Method for producing crystalline lactose produced by biotechnological production process

Abstract

The present invention relates to a method for producing crystalline lactose, more particularly lactose obtained from a biotechnological production process.

Description

[0001] This invention relates to a method for producing crystalline lactose, and more particularly lactose obtained from a biotechnological production process.

[0002] Lactose, as a disaccharide, has diverse applications in the food and pharmaceutical industries (Hebbink GA, Dickhoff BHJ. Chapter 5 - Application of lactose in the pharmaceutical industry. In: Paques M, Lindner CBT-L, eds. Academic Press, 2019. pp. 175-229. Available at https: / / www.sciencedirect.com / science / article / pii / B9780128117200000052; Hettinga KA. Chapter 6 - Lactose in the dairy production chain. In: Paques M, Lindner CBT-L, eds. Academic Press, 2019. pp. 231-66. Available at https: / / www.sciencedirect.com / science / article / pii / B9780128117200000064). Currently, it is isolated from mammalian milk, primarily cow's milk. Due to the environmental impact of this production process, there is a need for alternative synthesis methods that can compete with well-established extraction methods from milk at a lower production cost.

[0003] Mammals synthesize lactose through lactose synthase or β-1,4-galactosyltransferase (which transfers galactose to glucose, with UDP-galactose as the donor substrate and glucose as the acceptor substrate). This principle has been modified in microorganisms, replicating the mammalian biosynthetic pathway (Mao Z, Shin HD, Chen RR. Engineering the E. coli UDP-glucose synthesis pathway for oligosaccharide synthesis. Biotechnol Prog [Internet]. Jan [cited in 2016 Mar 13], 22(2):369-74. available at http: / / www.ncbi.nlm.nih.gov / pubmed / 16599548), and the synthesizing enzyme has previously been applied in vitro to synthesize lactose by adding expensive UDP-galactose as a donor (Lau K, Thon V, Yu H, Ding L, Chen Y, Muthana MM et al., Highly efficient chemoenzymatic synthesis of [small beta]1-4-linked galactosides with promiscuous bacterial [small beta]1-4-galactosyltransferases. Chem Commun [Internet]. 2010, 46(33):6066-8. (This can be found at http: / / dx.doi.org / 10.1039 / C0CC01381A).

[0004] One challenge with these methods is the energy requirement for lactose synthesis. Synthesizing UDP-galactose from UDP-glucose requires UTP, which must either be added to the reaction mixture or synthesized by the microbial host. After lactose synthesis, UDP is released; it is either lost as a substrate or recycled back to UTP via ATP (another expensive biochemical intermediate). Furthermore, galactose needs to be added or synthesized, which is currently still derived from lactose, and no alternative synthetic methods have been identified.

[0005] One challenge is purifying lactose from biosynthetic environments containing products not naturally present in lactose isolated from mammalian milk (e.g., culture medium components, buffer components, substrates, precursors, cells, proteins, DNA, lipids, cell debris, enzymes, chemicals, defoamers, etc.). Among these are, in particular: sugars such as glucose, fructose, UDP-glucose, UDP-galactose, galactose, and sucrose; salts such as phosphate buffers, sulfates, ammonia, magnesium, and calcium; proteins; and cells or cell components, which typically interfere with the purification of biotechnologically produced lactose. These impurities can inhibit nucleation and crystal growth, and the impurities themselves can potentially be incorporated into the crystals, thus affecting the purity of the final product. Therefore, purifying lactose from biotechnological processes to a commercially acceptable purity is far from straightforward.

[0006] From a purely energy and cost perspective, even though it is more environmentally friendly, existing technologies cannot compete with lactose extraction and production. Invention Overview

[0008] The objective of this invention is to provide a method for producing biotechnologically generated crystalline lactose, which solves the problem of separating such lactose with high purity and at a reasonable production cost. This invention provides a novel method for efficiently producing lactose, wherein the lactose is preferably first separated from its biotechnological production environment and then crystallized. According to the invention, this and other objectives are achieved by providing a method for producing crystalline lactose, wherein the lactose is produced via a biotechnological production process, wherein the method includes steps of drying and / or crystallizing the lactose.

[0009] Further benefits of the teachings of this invention will become apparent to those skilled in the art upon reading this invention.

[0010] definition

[0011] The vocabulary used in this specification to describe the invention and its various embodiments should be understood not only in its commonly defined meaning, but also, through the specific definitions herein, to include structures, materials, or actions that extend beyond their commonly defined meanings. Therefore, if an element can be understood to include more than one meaning in the context of this specification, its use in the claims must be understood to be superior to all possible meanings supported by this specification and by the vocabulary itself.

