with the possibility of re-use of the biocatalyst, process continualization and easier product isolation. Immobilization of enzymes and cells by encapsulation into goal carriers is one method that allows the biocatalyst to be concentrated in the carrier. This technique involves enclosing the biocatalyst in natural or synthetic gels.

To date, immobilization of β-galactosidase to and on various carriers such as alginate, chitosan, lectin, cellulose, cotton fibers, Sepabeads® or an inorganic carrier (SiO2.11H2C) is known. A suitable method of immobilization is the encapsulation of β-galactosidase into the polyvinyl chloride gel. This has several advantages over other carriers. In particular, it is inexpensive, non-toxic, biologically minimally degradable, has excellent physico-mechanical properties and exhibits long-term stability (Pat. DE 198 27 552.8). Enzymes isolated from various microbial sources such as Kluyveromyces lactis or Aspergillus oryzae can be immobilized.

The preparation of D-galactose by the method of the present invention is based on the production process by simultaneous saccharification and fermentation by immobilized biocatalysts, in which the lactose hydrolysis and the selective fermentation of the glucose generated are carried out in a single reactor at the same time. Process control in the SSF system allows cost reduction and acceleration of the D-galactose preparation process. The costs required for fermentation equipment, for procuring the enzyme (immobilized enzyme is reused) and substrates for growth of the microorganism, which is also reused immobilized in the matrix, will be reduced, which facilitates product purification by omitting the centrifugation step to remove biomass. Acceleration and streamlining of the preparation of D-galactose in the SSF system is achieved by reducing the time required for growth of the microorganism, reducing enzyme inhibition by the product by concomitant utilization of the resulting glucose and concentrating the biocatalysts by immobilization.

SUMMARY OF THE INVENTION

The essence of the process for the production of D-galactose according to the invention is that immobilized β-galactosidase enzyme and immobilized yeast are selectively cultured which selectively convert D-glucose to ethanol and do not ferment D-galactose, preferably of the genus Saccharomyces, in an aqueous nutrient medium at 10 to 40 ° C, a pH of 3.0 to 7.0 in the medium in the presence of lactose at a concentration of 5 to 25% by weight, whereby D-galactose is obtained by crystallization upon completion of the culture and separation of the fermented medium. Ethanol-producing yeasts that do not ferment D-galactose, preferably the Saccharomyces oviformis S5 strain, can be used as production microorganisms. This strain is deposited at the Department of Biochemical Technology FCHPT STU Bratislava and shows high selectivity of D-glucose to ethanol conversion with minimal formation of organic acids and other undesirable substances.

Suitable matrices such as alginate, pectin, chitosan, and the like can be used for immobilization. It is suitable to use polyvinyl alcohol with its advantages. Yeast preparation prior to mobilization can be accomplished by standard solid or liquid culture medium procedures. In liquid media, it is advantageous to induce a suitable physiological state of the producer at the end of the exponential growth phase by repeated passage. The prepared biomass is separated from the liquid fraction e.g. by centrifugation and transferred to the polyvinyl alcohol solution. To prepare immobilized β-galactosidase, a commercially available enzyme is added to the polyvinyl alcohol solution. The suspension is suitably transferred to a solid support, followed by crosslinking of the gel by drying over a temperature range of 15-60 ° C. The immobilisates are then cured in aqueous sodium sulfate (potassium in the case of enzyme) solution and stored in a suitable storage medium.

The fermentation medium contains lactose in an amount of 5-25%. Mineral and nutrient components are added to the medium to support yeast vitality and ethanol production. During the fermentation itself, the pH is maintained at the desired concentration by the addition of KOH solution. The cultivation takes place without aeration, the medium is mixed gently, which promotes the release of carbon dioxide and accelerates fermentation. The immobilized biocatalysts are fed to the production fermenter in an amount of from 5 to 20% of the total volume.

Fermentation is carried out until D-glucose is depleted from the medium. The fermented medium, which can be collected once, gradually and continuously, is separated from the immobilizates, e.g. filtration. The immobilisates are then used for further fermentation or stored in a storage medium at 4 ° C. Conventional production medium containing 2% by weight can also be used for the storage of immobilisates. glucose.

D-galactose can be prepared from the fermented medium after separation of the immobilized biocatalysts by conventional industrial processes (decolorization, desalting, crystallization, etc.). The ethanol produced during the cultivation, which can be obtained from the fermented medium by conventional industrial processes, such as e.g. distillation or extraction may be used during the crystallization of D-galactose.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

800 ml of a lactose solution of 200 g.dm < -1 > of lactose in 0.1 M phosphate (potassium) buffer at pH 6.5 are prepared. Was added 1.2 ml of β-galactosidase activity 85 U.ml ^first After 12 hours of hydrolysis at 30 ° C, the lactose is hydrolyzed to more than 95%. The residual lactose concentration is below 10 g.dm ³ .

To the resulting glucose-galactose syrup (the content of D-glucose and D-galactose is 95 g.dm &^lt; ^{3 >} ), the salts of the yeast extract are added to the following concentrations: 3 g.dm &^lt; ³ > 12SO4, 0.5 g.dm < ^{3 >} MgSO4 > 4.7H2O and sterilized at 120 [deg.] C. for 20 minutes. 100 ml glucose inoculum medium (100 g.dm- ³ glucose, 5 g.dm- ³ yeast extract, 1 g.dm- ³ (NH4ESO4, 1 g.dm- ³ KH2PO4, 0.5 gdm- ³ MgSO4.7H2O, The pH is adjusted to 6.5) in 250 ml culture flasks inoculated with Saccharomyces slant agar and cultured statically with intermittent stirring for 24 hours at 30 ° C. The thus produced submerged culture of the producing microorganism is used as an inoculum. syrup is seeded with 80 ml (10%, v / v) inoculum medium. the fermentation was carried out at 30 ° C without aeration and the excess pressure, the medium was gently stirred (150 rpm in ^1). After 36 hours of fermentation, the fermentation medium below 1 g.dm &^lt; ^{3 > of} glucose and about 45 g.dm &^lt; ^-3 > of ethanol The amount of D-galactose does not change during cultivation.

