DE10357707A1 - Process for the continuous production of galactosyl oligosaccharides - Google Patents

Abstract

The invention relates to a bioreactor system with an integrated membrane filtration unit and a process for the continuous production of concentrated solution of galactosyl-oligosaccharides.

Description

The Invention includes a continuous process for preparation a galactosyl oligosaccharide solution and a corresponding Bioreactor system with an integrated membrane filtration unit to carry out of the procedure. In the method according to the invention is alternatively lactose solution and / or whey used as a substrate, previously a concentration process has gone through. The process is characterized by the emergence a disposable one Product and by preventing large amounts of by-products out.

state of research

Galactosyl oligosaccharides become positive nutritional physiological Attributed properties, they promote, for example selectively the growth of as useful distinguished bacteria (bifidobacteria) of the intestine, improve the Calcium and magnesium absorption, the elimination of toxic Connections and support the liver function. Galactosyl oligosaccharides are not digestible Carbohydrates, built up in the form of a galactosyl galactose chain with a glucose unit at the end. They arise in the enzymatic Conversion of lactose by means of β-galactosidase, the one transferase activity has.

This results in a mixture of different oligosaccharides, lactose, glucose (Glu) and galactose (Gal). The composition of the galactosyl-oligosaccharide fraction varies in the chain length (N = 1 to 7) and the kind of compound of the monomers. The general chemical structure of galactosyl oligosaccharides is as follows:

For the preparation of galactosyl oligosaccharides numerous methods are described, which are usually carried out in a batch process, but all of which lead to lower yields of galactosyl oligosaccharides. As a reason, it is often described that the oligosaccharides rehydrolyze in the presence of the catalyst enzyme before they can be harvested, so that the overall yield of oligomers is reduced. It is believed that the poor yield results from enzymatic lactose hydrolysis using β-galactosidase as by-products of monosaccharides which have a disruptive effect of the reaction, with the monosaccharide glucose acting as a competing acceptor for β-galactosyl transfer. In addition, the monosaccharides inhibit the activity of β-galactosidase, so that there is a reduction in the speed of the transfer reaction. As a solution, Japanese Patent Laid-Open No. 62-130695 and the US 4435389A to consume the monosaccharides glucose and galactose by additional addition of yeast. The disadvantage here is that the yeasts, as well as the soluble enzyme ultimately remain in the product and must be deactivated or removed in an additional step. In the DE 68920814T2 It is proposed that a part of the glucose resulting from the treatment with β-galactosidase is converted into fructose by addition of glucose isomerase. Also here both enzymes remain in the product and must be deactivated or removed.

Currently There is no known method of producing galactosyl oligosaccharides in a continuous process in which the galactosyl oligosaccharides protected against hydrolysis are enriched and in high product yield.

The Galactosyl oligosaccharides are used commercially as a food supplement Child nutrition area, as a dietary Foods in functional and prebiotic foods.

task The present invention is the disadvantages described in Prior art eliminate and a continuous process for preparing a concentrated solution of galactosyl oligosaccharides provide.

Furthermore It is an object of the invention to provide a bioreactor system, this is a continuous operation for producing a concentrated solution on galactosyl oligosaccharides.

The Task is achieved by the requirements solved.

• – es durch einen zusätzlichen Aufkonzentrierungsschritt die Ausbeute an Galactosyl-Oligosacchariden steigert,
• – durch individuelle Temperatur- und pH-Regelung des Prozessvorganges und kontinuierliche Abtrennung von Monosacchariden die Reaktionsgeschwindigkeit gesteigert,
• – durch den kontinuierlichen Rückhalt des Enzyms den Abbau der entstehenden Galactosyl-Oligosaccharide verhindert,
• – ein kontinuierliches Verfahren darstellt, dass einen längeren Betrieb des Bioreaktorsystems ermöglicht und das Enzym aus dem Produkt zurückhält.
• – als Substrate alternativ, Laktose-Lösung bzw. hochkonzentrierte Laktoselösung oder Molke bzw. hochkonzentrierte Molke-Lösung oder eine Kombination von Laktose-Lösung mit Molke-Lösung verwendet wird.