[0012] The various aspects and embodiments of the invention disclosed herein should be understood not only in the order and context specifically described herein, but also in any order and any combination thereof. Each embodiment identified herein may be combined together unless otherwise stated. All publications, patents, and patent applications mentioned herein are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated herein in its entirety by reference. Unless otherwise stated, all words used in the singular shall be considered to include the plural, and vice versa. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures described herein in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's instructions.

[0013] Embodiments of the invention have been disclosed in this specification, and although specific terminology has been used, these terms are for descriptive purposes only and not for limiting purposes. The scope of the invention is set forth in the following claims. It must be understood that illustrative embodiments have been described for purposes of example only and should not be construed as limiting the invention. It will be apparent to those skilled in the art that changes, other embodiments, modifications, details, and uses may be made consistent with the wording and spirit of the disclosure herein and within the scope of this disclosure, the scope of which is limited only by the claims and interpreted in accordance with patent law, including the doctrine of equivalents. In the following claims, reference characters used to designate claim steps are provided for ease of description only and are not intended to imply any particular order of performing the steps unless otherwise specified.

[0014] Throughout this application, unless otherwise expressly stated, the features “synthesize” and “produce” are used interchangeably. Throughout this application, unless otherwise expressly stated, the expression “capable of…” is preferably replaced with the active voice of the verb, and vice versa. For example, the expression “capable of expressing” is preferably replaced with “expressing,” and vice versa, i.e., “expressing” is preferably replaced with “capable of expressing.” Throughout this document and in its claims, the verbs “comprising,” “having,” and “including,” and their inflections, are used in their non-limiting sense to mean items included following the word, but do not exclude items not specifically mentioned. The verb “consistently of…” means that the solution or composition defined herein may contain additional components besides those specifically identified, which do not alter the distinctive features of the invention. These additional compounds may be unavoidable byproducts, such as those generated during the production of the lactose of this invention, and compounds introduced into the process stream (from which lactose is recovered) but cannot be removed therefrom. Regarding spray-dried powders, the term "consistently composed of" includes spray-dried powders containing at least 80%-wt, at least 85%-wt, at least 90%-wt, at least 93%-wt, at least 95%-wt, or at least 98%-wt of lactose with respect to the dry matter of the spray-dried powder. The term "consistently composed of" is similarly used with respect to spray-dried powders, process streams, and solutions containing lactose. Throughout this document and in its claims, unless otherwise specified, the verbs "comprising," "having," and "including," and their variations, may preferably be replaced by "consisting of" (and its variations) or "consistently composed of" (and its variations), and vice versa. Furthermore, reference to an element by the indefinite article "a" or "an" does not preclude the possibility of the presence of more than one of the elements, unless the context explicitly requires the presence of only one element. Therefore, the indefinite article "a" or "an" often means "at least one (kind)." Throughout the document and in the claims, unless otherwise expressly stated, the articles “a” and “an” are preferably replaced with “at least one,” more preferably with “at least two,” even more preferably with “at least three,” even more preferably with “at least four,” even more preferably with “at least five,” even more preferably with “at least six,” and most preferably with “at least two.” When used in association with a numerical value (e.g., “about 10”) or with a range (e.g., “about x to approximately y”), the term “about” or “approximately” preferably means that the value or range is interpreted as being as accurate as the method used to measure it. Unless a specific error limit is specified, when used in association with a numerical value, the expression “about” or “approximately” is interpreted as having the same rounding as the given value.Throughout this document and its claims, unless otherwise stated, the expression "x to y" (where x and y represent numerical values) refers to a range of values, where x is the lower limit of the range and y is the upper limit of the range. In this document, x and y are also included within the range.

[0015] Throughout this application, unless otherwise expressly stated, the term "β-1,4-galactosyltransferase" should be understood to mean an enzyme capable of transferring galactose from UDP-galactose to glucose to form lactose. Such β-1,4-galactosyltransferases are preferably lactose synthases. An example of such galactosyltransferases is from Neisseria meningitidis (…). Neisseria meningitidis The N-acetylglucosamine β-1,4-galactosyltransferase GalT (Uniprot ID Q51116, sequence version 2023_02, May 3, 2023).

[0016] The term "biotransformation" should be understood as the transformation of products by resting cells or permeabilized cells.