The cell mass of the microorganism is removed from the medium by centrifugation. After discoloration and removal of residual minerals and organic acids by percolation through cation exchange anion exchange columns, the D-galactose solution is concentrated to a syrup and crystallized in the presence of ethanol. The high purity D-galactose thus prepared contains less than 1% impurities.

Example 2

The immobilized yeast in the PVA gel was prepared as follows. The inoculum is prepared in two steps of biomass propagation. In a first step, 50 ml of glucose inoculum medium of the same composition as in Example 1 is inoculated in a 100 ml Erlenmeyer flask from slanted agar and cultured statically (24 h) at 30 ° C. The biomass thus prepared is used as an inoculum (5%, v / v) in a 250 ml inoculation medium (500 ml Erlenmeyer flask) and cultured under the same conditions. The biomass is separated in the exponential phase by centrifugation (2000 rpm in ^1, 15 minutes) and is resuspended in saline.

A PVA solution is prepared containing 100 g of PVA (polyvinyl alcohol), 60 g of PEG (polyethylene glycol) per 790 ml of distilled water. 50 ml of a homogeneous suspension of biomass are added to the solution thus prepared so that the final cell concentration is 0.7 g of cells per dm ³ gel. The mixture of cells in the PVA solution thus prepared is dropped onto a hard support and subsequently the polyvinyl alcohol crosslinks in a dry air stream in a temperature gradient. The immobilizates are then transferred to a sodium sulfate curing solution (0.1 M) for 30 to 60 minutes.

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The biomass immobilized in PVA gel is propagated in a bioreactor of 3 dm propagation medium and 600 g of immobilisates at 30 DEG C. and stirring 200 otmin ^first When glucose utilization reaches 95%, the medium is separated and the immobilisates are washed with sterile distilled water and then placed in fresh propagation medium. After 8 cycles, the biomass concentration in the immobilisates is 160 mg.g ^-1 .

Immobilized enzyme: When immobilizing the enzyme, 50 ml of enzyme are added per 1 dm ^{3 of} PVA solution. Drying is the same as for yeast immobilization. However, 0.1 M potassium sulfate is used as the curing solution. The immobilizates thus prepared are transferred to 0.1 M potassium phosphate buffer.

800 ml of sterile lactose medium is prepared having the following composition: 200 g lactose, 1 g yeast extract, 1 g (NH4 SCh, 1 g KH2PO4 and 0.5 g MgSCLTThO per liter distilled water and the pH is adjusted to 6.5. 40 g (5%, w / v) of immobilized yeast (S. cerevisiae), adjusted the pH in the medium and maintained by the automatic addition of a 2 M KOH solution, and 60 g (7.5%, w / v) of immobilized enzyme were added. the process is carried out at 30 ° C without aeration and the excess pressure, the medium was gently stirred (150 rpm in ^1). After 15 h, the lactose hydrolyzed to more than 95%, and the residual glucose concentration of less than 1 g.dm ^'3.

The cell mass residues of the microorganism are removed from the medium by means of activated carbon. Furthermore, D-galactose is isolated as in Example 1.

Example 3

To 800 ml of sterile lactose medium is added 70 g (8.75%, w / v) of immobilized yeast (S. cerevisiae), the pH in the medium is adjusted and maintained by the automatic addition of 2 M KOH solution. 105 g (13.13%, w / v) of immobilized enzyme are added. The composition of the media, the fermentation conditions and the isolation of D-galactose are analogous to Example 2. After 9 hours, the lactose is hydrolyzed to more than 95% and the residual glucose concentration is less than 1 g.dm &^lt; ^-3 &^gt;.

Example 4

The fermentation procedure is the same as in Example 2. After completion of the first SSF (15 h, 95% lactose hydrolysis,) the immobilizates are reused in subsequent cycles. The fermented medium is separated and fresh sterile lactose medium is added. After five conversions, the conversion time is stable and the relative activity of the enzyme does not decrease. Approximately 320 g of D-galactose is prepared in 4 dm ^{3 of} fermented medium in 75 h. After 310 hours (20 conversions) in this system, approximately 1.3 kg of D-galactose was prepared from 15 dm ^{3 of} fermented medium. The relative activity decrease of the immobilized systems is less than 5%.

Industrial usability

The invention is useful in the production of D-galactose, which has wide application in biochemistry, microbiology, cosmetics, as well as a starting component for the production of other rare carbohydrates. It also serves as a sweetener, stabilizer in injectable medical solutions, or a replacement of phenols in resins.

Claims (3)

PATENT CLAIMS Process for the production of D-galactose, characterized in that immobilized β-galactosidase enzyme is cultivated submerged and the immobilized yeast selectively convert D-glucose to ethanol and do not ferment D-galactose, preferably of the genus Saccharomyces in an aqueous nutrient medium at a temperature of 10 to 40 ° C, a pH of 3.0 to 7.0 in the medium in the presence of lactose at a concentration of 5 to 25% by weight, wherein after the cultivation and separation of the fermented medium, D-galactose is separated from ethanol by crystallization. A method according to claim 1, characterized in that the immobilized β-galactosidase enzyme and the immobilized yeast are separated from the liquid fraction and reused for the production of D-galactose in further fermentation cycles. Production method according to claims 1 and 2, characterized in that the strain Saccharomyces oviformis S5 is used for the production of D-galactose.