The inventive method improves known methods, since

• It increases the yield of galactosyl oligosaccharides by an additional concentration step,
• - Increased by individual temperature and pH control of the process operation and continuous separation of monosaccharides, the reaction rate,
• - prevents the degradation of the resulting galactosyl oligosaccharides by the continuous retention of the enzyme,
• Represents a continuous process that allows for longer operation of the bioreactor system and retains the enzyme from the product.
• - As substrates alternatively, lactose solution or highly concentrated lactose solution or whey or highly concentrated whey solution or a combination of lactose solution with whey solution is used.

The the following drawing explains the individual steps of the process according to the invention:

1 Concept for the continuous production of galactosyl oligosaccharides in the enzyme membrane reactor with native β-galactosidase, enzyme retention using UF membranes and concentration by chromatography

2 Concept for the continuous production of galactosyl-oligosaccharides in the enzyme membrane reactor with native β-galactosidase, integrated enzyme retention by means of UF membranes and water removal from the reaction as well as the separation of the monosaccharides by chromatography

3 Concept for the continuous production of galactosyl-oligosaccharides in the enzyme membrane or fixed bed reactor with immobilized β-galactosidase, integrated enzyme retention and water removal from the reaction as well as the separation of the monosaccharides by chromatography

4 Model representation of the bioreactor system according to the invention.

The bioreactor system according to the invention ( 4 ) is characterized by a reactor 1 from, for example, is a stirred tank reactor. The reactor contains all inflows and outflows required for the functional sequence 2a . 2 B . 2c , a computer-controlled measuring and control unit 3 which measures or controls the fermentation parameters in the reactor and optimally adjusts, for example, pH value, temperature, oxygen partial pressure, feed of substrate and enzyme, and sequence of the products depending on or independently of one another. The bioreactor system includes a reactor 1 upstream device for the concentration of the substrates 5 , which consists for example of a nanofiltration device or a heating / cooling device. Substrate, for example, from a container with substrate 6 fed. The reactor is followed by a likewise temperature-controlled membrane filtration unit 4 at. The membrane filtration unit 4 is alternatively also integrated in the Rührkesselre actuator (not shown). It is an inorganic or organic ultrafiltration membrane with a pore diameter of 5 to 50 nm. In particular, it is a membrane that is steam sterilizable, so that it is operated sterile in the integrated process. Especially suitable are ceramic Membranes made of bio-inert materials such as zirconium oxide, titanium oxide, aluminum oxide or a combination of these materials, as these fulfill the requirements for sterility and have a long service life in the process. Due to the hydrophilic properties of the ceramic membranes is a much gentler separation of the product. In the membrane filtration unit 4 For example, the enzyme β-galactosidase used in the reaction is retained and is therefore in the retentate, while the main product galactosyl-oligosaccharides, but also glucose, galactose and lactose, can pass through the membrane and be in the filtrate. Filtrate is placed in a separate container 9 collected and retentate via a reflux 2 B back to the reactor 1 optionally supplied by means of pump P2.

In one embodiment of the invention, the membrane filtration unit filters in a continuous feed from the reactor 1 optionally supernatant coming from the reactor via a pump P1. The enzyme-containing retentate from the membrane filtration unit flows via a reflux 2 B back to the reactor or is collected in a waste container and discarded.

In order to prevent the hydrolysis of the galactosyl oligosaccharides, the bioreactor system includes following the membrane filtration unit 4 in one embodiment additionally a chromatography device 7 for separating the monosaccharides and by-products, which is also used to concentrate the galactosyl oligosaccharides. Thus, the galactosyl oligosaccharides contained in the filtrate of glucose, galactose and lactose are separated and significantly concentrated. Galactose and lactose are subsequently returned to the reactor via feeds (not shown). Preferably, the chromatography device is a simulated moving bed device (SMB), as in DE 199 56010 described or a continuous annulare chromatography. In principle, a discontinuous chromatography with recycling of the intermediate fractions is also used.

The computer-controlled measuring and control unit 3 is controlled and controlled so that retentate, filtrate and reactor volumes are maintained at different process conditions such as temperature levels, pHs, or substrate or metabolite concentrations. The temperature control regulates and controls, for example, an individual temperature of the reactor. This is the reactor 1 maintained at an optimum temperature for the enzymatic reaction, for example 40 ° C. The galactosyl oligosaccharides are so after a short residence time in the tempered reactor 1 can be quickly removed and combined with glucose, galactose and unreacted lactose in the membrane filtration unit 4 Pass through the membrane and protected from further hydrolysis, so that a high space-time yield at high product concentration is made possible. Depending on the nature and character of the enzymatic reaction, the temperature difference is arbitrarily regulated and adjusted to optimal conditions.

