RU2797427C2 - Method for inhibiting the isomerization of a reducing saccharide during heat treatment - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method is proposed for inhibiting the isomerization of a reducing saccharide in an aqueous solution during heat treatment to sterilize an aqueous saccharide solution, which includes the step of acidifying an aqueous saccharide solution prior to heat treatment by adding at least one acid to an aqueous saccharide solution to pH from 1 to 6. Heat treatment involves heating an aqueous solution of saccharide to a temperature in the range of 115 to 150°C and maintaining this temperature for 60 minutes, or autoclaving an aqueous saccharide solution at 121.1°C for 15 to 30 minutes, or heating the aqueous solution to a temperature in the range of 130 to 150°C and maintaining this temperature for up to 5 minutes, or exposing the aqueous solution to a temperature in the range of 71.5 to 74°C for a period of 15 to 30 seconds. The reducing saccharide is lactose.

EFFECT: invention provides high efficiency in implementation.

8 cl, 6 dwg, 5 tbl, 3 ex

Description

The present invention relates to methods involving heat treatment of an aqueous solution containing at least one reducing saccharide. More specifically, this invention relates to methods for preventing the isomerization of a reducing saccharide in an aqueous solution by thermally treating an aqueous solution containing the reducing saccharide.

PRIOR ART

Maintaining a sterile environment is often a prerequisite for cell culture in biotech manufacturing processes. However, these processes are often prone to the growth of alien bacteria, fungi or viruses. Alien contamination has a serious impact on the production process, as it impairs productivity (reduced production efficiency due to degradation or modification of feedstock, desired product, or desired biomass, and long bioreactor shutdown periods to remove contamination), product quality, and product safety (through contamination of the final product due to the foreign growth itself and/or due to metabolites produced by undesirable microorganisms).

Prevention of alien growth on a biological product, as well as during its production, is challenging due to the ubiquitous nature of microorganisms and the many entry points for microbes into the manufacturing process. Various methods have been developed for sterilizing equipment (eg, the interior of a fermenter), growth media, and any additives to growth media that need to be used in a biotech manufacturing process.

Several methods are known for sterilizing compounds or compositions, and they are used to eliminate alien microorganisms from raw materials to be used in biotech manufacturing processes. Such methods include gas sterilization (for example, with ozone or ethylene oxide), radiation sterilization (for example, ultraviolet radiation or gamma radiation), bright light/pulsed light sterilization, and sterilizing filtration of liquids and solutions, in particular, liquids and solutions that are heat sensitive or contain at least one heat sensitive compound.

Heat treatment of any heat-resistant materials is known to be the most reliable and effective sterilization method, and moist heat is most often used to achieve heat sterilization. Moist heat (steam) sterilization involves exposing the components to be sterilized to pressurized steam for a period of time. Typical wet heat sterilization protocols that are used to sterilize equipment and raw materials are exposing them to high pressure saturated steam at 115°C - 140°C for about 3-60 minutes. These sterilization protocols may vary, depending on the bioburden and the nature of the raw material, solution or surface to be sterilized.

As such, damage to a component by an inappropriate sterilization process can affect the quality, safety, or productivity of the biotech manufacturing process, and thus should be avoided. Especially liquids can undergo different chemical reactions due to sterilization methods using heat, radiation or chemicals.

The complexity of such chemical reactions is illustrated by the heat treatment of milk, which is commonly used to reduce the microbial load or to inactivate enzymes in the milk and hence extend the shelf life of milk. Maillard reactions are known to occur, which affect the color and taste of milk, when it is traditionally sterilized at 121°C for 20 minutes. In addition, it is known that due to the heat treatment of milk, denaturation or inactivation of proteins or vitamins occurs, as well as reactions of aldosaccharides with amino acids or substances containing an amino group. In addition, it is also well known that lactose (4-O-β-D-galactopyranosyl-D-glucopyranose, CAS number: 63-42-3) - an important component of milk - isomerizes to lactulose (4-O-β-D- galactopyranosyl-D-fructofuranose, CAS number: 4618-18-2) due to heat treatment and further degraded to glucose and galactose, as well as to other degradation products such as acids. This type of isomerization is favored by basic pH and is also known as the Lobri de Bruijn-Alberd van Eckenstein transformation.

In an attempt to limit the undesirable effect of heating on the quality and composition of milk, alternative sterilization methods are used, such as "Ultra high temperature treatment", in which milk is subjected to a temperature of 140 ° C for a period of a couple of seconds to several minutes, or "low temperature pasteurization", at which the milk is heated to a temperature up to 74°C. However, heat-mediated adverse reactions also occur during these alternative sterilization methods, albeit to a lesser extent.

This could be further improved by adjusting the pH values of raw skimmed milk, having an initial pH of 6.70, to values from 6.59 to 6.72 before sterilizing it at 120° C. for 10 minutes. This resulted in 28% less lactulose production at pH 6.59 and 9% more lactulose production at pH 6.72 compared to lactulose production in the original milk.

In the biotechnological production of human milk oligosaccharides such as 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, 3'-sialyllactose, or 6'-sialyllactose, lactose is typically used as the initial acceptor molecules for further glycosylation steps leading to the production of the desired HMO (human milk oligosaccharide). To prevent foreign growth in the fermentation broth, the supplied lactose must be sterilized. However, the presence of lactulose in fermentation broth should be avoided as it is a known laxative that should not be present in Level 1 formula milk or any other nutritional product supplemented with these HMOs. In addition, lactulose could be used by HMO producing bacteria as an alternative acceptor molecule, thus leading to oligosaccharides that are not naturally present.