[0017] Throughout the application, unless otherwise expressly stated, the terms “UDP-glucose-4-epimerase” and “UDP-galactose-4-epimerase” are used interchangeably.

[0018] The terms “glucose isomerase,” “xylose isomerase,” and “fructose isomerase” are used in this field to refer to enzymes that convert fructose into glucose, and vice versa.

[0019] Protein or peptide sequence and functional information can be provided by a wide range of resources on protein sequence and annotation data, such as the Universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49(D1), D480-D489). UniProt contains an expertly and richly managed protein database called the UniProtKnowledgebase (UniProtKB), along with UniProtReference Clusters (UniRef) and the UniProt Archive (UniParc). The UniProt identifier (UniProtID) is unique for each protein present in the database. Throughout the application, the peptide sequence is represented by the UniProt ID. Unless otherwise stated, the UniProt ID of the described protein corresponds to its sequence version 01 present in the UniProt database (www.uniprot.org) version release 2023_02 on May 3, 2023.

[0020] Those skilled in the art will understand that, for the databases used herein, the content of each database is fixed at each publication and will not be changed. When the content of a particular database is changed, that particular database accepts a new release version with the new publication date. All release versions of each database, with their respective publication dates and specific content noted on those specific publication dates, are available and known to those skilled in the art. Brief description of the attached diagram

[0022] Figure 1 The diagram shows the production of lactose using sucrose as a raw material or carbon source.

[0023] Figure 2 The production of lactose using trehalose as a carbon source or raw material is shown.

[0024] Figure 3 The diagram shows the production of lactose using sucrose and glucose as raw materials or carbon sources. Invention Details

[0026] In a first aspect, the present invention provides a method for producing crystalline lactose, wherein the lactose is produced via a biotechnological production process, wherein the method includes steps of drying and / or crystallizing the lactose.

[0027] In a preferred embodiment of the process for producing lactose by a biotechnological means, the process is a fermentation process, a biotransformation process, or an enzymatic process. The fermentation process used in this invention can be a microbial fermentation process, but it can also be a cell culture fermentation process.

[0028] The cells used in such fermentation or biotransformation processes are selected from a list consisting of microorganisms, plant cells, animal cells, insect cells, or protozoan cells, more preferably microorganisms, more preferably bacteria or yeast, even more preferably bacteria, and even more preferably cells belonging to the genus Escherichia ( ). Escherichia ) or Bacillus spp. ( Bacillus More preferably, the cells are bacteria belonging to the genus *Escherichia*, and even more preferably, the cells are *Escherichia coli*. Escherichia coli More preferably, the cells are Escherichia coli K-12 strain, and most preferably, the cells are Escherichia coli MG1655.

[0029] Throughout the application and claims, unless otherwise specified, the microorganism is preferably bacteria, yeast or fungus, more preferably bacteria or yeast, and most preferably bacteria.

[0030] Throughout the application and claims, unless otherwise specified, the bacteria preferably belong to the phylum Proteobacteria, Firmicutes, Cyanobacteria, or Deinococcus-Thermus. The bacteria belonging to Proteobacteria preferably belong to the family Enterobacteriaceae, and more preferably to the species *Escherichia coli*. Escherichia coli The bacteria preferably refer to any strain belonging to the species *Escherichia coli*, such as, but not limited to, *Escherichia coli* B, *Escherichia coli* C, *Escherichia coli* W, *Escherichia coli* K12, and *Escherichia coli* Nissle. More particularly, the bacteria refer to cultured *Escherichia coli* strains – named *Escherichia coli* K12 strains – which are well adapted to laboratory environments and, unlike wild-type strains, have lost their ability to proliferate in the gut. Well-known examples of *Escherichia coli* K12 strains are K12 wild-type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111, and AA200. Therefore, the present invention preferably specifically relates to *Escherichia coli* K12 strains, more preferably *Escherichia coli* MG1655 strains. The bacteria belonging to the phylum *Cladotomyces* preferably belong to the genus *Bacillus*, preferably from the genus *Bacillus* (…). Bacillus Bacillus species, such as Bacillus subtilis ( Bacillus subtilis ) or Bacillus amyloliquefaciens ( B. amyloliquefaciens The bacteria belonging to the phylum Actinobacteria preferably belong to the family Corynebacteriaceae, which includes the member Corynebacterium glutamicum (…). Corynebacterium glutamicum ) or non-fermenting Corynebacterium ( C. afermentans ), or belong to the family Streptomycetaceae, which has the member Streptomyces griseus ( Streptomyces griseus ) or Streptomyces freundii ( S. fradiae ).