• a) Aufkonzentrierung der Substrate
• b) Zufuhr der hochkonzentrierten Substrate in das Bioreaktorsystem
• c) Zufuhr eines Enzyms zum enzymatischen Umsatz der Substrate
• d) Rückhaltung des Enzyms und Filtration der entstehenden Produkte
• e) Abtrennung von Wasser während der Reaktion durch Verdampfung während der Reaktion.
• f) Chromatographie des Filtrates und Trennung der Galactosyl-Oligosaccharide von Monosacchariden und Laktose
• g) Trocknung der Galactosyl-Oligosaccharide

The process for preparing concentrated solutions of galactosyl oligosaccharides in a bioreactor system consists of the following steps:

• a) concentration of the substrates
• b) Supply of highly concentrated substrates in the bioreactor system
• c) supply of an enzyme to the enzymatic conversion of the substrates
• d) retention of the enzyme and filtration of the resulting products
• e) Separation of water during the reaction by evaporation during the reaction.
• f) Chromatography of the filtrate and separation of the galactosyl oligosaccharides from monosaccharides and lactose
• g) Drying of the galactosyl oligosaccharides

The enzyme used to produce galactosyl oligosaccharides by enzymatic conversion of lactose is β-galactosidase derived from various microorganisms. Fungi are known, such as Aspergillus oryzae and Aspergillus niger, yeasts such as Bullera singularis, Candida and Klyveromyces lactis or bacteria such as Bacillus circulans, Lactobacillus bulgaricus, Streptococcus thermophilus. Commercially available enzymes are Maxilact ^® Gist Brocades, Lactase F ^® Amano Pharmaceutical Co., lactase Y400 ^® from Vakult Honsha, Lactozym ^® from Novo Industri, Godo-YNL ^® from Godo Shusei and Biolacta ^® by Daiwa Chemicals ,

The amounts used are 1-100 Units per gram of lactose, with the enzyme in solution or immobilized form is used. Alternatively, all. Microorganisms containing β-galactosidase used in the reaction.

Lactose, which is reacted with β-galactosidase, comes from whey and / or lactose solution, wherein it is preferred to use a highly concentrated lactose solution as possible. This is done in a Aufkon centering step. At temperatures above 93.5 ° C, supersaturated lactose solutions predominantly crystallize the β-lactose. Beta-lactose is converted by the β-galactosidase with the same affinity as alpha-lactose, but has a higher solubility. Therefore, highly concentrated beta-lactose solution is preferably used. When using whey, it is preferred to first desalt the deproteinized whey via nanofiltration and to concentrate the lactose. In the method according to the invention, therefore, on the one hand highly concentrated lactose solution or concentrated whey solution, but also one combination of lactose and whey solution are successfully used.

at the enzymatic hydrolysis of lactose by means of β-galactosidase arise in first and foremost the monosaccharides glucose and galactose. By the continuous invention Procedure is possible the yield of galactosyl oligosaccharides up to 45% of the total to increase the saccharides, however, prepare those from the accompanying reaction always resulting monosaccharides in the application of the products as a dietary supplement certain technical problems. Monosaccharides are especially prone to they are present as a powder for water absorption and lumping and are no longer pourable. To address this problem in the production of galactosyl oligosaccharides prevent and produce a non-hygroscopic powder form, sees the method according to the invention An additional Step, the continuous chromatographic separation and workup the galactosyl oligosaccharides of the monosaccharides, wherein Galactosyl oligosaccharides, which have a wider scope in the Food industry, can be concentrated. The Galactosyl oligosaccharide solution is alternatively dried in a final step to the powder form.

The Separation of water during the reaction by evaporation is carried out by means of an evaporator e.g. Falling film or thin film evaporator, which is integrated into the reaction cycle or through a membrane evaporation unit, which is integrated into the reaction cycle.