As an alternative to heat treatment, the lactose solution can be sterilized by filtration. Typically sterilizing filtration is subjected to solutions containing from 1 mm to 1 M lactose. However, the implementation of sterilization of a large amount of solution by filtration, which is necessary for production on an industrial scale, is expensive, time consuming and less reliable compared to heat sterilization. Therefore, a reliable, more convenient way to sterilize lactose without or with only a tiny amount of lactose converted to lactulose is needed.

This goal was achieved by a method in which the acidic pH of the lactose solution is adjusted before and/or during exposure to heating of the lactose solution, although the idea of acidifying the sugar solution before heat treatment can also apply to saccharides other than lactose.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method for inhibiting the isomerization of a reducing saccharide in an aqueous solution containing said reducing saccharide during heat treatment of said aqueous solution by acidifying the aqueous solution before and/or during its heat treatment.

In a second aspect, the present invention provides a heat-treated aqueous solution containing at least one reducing saccharide.

In a third aspect, the present invention provides the use of a heat-treated aqueous solution containing at least one reducing saccharide in the biotechnological production of a biological product.

In a fourth aspect, the present invention provides methods for producing a biological product using a thermally treated aqueous solution containing at least one reducing saccharide.

In a fifth aspect, the present invention provides a biological product obtained by biotechnological production using a heat-treated aqueous solution containing at least one reducing saccharide.

In another aspect, according to this invention, the use of a biological product produced by biotechnological production using a thermally treated aqueous solution containing at least one reducing saccharide, for the manufacture of a drug.

In another further aspect, the present invention provides a formulation comprising a biological product that has been biotechnologically produced using a heat-treated aqueous solution containing at least one reducing saccharide.

BRIEF DESCRIPTION OF GRAPHICS

FIG. 1 shows chromatograms of an aqueous solution containing lactose, (A) before heat sterilization and (B) after heat sterilization. The aqueous solution was not acidified before heat sterilization. FIG. 1B shows a chromatogram of various specific saccharides used as standards.

FIG. 2 shows chromatograms of an aqueous solution containing lactose, (A) before heat sterilization and (B) after heat sterilization. The aqueous solution was acidified prior to heat sterilization by adding sulfuric acid to this aqueous solution containing lactose. FIG. 2B shows the chromatogram of various specific saccharides used as standards.

DETAILED DESCRIPTION OF THE INVENTION

According to the first aspect, a method is provided for inhibiting the isomerization of a reducing saccharide in an aqueous solution containing said reducing saccharide (aqueous saccharide solution) during heat treatment of said aqueous saccharide solution, the method comprising the step of acidifying the aqueous saccharide solution before and/or during its heat treatment.

The term "reducing saccharide" as used herein refers to any sugar or saccharide that is capable of acting as a reducing agent because it has a free aldehyde group. This reducing saccharide includes monosaccharides, disaccharides and oligosaccharides. All monosaccharides are reducing sugars and can be classified into aldoses, which have an aldehyde group, and ketoses, which have a ketone group. Ketoses must first tautomerize to aldoses before they can act as reducing sugars. Disaccharides are formed from two monosaccharide residues, and oligosaccharides are formed from three to seven monosaccharide residues.

Disaccharides and oligosaccharides can be classified as either reducing or non-reducing. Reducing disaccharides like lactose and maltose have only one of their two anomeric carbons involved in a glycosidic bond, meaning that they can be converted to open chain forms with an aldehyde group.

In a further and/or alternative embodiment, the reducing saccharide is selected from the group consisting of aldoses, disaccharides and oligosaccharides.

The term "aldose" in the context of this document refers to monosaccharides that contain only one aldehyde group per molecule. Examples of aldoses are: D-(+)-glyceraldehyde, D-(-)-erythrose, D-(-)-threose, D-(-)-ribose, D-(-)-arabinose, D-(+)- xylose, D-(-)-lyxose, D-(+)-allose, D-(+)-altrose, D-(+)-glucose, D-(+)-mannose, D-(-)-gulose, D-(-)-idose, D-(+)-galactose and D-(+)-talose.

In a further and/or alternative embodiment, the disaccharide is selected from the group consisting of lactose, maltose, trehalose, cellobiose, chitobiose, coibiose, nigerose, isomaltose, sophorose, lamaribiose, gentibiose, turanose, matulose, palatinose, gentibiose, mannobioses, melibiose, chalk. ibiulose, rutinoses, rutinuloses and xylobioses.

The term "oligosaccharide" in the context of this document refers to saccharides consisting of 3, 4, 5, 6 or 7 monosaccharide residues and thus includes trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides and heptasaccharides.

To obtain an aqueous saccharide solution, a certain amount of at least one reducing saccharide is dissolved in a source of water. Said water may be selected from the group consisting of distilled water, bidistilled water, deionized water, ground water, river water, sea water, tap water, municipal water and water containing salts. The term "water containing salts" in the context of this document refers to an aqueous solution of one or more than one salt. In a further and/or alternative embodiment, the aqueous saccharide solution does not contain one or more selected from the group consisting of proteins, polypeptides, nucleic acids (such as DNA and/or RNA), and lipids (such as fatty acids, mono-, di- and/or triglycerides).

In one embodiment of the method, the aqueous saccharide solution is acidified to a pH of about 1 to about 6, preferably to a pH of about 2 to about 5, and more preferably to a pH of about 3 to about 4. A pH of about 3 to about 5 in an aqueous saccharide solution has been found to be particularly preferred since isomerization of the reducing saccharide is inhibited or even prevented, while the formation of degradation products of said reducing saccharide is negligible.

In a further and/or alternative embodiment, the aqueous saccharide solution is acidified by adding an acid to the aqueous saccharide solution. This acid may be any acid that does not lead to an undesirable chemical reaction with the reducing saccharide. An example of such an undesirable chemical reaction is the formation of mucic acid when nitric acid is added to an aqueous solution of lactose.