[0031] Throughout the application and claims, unless otherwise specified, the yeast cells preferably belong to the phylum Ascomycota, Basidiomycota, Deuteromycota, or Zygomycetes. The yeast cells preferably belong to the genus *Saccharomyces*. Saccharomyces (Has members such as brewer's yeast) Saccharomyces cerevisiae ), saccharin yeast ( S. bayanus ), Blakely yeast ( S. boulardii ), Pichia pastoris ( Pichia (Has members such as Pichia pastoris) Pichia pastoris ), abnormal Pichia pastoris ( P. anomala ), Klufer Pichia pastoris ( P. kluyveri ), genus *Komagata* ( Komagataella ), Hansenula genus ( Hansunella Kluyveromyces ( ) Kluyveromyces (Has members such as Kluyveromyces lactis) Kluyveromyces lactis ), Max Kluyveromycin ( K. marxianus ), heat-resistant Kluyveromycin ( K. thermotolerans ), Yersinia ( Yarrowia (For example, Yersinia lipophila) Yarrowia lipolytica )), Pseudomonas ( Eremothecium ), Zygosaccharomyces ( Zygosaccharomyces ), Stamozymes ( Starmerella (For example, bumblebee Stamozyme) Starmerella bombicola )) or Debali yeast ( Debaromyces The yeast cells are more preferably selected from Pichia pastoris, Yersinia lipolytica, Saccharomyces cerevisiae, and Kluyveromyces lactis.

[0032] Throughout the application and claims, unless otherwise specified, the fungus preferably belongs to the genus Rhizopus (Rhizopus). Rhizopus ), genus *Dendrocalamus* Dictyostelium ), Penicillium ( Penicillium Mucor ( ) Mucor ) or Aspergillus ( Aspergillus ).

[0033] Throughout this application and claims, unless otherwise specified, plant cells include cells of flowering and non-flowering plants, as well as algal cells, such as Chlamydomonas and Chlorella. Preferably, the plant cells are tobacco, alfalfa, rice, cotton, rapeseed, tomato, corn, maize, or soybean cells.

[0034] Throughout the application and claims, unless otherwise specified, the animal cells are preferably derived from non-human mammals (e.g., cattle, bison, pigs, sheep, mice, rats), birds (e.g., chickens, ducks, ostriches, turkeys, pheasants), fish (e.g., swordfish, salmon, tuna, sea bass, trout, catfish), invertebrates (e.g., lobsters, crabs, shrimp, clams, oysters, mussels, sea urchins), reptiles (e.g., snakes, alligators, turtles), amphibians (e.g., frogs), or insects (e.g., flies, nematodes), or genetically modified cell lines derived from human cells (excluding embryonic stem cells). The human and non-human mammalian cells are preferably selected from a list consisting of the following: epithelial cells, such as mammary epithelial cells, embryonic kidney cells (e.g., HEK293 or HEK 293T cells), fibroblasts, COS cells, Chinese hamster ovary (CHO) cells, mouse myeloma cells, such as N20, SP2 / 0 or YB2 / 0 cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, such as those described in WO21067641.

[0035] Throughout the application and claims, unless otherwise specified, the insect cells are preferably derived from the fall armyworm (Pterocarya stenoptera). Spodoptera frugiperda (e.g., Sf9 or Sf21 cells), silkworm ( Bombyx mori ), cabbage moth ( Mamestra brassicae ), Powdered Noctuid moth ( Trichoplusia ni (e.g., BTI-TN-5B1-4 cells) or Drosophila melanogaster ( Drosophila melanogaster (e.g., Drosophila S2 cells).

[0036] Throughout the application and claims, unless otherwise specified, the protozoan cell is preferably Leishmania lizarda ( Leishmania tarentolae )cell.

[0037] In another preferred embodiment, the cells used in the biotechnology production process according to the present invention are single cells. More preferably, the cells used in the biotechnology production process according to the present invention are isolated cells.

[0038] In a preferred embodiment, when the process is a fermentation or bioconversion process, a biomass removal step follows the fermentation or bioconversion. Preferably, such a biomass removal step includes at least one of centrifugation, microfiltration, and / or ultrafiltration.

[0039] Lactose produced via a biotechnological process can be generated by using i) a synthase and ii) uridine diphosphate (UDP) for the formation of UDP-glucose. In such a process, UDP is preferably added in a catalytic amount, more preferably at a concentration of 0.1 mM to 10 mM. In an alternative embodiment, UDP is preferably added in a stoichiometric amount. Preferably, such a process further includes using a UDP-glucose-4-epimerase to convert the UDP-glucose to UDP-galactose. Alternatively or preferably, the UDP-galactose is a galactose donor for the formation of lactose by β1,4-galactosyltransferase.