2 shows the integrated water removal in the inlet of membrane filtration unit to the reactor 2 B by means of vacuum evaporation. This already gives the first anhydrous product.

embodiments

The Membrane reactor system consists of a conventional stirred tank reactor combined with a membrane filtration unit. The reactor in which the native enzyme is flowing continuously the lactose solution (Feed) too. In the membrane module, the enzyme is retained and returned to the reactor fed again (Retentate), thus again for the reaction available to stand. The resulting products, galactosyl oligosaccharides, Glucose and galactose as well as the unreacted lactose may be the Membrane happen and are in the filtrate (permeate). As a substrate for the enzymatic lactose hydrolysis by means of β-galactosidase is lactose solution and / or Whey used as a byproduct in dairies as a byproduct huge Quantities accumulate. Although whey has only a low lactose concentration, can the yield of galactosyl oligosaccharides can be increased so that the implementation of the method is also possible on an industrial scale.

The Device for the concentration of the substrates, in the case of Whey as substrate a nanofiltration device, in the case of Lactose as substrate a heating / cooling device.

The membrane filtration unit is, for example, a hydrophilicized PES flat membrane in a membrane module type TTP-90NR0019 from Amafilter with a membrane area A = 64 cm ² . Alternatively, a ceramic membrane from Atech Innovations is used in a membrane module type M05 from Amafilter with a membrane area A = 0.1 m ² . The membrane filtration unit used has the following specification:

The Chromatography device is a simulated moving bed device (SMB).

Lactose as Substrate for producing a concentrated solution of galactosyl oligosaccharides

• a) Aufkonzentrierung der Substrate Aufkonzentrierung der Laktose (20%–45%), die hier als Substrat diente, in einem 5 mmol Kaliumphosphatpuffer. Die entsprechende Menge Laktose (200–450 g/l) wurde im Puffer gelöst und in 5 Liter-Gefäßen vorgelegt. Erhitzen der Laktoselösung bestehend aus Alpha-Laktose auf 93,5°C in einer Heizvorrichtung, wodurch eine Umlagerung der alpha- zur Beta-Laktose erfolgt, die eine höhere Löslichkeit besitzt, als Alpha-Laktose. Abkühlen der Substrate auf entsprechendes Temperaturoptimum des eingesetzten Enzyms (35–55°C)
• b) Zufuhr der hochkonzentrierten Laktose-Lösung in den 5 l Rührkesselreaktor
• c) Zufuhr der β-Galaktosidase zum enzymatischen Umsatz der Laktose-Lösung bezogen auf die Menge der Laktose, die kontinuierlich umgesetzt werden soll

The fermentation process using lactose takes place under the following conditions:

• a) Concentration of the substrates Concentration of the lactose (20% -45%), which served as substrate here, in a 5 mmol potassium phosphate buffer. The appropriate amount of lactose (200-450 g / l) was dissolved in the buffer and placed in 5 liter containers. Heating the lactose solution consisting of alpha-lactose to 93.5 ° C in a heating device, resulting in a rearrangement of the alpha to beta-lactose, which has a higher solubility, than alpha-lactose. Cooling of the substrates to a corresponding temperature optimum of the enzyme used (35-55 ° C)
• b) Supply of highly concentrated lactose solution in the 5 l stirred tank reactor
• c) supplying the β-galactosidase to the enzymatic conversion of the lactose solution based on the amount of lactose to be reacted continuously

The β-galactosidase, for example, comes from the following manufacturers:

• d) Rückhaltung des Enzyms und Filtration der entstehenden Produkte

The optimum reaction conditions in the reactor for the production of galactosyl oligosaccharides, when using different membranes and enzymes, are, for example:

• d) retention of the enzyme and filtration of the resulting products

In the membrane filtration unit, the enzyme is retained and returned to the reactor fed back (retentate), so again for the reaction available to stand. The resulting products, galactosyl oligosaccharides, Glucose and galactose as well as the unreacted lactose may be the Membrane happen and are in the filtrate (permeate)

The Filtration takes place over the membrane filtration unit, for example, PES 50 at a transmembrane pressure (TMP) of 1.5 bar. The retentate is returned to the reactor.