This acid may be selected from the group of organic acids and inorganic acids, provided that the inorganic acid is not nitric acid (or nitrous acid), if the reducing saccharide is galactose or galactose-containing saccharide, since the oxidation of galactose or galactose-containing compounds such as lactose , nitric acid leads to mucinic acid.

In a further and/or alternative embodiment, the at least one acid for acidifying the aqueous saccharide solution is an inorganic acid or a mineral acid. An inorganic acid is a suitable inorganic acid which, in an amount to be added to aqueous saccharide solutions, does not spontaneously react with the saccharide. For example, the addition of nitric acid to an aqueous solution of lactose can lead to the formation of mucic acid. The inorganic acid is preferably selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, boric acid, hydrofluoric acid, hydrobromic acid, perchloric acid, periodic acid and carbonic acid.

In one embodiment, in which the inorganic acid is carbonic acid, the aqueous saccharide solution may be acidified by gassing the aqueous saccharide solution with carbon dioxide in a pressurized container.

In a further and/or alternative embodiment, the at least one acid for acidifying the aqueous saccharide solution is an organic acid. The organic acid may be selected from the group consisting of monocarboxylic acids, dicarboxylic acids and tricarboxylic acids.

In a further and/or alternative embodiment, the monocarboxylic acid is selected from the group consisting of carboxylic acid, formic acid (methanoic acid), acetic acid (ethanoic acid), propionic acid (propanoic acid), butyric acid (butanoic acid), and valeric acid (pentanoic acid). acid), but is not limited to them.

In a further and/or alternative embodiment, the dicarboxylic acid is selected from the group consisting of oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, malic acid, fumaric acid, glutaconic acid, muconic acid and citraconic acid.

In a further and/or alternative embodiment, the tricarboxylic acid is selected from the group consisting of citric acid, isocitric acid, and aconitic acid.

Adjusting the pH of the saccharide solution to an acidic value allows heat treatment of the reducing saccharide in aqueous solution without or at least reduced isomerization of the reducing saccharide.

The term "heat treatment" as used herein encompasses heating or heating and/or maintaining an aqueous saccharide solution at an elevated temperature, i.e. above room temperature (21°C). Thermal treatment of the aqueous saccharide solution includes heating the aqueous saccharide solution after acidification and/or during acidification to a temperature of about 30°C, about 40°C, about 50°C, about 60°C, about 70°C, or even about 80°C. and also includes maintaining the aqueous saccharide solution at that temperature - possibly after it has been heated to even higher temperatures - for an extended period of time, i.e. for several hours or even days, such as, but not limited to, for about 20 hours, about 30 hours, about 40 hours, about 50 hours, about 60 hours, about 72 hours, or even longer.

Heating and/or maintaining an aqueous solution containing the reducing saccharide at such elevated temperatures without isomerization or with significantly reduced isomerization of the reducing saccharide offers numerous advantages, such as, for example, the possibility of dissolving more of the saccharide in a given amount of water, thereby increasing the concentration saccharide in the aqueous solution and reducing the volume of the aqueous saccharide solution to be added to the batch to obtain the desired final saccharide concentration. Additionally and/or alternatively, the viscosity of the aqueous saccharide solution can be lowered by heating/maintaining the aqueous saccharide solution at an elevated temperature, thereby facilitating circulation and/or pumping of the aqueous saccharide solution, for example through a membrane filter.

The term "heat treatment" as used herein also includes heating and/or maintaining an aqueous saccharide solution for some time at an elevated temperature which is suitable for sterilizing the aqueous saccharide solution. Therefore, "heat treatment" also includes heating an aqueous saccharide solution to a temperature in the range of from about 115°C to 150°C, but not limited to, and maintaining this temperature for up to about 60 minutes.

In one embodiment of the heat treatment to sterilize the aqueous saccharide solution, the aqueous saccharide solution is sterilized by autoclaving. Autocupping is one of the most important microbial destruction methods that uses saturated superheated steam. The condensation of steam on the object to be sterilized releases energy that causes irreversible damage to microorganisms. To this end, the internal volume of the autoclave is ventilated during the initial temperature increase. When this is done, atmospheric air is displaced from the internal volume and replaced by saturated, superheated steam. Ventilation occurs using a flow process or through fractional ventilation; as soon as ventilation is completed, the vent valve closes. This marks the start of the compensation time. After this period, each point of the object to be sterilized reaches the required temperature due to the saturated steam effect. After that, the actual sterilization phase begins. The duration of sterilization depends on both the microbial load and the sterilization temperature. Autocupping at 121.1°C (250°F) for 15 minutes to 30 minutes is considered standard. Vegetative forms, including prokaryotic and eukaryotic organisms, as well as viruses/bacteriophages, can usually be inactivated within minutes at temperatures of 65°C-100°C, while survival forms such as spores may need to be handled at temperatures up to 140°C. WITH. Prions require at least 30 minutes at 132°C-134°C and 300 kPa pressure to be inactivated or destroyed. The subsequent cooling phase, and thus the end of the autoclaving cycle, starts after the sterilization time.

In an alternative embodiment of the heat treatment to sterilize the aqueous saccharide solution, the aqueous saccharide solution is sterilized by a method referred to as "Ultra High Temperature Treatment", a continuous sterilization method that includes heating the aqueous solution to a temperature of 130°C-150°C with 140°C as the main point. The appropriate holding time may vary from 8 to 40 seconds, sometimes up to 5 minutes, depending on the properties of the solution to be sterilized.