[0040] Alternatively or preferably, the process may further include using β1,4-galactosyltransferase to convert glucose and UDP-galactose to form lactose and UDP. Preferably, the β1,4-galactosyltransferase uses glucose as an acceptor and UDP-galactose as a donor. In such a process, preferably, the UDP released by the β1,4-galactosyltransferase is recycled for use by the synthase to form UDP-glucose. Thus, the generated UDP can in turn be used in a reaction catalyzed by sucrose synthase.

[0041] In a preferred embodiment of the process for biotechnologically producing lactose, the synthase used also forms glucose or fructose. Preferably, in the process for biotechnologically producing lactose in which the synthase also forms fructose, the fructose is converted into glucose.

[0042] In a preferred embodiment of the process for producing lactose in a biotechnological manner, the synthase is sucrose synthase or trehalose synthase.

[0043] Preferably, the sucrose synthase is selected from the GT4 glycosyltransferase subfamily, or from the KEGG enzyme class EC2.4.1.13. Alternatively, the sucrose synthase is selected from the list of the following: from soybean ( Glycine maxThe sucrose synthase (Uniprot ID P13708, sequence version 2023_02 from May 3, 2023) is from Arabidopsis thaliana ( Arabidopsis thaliana The sucrose synthase (Uniprot ID P49040, sequence version 2023_02, May 3, 2023) from a species of the genus Anabaena ( Anabaena Sucrose synthase (Uniprot ID Q9ZEV2, sequence version 2023_02, May 3, 2023) of sp. PCC 7119 (ATCC number 29151), from *Thiobacillus thermophilus* (sp.) ... is derived from *Thiobacillus thermophilus* (sp.) 7119 (ATCC number 29151). Acidithiobacillus caldus Sucrose synthase from ATCC 51756 (Uniprot ID A0A059ZV61, sequence version 2023_02, May 3, 2023), or containing sucrose synthase from soybean (Uniprot ID P13708, sequence version 2023_02, May 3, 2023), sucrose synthase from Arabidopsis thaliana (Uniprot ID P49040, sequence version 2023_02, May 3, 2023), sucrose synthase from Anabaena species PCC 7119 (ATCC number 29151) (Uniprot ID Q9ZEV2, sequence version 2023_02, May 3, 2023), or sucrose synthase from Thiobacillus thermophilus ATCC 51756 (Uniprot ID Q9ZEV2, sequence version 2023_02, May 3, 2023). Any protein sequence that has 80% or more sequence identity with any of the sequences in IDA0A059ZV61 (sequence version 2023_02 of May 3, 2023) and has sucrose synthase activity.

[0044] Preferably, the trehalose synthase is selected from the GT4 glycosyltransferase subfamily, or from the KEGG enzyme class EC2.4.1.245. Alternatively, the trehalose synthase is selected from the list of the following: from Thermococcus Bergensis (… Thermococcus bergensis Trehalose synthase from strain T7324 (Uniprot ID Q7LYW5, sequence version 2023_02 from May 3, 2023), and from *Porciniella horikoshi* (… Pyrococcus horikoshiiTrehalose synthase (Uniprot IDO58762, sequence version 2023_02 of May 3, 2023) from strain ATCC700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3, or trehalose synthase (Uniprot ID Q7LYW5, sequence version 2023_02 of May 3, 2023) from *Thermococcus Bergensis* (strain T7324) or trehalose synthase (Uniprot ID) from *Porcine horikoshi* (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3). Any protein sequence that has 80% or more sequence identity with any of O58762 (sequence version 2023_02 of May 3, 2023) and has trehalose synthase activity.