• e) Chromatographie des Filtrates und Trennung der Galactosyl-Oligosaccharide von Monosacchariden Die Galactosyl-Oligosaccharide werden mit Hilfe der SMB-Chromatographie von den Neben- und Ausgangsprodukten Glucose, Galactose und Laktose abgetrennt, aufkonzentriert und in einem Gefäß aufgefangen.
• f) Trocknung der Galactosyl-Oligosaccharide

The yield of galactosyl oligosaccharides can be up to 45% of the total amount of saccharides, depending on the enzyme source and substrate concentration. Example:

• e) Chromatography of the Filtrate and Separation of the Galactosyl Oligosaccharides from Monosaccharides The galactosyl oligosaccharides are separated from the by-products and starting products glucose, galactose and lactose by means of SMB chromatography, concentrated and collected in a vessel.
• f) Drying of the galactosyl oligosaccharides

The Galactosyl oligosaccharide solution is dried to powder.

1

reactor

2a

Intake from the substrate container to the reactor

2 B

Intake from membrane filtration unit to the reactor

2c

procedure from the reactor to the membrane filtration unit

3

measurement and control unit of the reactor

4

Membrane filtration unit

5

contraption for concentration of the substrate

6

Container with substratum

7

chromatography

9

Vessel with filtrate

Claims (20)

Process for the preparation of a concentrated solution of galactosyl-oligosaccharides in a bioreactor system combined with a membrane filtration unit and an evaporation device characterized by the steps: a) concentration of the substrates b) supply of the highly concentrated substrates into the bioreactor system c) supply of an enzyme to the enzymatic conversion of the substrates d Retention of the enzyme and filtration of the resulting products e) Separation of water by evaporation during the reaction f) Chromatography of the filtrate and separation of the galactosyl oligosaccharides from monosaccharides and Lactose g) Drying of the galactosyl oligosaccharides Method according to claim 1, characterized in that as a substrate lactose solution and / or whey is used. Method according to the previous Claims, characterized in that the concentration of lactose by Heating to 93.5 ° C and rearrangement of alpha to beta-lactose occurs. Method of the preceding claims, characterized that the concentration of whey by nanofiltration takes place. Method according to the previous Claims, characterized in that in the enzymatic reaction the isolated enzyme β-galactosidase or a β-galactosidase producing microorganism is used. Method according to the previous Claims, characterized in that the enzymatic reaction with β-galactosidase at 37 ° C takes place. Method according to the previous Claims, characterized in that the retention of the enzyme β-galactosidase in a membrane filtration unit takes place. Method according to the previous Claims, characterized in that for concentration of the products while the reaction water is removed by evaporation Method according to the previous Claims, characterized in that the chromatography of the filtrate and Separation of the galactosyl oligosaccharides of monosaccharides by SMB chromatography or annular chromatography or batch Chromatography with recycling of Intermediate fractions takes place. Method according to the previous claims characterized in that process parameters such as temperature and / or pH especially for concentrating the substrate individually be managed. Bioreactor system for producing a concentrated solution of Galactosyl oligosaccharides at least consisting of a device for concentration of the substrates, a reactor with measuring and control unit, is fed into the continuously concentrated substrate, a membrane filtration unit that retains enzyme and removes filtrate and a chromatography device wherein the concentration device the substrates upstream of the reactor and the reactor upstream of the chromatography apparatus is so that a continuous flow is ensured. Bioreactor system according to claim 11, characterized in that the measuring and control unit temperature, pH, feeds and residence times controls and regulates. Bioreactor system according to claim 11 and 12, characterized in that the membrane filtration unit an inorganic or organic ultrafiltration membrane. Bioreactor system according to claim 11 to 13, characterized in that the membrane filtration unit a ceramic membrane of bioinertem material. Bioreactor system according to claim 11 to 14, characterized in that the membrane filtration unit is steam sterilizable. Bioreactor system according to claim 11 to 15, characterized in that the chromatography device is a simulatedmoving-bed chromatography. Use of a bioreactor system according to claims 11 to 16 for the production a concentrated solution on galactosyl oligosaccharides. Use of a method according to claims 1 to 9 for the preparation of a concentrated solution Galactosyl oligosaccharides. Use of a bioreactor system according to claims 11 to 16 for the production of galactosyl oligosaccharides in powder form. Use of a method according to claims 1 to 9 for the production of galactosyl oligosaccharides in powder form