In yet another embodiment of the heat treatment to sterilize the aqueous saccharide solution, the aqueous saccharide solution is subjected to high temperature/short time (HTST) pasteurization in which the solution is heated to a temperature of 71.5°C to 74°C, preferably to 72°C, for from about 15 seconds to about 30 seconds, and driven in a controlled continuous flow while undergoing said heat treatment at the same time.

In yet another embodiment of the heat treatment to sterilize the aqueous saccharide solution, the aqueous saccharide solution is subjected to "flash pasteurization" in which the aqueous saccharide solution is subjected to a temperature of 71.7° C. for 15 seconds.

Acidifying the aqueous solution of the reducing saccharide prior to subjecting the aqueous saccharide solution to any of these heat treatments and/or during its heat treatment to sterilize the aqueous saccharide solution inhibits or even prevents the isomerization of the reducing saccharide in the aqueous solution during its heat treatment.

Thus, according to the second aspect, there is provided a thermally treated aqueous solution containing at least one reducing saccharide obtained by the method according to the first aspect, i.e. a method for inhibiting isomerization of said reducing saccharide in an aqueous solution of said reducing saccharide, comprising acidifying the aqueous saccharide solution before and/or during thermal treatment of said aqueous saccharide solution.

The thermally treated aqueous solution containing at least one reducing saccharide, obtained by acidifying this aqueous saccharide solution before and/or during its thermal treatment, does not contain or contains at least smaller amounts of undesirable isomerization products of the specified at least one reducing saccharide compared to with a similar aqueous solution of the same reducing saccharide that has not been acidified to an identical heat treatment.

In one embodiment of the second aspect, the aqueous solution containing the reducing saccharide is a sterile aqueous solution. This sterile aqueous solution containing the reducing saccharide is prepared by the method of inhibiting the isomerization of said reducing saccharide as previously described herein, including thermally treating said aqueous saccharide solution to sterilize said aqueous saccharide solution. Thus, the aqueous solution was heat-sterilized.

In a further and/or alternative embodiment, the aqueous solution containing the reducing saccharide is at an elevated temperature, i. temperature of about 30°C, about 40°C, about 50°C, about 60°C, about 70°C, or even about 80°C, and contains a reducing saccharide in an amount that is higher than the amount of said reducing saccharide that can be dissolved in water at room temperature. For example, an aqueous solution is provided containing lactose at a concentration greater than or equal to 10 mM, preferably at a concentration greater than or equal to 100 mM, more preferably at a concentration greater than or equal to 0.66 M, most preferably at a concentration greater than or equal to 1 M if the temperature of the acidified aqueous lactose solution is maintained at from about 40°C to about 60°C.

An aqueous saccharide solution containing at least one reducing saccharide, such as, for example, lactose, in an amount that is higher than the amount of saccharide that can be dissolved in water at room temperature, may be a sterile aqueous lactose solution that has been sterilized through heat treatment for sterilization, as previously described herein, and provided cooling to the desired elevated temperature at which this sterile aqueous saccharide solution is maintained.

According to a third aspect of the present invention, the use of a thermally treated aqueous solution containing a reducing saccharide as described hereinbefore in the biotechnological production of a biological product is provided. Production of a biological product

In one embodiment, the use of a thermally treated aqueous solution containing a reducing saccharide in the biotechnological production of a biological product includes use in a biocatalytic production process.

It is understood that the term "biocatalytic production process" as used herein refers to a process for the production of a biological product in which one or more purified or isolated enzymes are brought into contact with one or more educts in an in vitro reaction to converting one or more educts to the desired biological product.

In an alternative embodiment, the use of a thermally treated aqueous solution containing a reducing saccharide includes use in an enzymatic manufacturing process. The term "enzymatic production process" in the context of this document refers to a process in which microorganisms are grown in a medium or broth in order to produce a biological product or a special product that is synthesized by microorganisms.

The use of a thermally treated aqueous solution containing at least one reducing saccharide, wherein said aqueous saccharide solution has been acidified as previously described herein, prior to and/or during thermal treatment of the aqueous saccharide solution, is beneficial in, among other things, that no or fewer undesired isomerization products of the reducing saccharide are present in the aqueous saccharide solution, and that no or less undesirable isomerization products are supplied to the biotechnological process compared to a similar aqueous saccharide solution that has not been acidified prior to its heat treatment. Thus, it is not necessary to remove unwanted isomerization products from the thermally treated aqueous saccharide solution before it can be used in a biotechnological production process.

According to a fourth aspect, the present invention provides methods for the biotechnological production of a biological product.

In one embodiment of a method for the biotechnological production of a biological product, the method is a biocatalytic production method. This method includes the following steps:

- providing at least one purified enzyme;

- contacting at least one purified enzyme with one or more educts in the presence of a thermally treated aqueous saccharide solution that has been acidified as described herein before, before and/or during thermal processing, with a reaction to convert one or more educts to the desired biological product; And

- purification of this biological product is possible.

In one embodiment, the at least one reducing saccharide of the aqueous saccharide solution is an extraction product of a biocatalytic manufacturing process.

In an alternative embodiment of the biotechnological production method, the method is an enzymatic production method.

The terms "fermentation" or "enzymatic" refer to the volumetric growth of microorganisms on or in a growth medium (fermentation broth) for the purpose of producing a particular chemical product - a "biological product". To this end, the cells of one or a limited number of strains of microorganisms are grown in a bioreactor (fermenter) under optimal conditions in order for these microorganisms to carry out the desired production with limited production of undesirable impurities. Environmental conditions inside the bioreactor, such as temperature, nutrient concentrations, pH, and dissolved gases (especially oxygen for aerobic fermentations), affect the growth and productivity of organisms and are therefore monitored, controlled, and adjusted if necessary.

Thus, the method of enzymatic production of a biological product includes the following steps:

- providing a cell that is capable of producing a biological product;

- culturing at least one cell in a fermentation broth containing and/or supplemented with a heat-treated aqueous solution containing at least one reducing saccharide, so that at least one cell produces a biological product; And

- it is possible to clean the biological product from the fermentation broth.

In one embodiment of the use according to the third aspect and/or the method according to the fourth aspect, said living cell is a prokaryotic cell or a eukaryotic cell. Suitable cells include yeast, bacteria, archaebacteria, fungi, insects, plant cells, and animal cells, including mammalian cells (such as human cells and cell lines).

In a further and/or alternative embodiment, the prokaryotic cell is a bacterial cell, preferably selected from a genus selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Enterococcus, Bifidobacterium, species of the genus Sporolactobacillus, species of the genus Micromomospora, species of the genus Micrococcus, species of the genus Rhodococcus and Pseudomonas. Suitable bacterial species are Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, Bacillus circulans, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum , Citrobacter freundii, Clostridium celiulolyticum, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum, Corynebacterium glutamicum, Enterococcus faecium, Enterococcus thermophiles, Escherichia coli, Erwinia herbicola (Pantoea agglomerans), Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus . homonas campestris.

In a further and/or alternative embodiment, the eukaryotic cell is a yeast cell, an insect cell, a plant cell, or a mammalian cell. The yeast cell is preferably selected from the group consisting of species of the genus Saccharomyces, in particular Saccharomyces cerevisiae, species of the genus Saccharomycopsis, species of the genus Pichia, in particular Pichia pastoris, species of the genus Hansenula, species of the genus Kluyveromyces, species of the genus Yarrowia, species of the genus Rhodotorula and species genus Schizosaccharomyces.

In one embodiment of the use according to the third aspect and/or the method according to the fourth aspect, said biological product is a human milk oligosaccharide. The human milk oligosaccharide may be selected from the group consisting of the following: 2'-fucosyl lactose, 3-fucosyl lactose, 2',3-difucosyl lactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto- N-fucopentaose I, lacto-N-neo-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neo-fucopentaose V, lacto-N-difucohexaose I, lacto-N -difucosylhexaose II, para-lacto-N-fucosylhexaose, fucosyl-lacto-N-sialylpentaose b, fucosyl-lacto-N-sialylpentaose c, fucosyl-lacto-N-sialylpentaose c, disialyl-lacto-N-fucopentaose, 3-fucosyl -3'-sialyllactose, 3-fucosyl-6'-sialyl lactose, lacto-N-neodifucohexaose I, 3'-sialyllactose, 6'-sialyllactose, sialyllacto-N-tetraoses LST-a, LST-b, LST-c and disialyllacto -N-tetraose.

In a further and/or alternative embodiment of the use according to the third aspect and/or the method according to the fourth aspect, said reducing saccharide is lactose. This embodiment has the particular advantage that lactose must be supplied to the living cell fermentation broth to produce the desired human milk oligosaccharide. With the ability to reduce or avoid the formation of lactulose during heat sterilization of an aqueous solution of lactulose by the method according to the first aspect, the need to remove lactulose from the HMO preparation is eliminated when the HMO preparation is to be used for the manufacture of a nutritional formula, especially a first level formula, medical nutrition or nutritional supplement.

The use of a heat-treated aqueous saccharide solution containing at least one reducing saccharide, wherein the aqueous saccharide solution has been acidified as previously described herein, prior to and/or during its heat treatment in an enzymatic manufacturing process provides further advantages. First, the present invention provides for the sterilization of an aqueous solution containing a reducing saccharide by heat sterilization methods without or with reduced isomerization of that reducing saccharide. Therefore, an aqueous solution containing a reducing saccharide should not be sterilized by sterilizing filtration, which is less reliable than heat sterilization (for example, with regard to the elimination of bacteriophages), in particular if large volumes of an aqueous saccharide solution, such as several cubic meters, are to be sterilized. . In addition, heat sterilization of large volumes is more economical than sterilizing filtration. Secondly, the present invention provides for the provision of aqueous saccharide solutions containing at least one reducing saccharide, wherein the concentrations of the at least one reducing saccharide are higher than the saturating concentration of the at least one reducing saccharide at room temperature, since the aqueous saccharide solution can be heat and maintain at an elevated temperature, i.e. at temperatures above room temperature. In addition, maintaining the aqueous saccharide solution at an elevated temperature reduces the viscosity of the aqueous saccharide solution, which in turn makes it easier to handle the aqueous saccharide solution, for example, by pumping the aqueous saccharide solution through a pipe or hose. Thirdly, the ability to provide an aqueous saccharide solution having an increased saccharide concentration allows for higher product yields in the enzymatic manufacturing process. This is because the volume of the fermenter is limited and the volume of the fermentation broth in the fermenter increases during the fermentation process due to the supply of, among others, an aqueous saccharide solution to the fermentation broth, the aqueous saccharide solution being required for the production of the desired biological product by the cultivated cells. The higher the concentration of saccharide in the aqueous saccharide solution, the smaller the volume of aqueous saccharide solution that must be added to the fermentation broth in order to obtain and/or maintain the desired concentration of at least one reducing saccharide in that fermentation broth. Thus, by using an aqueous saccharide solution according to the invention having an increased saccharide concentration in the fermentation broth, a higher saccharide concentration can be achieved in a given fermenter, or a given saccharide concentration can be maintained for a longer period of time, since the volume of the fermenter available for feeding is exhausted more slowly. . This, in turn, provides more reducing saccharide for the cells to produce the desired biological product and therefore increases the yield of the desired biological product that can be obtained in a single-batch fermentation. Therefore, the desired biological product can be obtained - at the end of the fermentation process - in an amount greater than or equal to 100 g/l in the fermentation broth, preferably in an amount greater than or equal to 150 g/l in the fermentation broth, more preferably in an amount greater than or equal to equal to 200 g/l in fermentation broth. For example, amounts of 2'-fucosyllactose greater than 100 g/l, namely about 150 g/l, were obtained using acidified 0.66 M aqueous lactose solution, which was sterilized by heat, and even higher yields can be obtained with further increase the concentration of lactose in the aqueous solution to be fed into the fermentation broth.

In yet another embodiment of the use according to the third aspect and/or the method according to the fourth aspect, at least one reducing saccharide is lactose and said biological product is selected from the group consisting of lactosucrose and lactobionic acid.

Thus, according to the fourth aspect, the present invention provides a biological product, which was obtained by one of the methods according to the fourth aspect.

In one embodiment, the biological product is a human milk oligosaccharide, preferably a human milk oligosaccharide, selected from the group consisting of the following: 2'-fucosyl lactose, 3-fucosyl lactose, N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-neo-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neo-fucopentaose V, lacto-N-difucohexaose I, lacto-N-difucosylhexaose II, para-lacto-N-fucosylhexaose, fucosyl-lacto-N-sialylpentaose b, fucosyl-lacto-N-sialylpentaose c, fucosyl-lacto-N-sialylpentaose c , disialyl-lacto-N-fucopentaose, 3-fucosyl-3'-sialyllactose, 3-fucosyl-6'-sialyl lactose, lacto-N-neodifucohexaose I, 3'-sialyllactose, 6'-sialyllactose, sialyllacto-N-tetraose LST -a, LST-b, LST-c and disialyl lacto-N-tetraose.

In an alternative embodiment, the biological product is selected from the group consisting of lactosucrose and lactobionic acid or derivatives of the aforementioned human milk oligosaccharides.

According to another aspect, this invention provides the use of a biological product for the manufacture of a drug.

In one embodiment of the use of a biological product for the manufacture of a formulation, the biological product is a human milk oligosaccharide selected from the group consisting of the following: 2'-fucosyl lactose, 3-fucosyl lactose, -N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-neo-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N- neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucosylhexaose II, lara-lacto-N-fucosylhexaose, fucosyl-lacto-N-sialylpentaose b, fucosyl-lacto-N-sialylpentaose c, fucosyl-lacto-N-sialylpentaose c, disialyl-lacto-N-fucopentaose, 3-fucosyl-3'-sialyllactose, 3-fucosyl-6'-sialyl lactose, lacto-N-neodifucohexaose I, 3'-sialyllactose, 6'-sialyllactose, sialyllacto-N-tetraose LST-a, LST-b, LST-c and disialyl lacto-N-tetraose. The formulation in this embodiment is selected from the group consisting of nutritional formulations, preferably a first level formula, clinical nutrition, and nutritional supplements.

Provided that the reducing saccharide is lactose, the isomerization inhibition method according to the first aspect can provide a heat-sterilized lactose solution with no or reduced amounts of lactulose for enzymatic production of human milk oligosaccharide. Said human milk oligosaccharide can then be used in the manufacture of a nutritional preparation, preferably a first level formula, that contains no or less epilactose, lactulose and/or a lactulose derivative such as fucosyllactulose (without the need to remove lactulose or its derivatives from the HMO preparation).

In another aspect, formulations are provided that contain at least one biological product that has been produced by a biotechnological manufacturing process as previously described herein. Said preparation is preferably selected from the group consisting of nutritional preparations, preferably a first level formula, medical nutrition and nutritional supplements.

The present invention will be described with respect to specific embodiments and with reference to the drawings, but the present invention is not limited thereto, but only by the claims. In addition, the terms first, second, and the like in the description and claims are used to distinguish between like elements, and not necessarily to describe a sequence, either in time or space, in ranking, or in any other way. It should be understood that the terms used in this manner are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein may operate in different sequences than those described or illustrated herein.

It should be noted that the term "comprising" used in the claims should not be interpreted as limited to the meanings listed below; it does not exclude other elements or steps. Thus, it should be interpreted as defining the presence of the claimed characteristics, integers, steps or components as they are referred to, but it does not preclude the presence or addition of one or more other characteristics, integers, steps or components. , or their groups. Thus, the scope of the expression "device comprising means A and B" should not be limited to devices consisting only of components A and B. It means that, with respect to the present invention, the only relevant components of the device are A and B.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular property, structure, or characteristic described in connection with a given embodiment is included in at least one embodiment of the present invention. Thus, the occurrences of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may refer to it. In addition, specific properties, structures, or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more than one embodiment.

Likewise, it should be understood that in describing exemplary embodiments of the invention, various features of the invention are sometimes grouped together in one embodiment, drawing, or description thereof for the purpose of streamlining the description of the invention and aiding in the understanding of one or more of the various inventive aspects. This mode of disclosure, however, should not be interpreted as suggesting that the claimed invention requires more features than are expressly listed in each claim. Rather, as the following claims reflect, inventive aspects are found in less than all of the features of a single embodiment disclosed hereinafter. Thus, the claims following the detailed description are thereby expressly incorporated into this detailed description, each claim having its own value as a separate embodiment of the present invention.

In addition, while some embodiments described herein include some but not other characteristics included in other embodiments, it is intended that combinations of characteristics of different embodiments are within the scope of this invention and form different embodiments, as would understood by those skilled in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

In addition, some of the embodiments are described herein as a method or combination of method elements that may be employed by a computer system processor or other means for performing a given function. Thus, the processor, with the necessary instructions for carrying out such a method or method element, forms a means for carrying out this method or method element. In addition, the device embodiment element described herein is an example of a means for carrying out the function performed by this element for the purpose of carrying out the invention.

Numerous specific details are set forth in the description and graphics provided herein. However, it is understood that embodiments of the present invention may be practiced without these specific details. In other cases, well-known methods, structures, and techniques have not been shown in detail in order not to interfere with the understanding of this description.

The present invention will now be described by way of a detailed description of several embodiments of the present invention. It is understood that other embodiments of the present invention may be arranged according to the knowledge of those skilled in the art without departing from the true spirit or technical idea of the present invention, the present invention being limited only by the terms of the appended claims.

Example 1 - Acidification of lactose with organic acids prior to heat sterilization

A 0.66 M lactose solution was prepared by dissolving 226 g of lactose in water. The final volume of this solution was 1 liter. Between 30° C. and 35° C., the pH was adjusted using 50% (w/v) citrate or 99% (v/v) acetic acid. This solution was then sterilized in a vertical autoclave (Systec VX-65, Linden, Germany) at 121°C for 20 minutes. Samples were taken before and after heat sterilization and kept frozen until analysis by high performance liquid chromatography (HPLC). HPLC was performed using a RID-10A refractive index detector (Shimadzu, Germany) and a Waters XBridge 3.5 µm (250 × 4.6 mm) amide column (Eschborn, Germany) connected to a Shimadzu HPLC system. Isocratic elution was performed using 30% solvent A (50% (v/v) acetonitrile in bidistilled water, 0.1% (v/v) NH ₄ OH) and 70% solvent B (80%- ny (v/v) acetonitrile in bidistilled water, 0.1% (v/v) NH ₄ OH) at 35°C and a flow rate of 1.4 ml min ^-1 . Samples were clarified by solid phase extraction on an ion exchange matrix (Strata ABW, Phenomenex). Ten microliters of sample (1:5 dilution) was applied to the column. Finally, the relative amount of sugars detected was determined. As shown in Tables 1 and 2, heat sterilization-induced isomerization of lactose decreased with decreasing pH values of solutions prior to heat treatment. The formation of lactulose could not be observed at pH 3.0 to 4.0 or pH 3.0 when acidifying with citrate or acetic acid, respectively. Acid-catalyzed degradation of lactose to its monosaccharides increased at lower pH values, but was not detected at pH 4.5.

Example 2 - Acidification of lactose with inorganic acids before heat sterilization A 0.66 M lactose solution was prepared by dissolving 226 g of lactose in water. The final volume of the solution was 1 liter. At a temperature of 30°C to 35°C, the pH was adjusted using 37% (v/v) hydrochloric acid, 50% (v/v) phosphoric acid, or 96% (v/v) vol.) sulfuric acid. This solution was then sterilized in a vertical autoclave (Systec VX-65, Linden, Germany) at 121°C for 20 minutes. Samples were taken before and after heat sterilization and kept frozen until analysis by high performance liquid chromatography (HPLC). HPLC was performed using a RID-10A refractive index detector (Shimadzu, Germany) and a Waters XBridge 3.5 µm (250 × 4.6 mm) amide column (Eschborn, Germany) connected to a Shimadzu HPLC system. Isocratic elution was performed using 30% solvent A (50% (v/v) acetonitrile in bidistilled water, 0.1% (v/v) NH ₄ OH) and 70% solvent B (80%- ny (v/v) acetonitrile in bidistilled water, 0.1% (v/v) NH ₄ OH) at 35°C and a flow rate of 1.4 ml min ^-1 . Samples were clarified by solid phase extraction on an ion exchange matrix (Strata ABW, Phenomenex). Ten microliters of sample (1:5 dilution) was applied to the column. Finally, the relative amount of sugars detected was determined. As shown in Tables 3, 4 and 5, heat sterilization-induced lactose isomerization was reduced due to increased acidification of solutions prior to heat treatment. The formation of lactulose could not be observed at pH from 3.0 to 3.5. In contrast, acid-catalyzed degradation of lactose to its monosaccharides increased at lower pH values but was absent at pH values of 4.0 to 4.5 or 4.0 to 5.0 when phosphoric acid and sulfuric or hydrochloric acid were used for acidification. acids, respectively.

The percentage of sugars (area under the curve - AUC) detected by HPLC is shown.

was carried out using 96% (v/v) sulfuric acid. The percentage of sugars (area under the curve - AUC) detected by HPLC is shown.

Example 3 Improved Process for the Production of 2'-fucosyllactose According to European Patent Application 16196486, a genetically modified strain of E. coli BL21 (DE3) ΔnagAb ΔwcaJ ΔfuclK ΔpfkA was used, overexpressing the enzymes for de novo synthesis of GDP-fucose (ManB, ManC, Gmd, WcaG), bifunctional Bacteroides fragilis L-fucokinase/L-fucose-1-phosphate guanyltransferase, wbgL 2-fucosyltransferase gene from E, coli:O126, lacY lactose permease gene, yberc0001_9420 sugar transporter from Yersinia bercovieri ATCC 43970, fructose-1,6-bisphosphate aldolase (fbaB) and heterologous fructose-1,6-bisphosphate phosphatase (fbpase) from Pisum sativum.

The strain E, coli was cultivated in a 3 l fermenter at 33°C in a mineral salt medium containing 3 g/l KH ₂ PO ₄ , 12 g/l K ₂ HPO ₄ , 5 g/l (NH ₄ ) ₂ SO ₄ , 0.3 g/l citric acid, 2 g/l MgSO ₄ × 7H ₂ O, 0.1 g/l NaCl and 0.015 g/l CaCl ₂ × 6H ₂ O with 1 ml/l trace element solution (54.4 g /l ammonium citrate Fe(III), 9.8 g/l MnCl ₂ × 4H ₂ O, 1.6 g/l CoCl ₂ × 6H ₂ O, 1 g/l CuCl ₂ × 2H ₂ O, 1.9 g /l H ₃ BO ₃ , 9 g / l ZnSO ₄ × 7H ₂ O, 1.1 g / l Na ₂ MoO ₄ × 2H ₂ O, 1.5 g / l Na ₂ SeO ₃ , 1.5 g / l NiSO ₄ x 6H ₂ O), 2% (v/v) glycerol as sole carbon and energy source, and 60 mM heat-sterilized lactose acidified to pH 3.0 with 96% (v/v) .) sulfuric acid before sterilization. The pH was maintained at 7.0 by titration with 25% ammonia. The fermenter was inoculated to OD ₆₀₀ (optical density at 600 nm) with a 0.1 preculture grown in the described medium but lactose-free. After leaving the feeding phase, indicated by an increase in dissolved oxygen, feeding with glycerol (60% v/v) was started, as well as 0.66 M lactose (acidified to pH 3.0 using 96% (v/v) lactose). /vol.) sulfuric acid before heat sterilization). The lactose concentration of 10-40 mM was maintained throughout the production phase of the fermentation process, adjusted according to HPLC analysis. Glycerol (60% v/v) was fed at flow rates of 6-8 ml/l/h (relative to initial volume). The fermentation was stopped when the maximum filling volume of the container was reached. At this point in the supernatant culture of the broth was determined by the titer of 2'-fucosyllactose 146 g/L.

Comparable titers of 2'-fucosyllactose were achieved during 2'-fucosyllactose fermentation processes using sterile-filtered lactose instead of acidified, heat-sterilized lactose. In addition, the type and amounts of by-products detected in the culture broth of fermentations carried out using sterile filtered or heat sterilized (acidified) lactose were comparable. None of the by-products such as lactulose, epilactose, fucosyllactulose or fucosyl epilactose, and other by-products that may result from the addition of heat-sterilized lactose, were detected in the broth when acidified, heat-sterilized lactose was allowed to ferment.

Claims (11)

1. A method for inhibiting the isomerization of a reducing saccharide in an aqueous solution containing said reducing saccharide during heat treatment to sterilize said aqueous saccharide solution, the method comprising the step of acidifying the aqueous saccharide solution prior to the heat treatment, wherein the aqueous saccharide solution is acidified by adding at least one acid in said aqueous saccharide solution to a pH value between 1 and 6; wherein the heat treatment comprises heating the aqueous saccharide solution to a temperature in the range of 115°C to 150°C and maintaining that temperature for up to 60 minutes, or autoclaving the aqueous saccharide solution at 121.1°C for a range of 15 to 30 minutes or heating the aqueous solution to a temperature in the range of 130 to 150°C and maintaining this temperature for up to 5 minutes, or exposing the aqueous solution to a temperature in the range of 71.5 to 74°C for a period of 15 to 30 seconds; and wherein the reducing saccharide is lactose. 2. The method of claim 1 wherein the aqueous saccharide solution is acidified to a pH of about 2 to about 5, and more preferably to a pH of about 3 to about 4. 3. The method according to claim 1 or 2, wherein the acid is an inorganic acid, preferably selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, boric acid, hydrofluoric acid, hydrobromic acid, perchloric acid, hydroiodic acid and carbonic acid. 4. The method according to claim 1 or 2, wherein the acid is an organic acid, preferably selected from the group consisting of monocarboxylic acids, dicarboxylic acids and tricarboxylic acids. 5. The use of a sterile aqueous solution containing at least lactose as a reducing saccharide, obtained by the method according to any one of paragraphs. 1-4 in a method for the biotechnological production of human milk oligosaccharide, said method comprising the following steps: - providing at least one cell that is capable of producing human milk oligosaccharide; - culturing at least one cell in a fermentation broth containing and/or supplemented with a sterile aqueous oligosaccharide solution, wherein lactose is in a sterile solution at a concentration greater than or equal to 0.66 M, so that at least one cell produces human oligosaccharide milk; And possibly purifying said human milk oligosaccharide from the fermentation broth. 6. Use according to claim 5, wherein the lactose is in a sterile aqueous solution at a concentration equal to or greater than 1M and the sterile aqueous solution is a wet heat sterilized aqueous solution. 7. Use according to claim 5 or 6, wherein the human milk oligosaccharide is obtained in an amount greater than or equal to 100 g/l, in a fermentation broth, preferably in an amount greater than or equal to 150 g/l, in a fermentation broth, more preferably in an amount , greater than or equal to 200 g/l, in the fermentation broth, at the end of the fermentation process. 8. Application according to any one of paragraphs. 5-7 wherein the human milk oligosaccharide is selected from the group consisting of the following: 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose , lacto-N-fucopentaose I, lacto-N-neo-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neo-fucopentaose V, lacto-N-difucohexaose I, lacto-N-difucosylhexaose II, para-lacto-N-fucosylhexaose, fucosyl-lacto-N-sialylpentaose b, fucosyl-lacto-N-sialylpentaose c, fucosyl-lacto-N-sialylpentaose c, disialyl-lacto-N-fucopentaose, 3-fucosyl-3'-sialyllactose, 3-fucosyl-6'-sialyllactose, lacto-N-neodifucohexaose I, 3'-sialyllactose, 6'-sialyllactose, sialyllacto-N-tetraoses LST-a, LST-b, LST- c and disialyl lacto-N-tetraose.

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