[0045] Preferably, the UDP-glucose-4-epimerase is selected from enzyme class EC 5.1.3.2, or from the list of the following: GalE from Escherichia coli K-12 MG1655 (Uniprot ID P09147, sequence version 2023_02, May 3, 2023), from Bifidobacterium longum subsp. longum (… Bifidobacterium longum subsp. longum GalE (Uniprot ID E8MF10, sequence version 2023_02, May 3, 2023) is from a species of the genus *Thermophyton* (*Thermophyton*). Pyrobaculum GalE (Uniprot ID A0A0K1E1Q5, sequence version 2023_02, May 3, 2023) of sp. WP30, is from Campylobacter jejuni subsp. ( Campylobacter jejuni subsp. jejuniGalE of serotype O:2 (Uniprot ID Q0P9C3, sequence version 2023_02, May 3, 2023), or containing GalE from *Escherichia coli* K-12 MG1655 (Uniprot ID P09147, sequence version 2023_02, May 3, 2023), GalE from *Bifidobacterium longum* subsp. *longum* (Uniprot ID E8MF10, sequence version 2023_02, May 3, 2023), GalE from *Thermoplasia* species WP30 (Uniprot ID A0A0K1E1Q5, sequence version 2023_02, May 3, 2023), or GalE from *Campylobacter jejuni* subsp. *jejuni* serotype O:2 (Uniprot ID...). Any protein sequence that has 80% or more sequence identity with any one of Q0P9C3 (sequence version 2023_02 of May 3, 2023) and has UDP-glucose-4-epimerase activity.

[0046] Preferably, the β1,4-galactosyltransferase is lactose synthase, or is selected from enzyme class EC 2.4.1.87, GalT from Neisseria meningitidis (Uniprot ID Q51116, sequence version 2023_02, May 3, 2023), or Pasteurella multocida (…). Pasteurella multocida GalT of M1404 (UniProt IDD0EAD4 sequence version 2023_02 dated May 3, 2023), or having GalT containing the same as that from Neisseria meningitidis (UniprotID Q51116, sequence version 2023_02 dated May 3, 2023) or the same as that from Pasteurella multocida ( Pasteurella multocida Any protein whose sequence has 80% or more sequence identity with GalT (UniProt ID D0EAD4 sequence version 2023_02, May 3, 2023) of M1404.

[0047] Preferably, the glucose isomerase is selected from the list of the following: from Streptomyces griseus ( Streptomyces murinus XylA (Uniprot ID P37031, sequence version 2023_02, May 3, 2023) is from a species of the genus Arthrobacter ( Arthrobacter XylA (Uniprot ID P12070, sequence version 2023_02, May 3, 2023) of sp. (strain NRRL B3728) is from ethanol-producing anaerobic bacteria ( Thermoanaerobacter ethanolicusXylA (Uniprot ID D2DK62, sequence version 2023_02, May 3, 2023), or any protein having a sequence containing 80% or more sequence identity with any one of the following: XylA from *Streptomyces gravidarum* (Uniprot ID P37031, sequence version 2023_02, May 3, 2023), XylA from a species of *Arthrobacter* (strain NRRL B3728) (Uniprot ID P12070, sequence version 2023_02, May 3, 2023), or XylA from *Aerobic ethanol-producing thermophilic bacteria* (Uniprot ID D2DK62, sequence version 2023_02, May 3, 2023) and having glucose isomerase activity.

[0048] In a preferred embodiment of the process for producing lactose in a biotechnological manner, the carbon source for conversion to lactose by the synthase is selected from the list of: sucrose, trehalose, a combination of sucrose and glucose, and even starch.

[0049] In a preferred embodiment of the process for biotechnologically producing lactose, the process uses a synthase, specifically sucrase synthase, and sucrose is used to initiate the conversion. The sucrose is converted to UDP-glucose and fructose using the sucrase synthase. The resulting UDP-glucose is then converted to UDP-galactose. The fructose is converted to glucose, and then the UDP-galactose and glucose are converted to lactose. Figure 1 An exemplary implementation of this can be found in [the text].

[0050] In an alternative preferred embodiment of the process for biotechnologically producing lactose, the process uses a synthase, specifically trehalose synthase, and trehalose is used to initiate the conversion. The trehalose is converted to UDP-glucose and glucose using the trehalose synthase. The resulting UDP-glucose is converted to UDP-galactose, which is then converted to lactose along with the glucose. Figure 2 An exemplary implementation of this can be found in [the text].

[0051] In another alternative preferred embodiment of the process for biotechnologically producing lactose, the process uses a synthase, specifically sucrose synthase, and initiates the conversion using sucrose and glucose. The sucrose is converted to UDP-glucose and fructose using the sucrose synthase. The resulting UDP-glucose is then converted to UDP-galactose. It is not necessary to convert fructose to glucose because glucose is also present as a carbon source. The UDP-galactose and glucose are then converted to lactose. Figure 3An exemplary implementation of this can be found in the text, in which the carbon source is underlined.

[0052] In a further preferred embodiment of the process for the biotechnological production of lactose, the process is the enzymatic production of lactose, using i) sucrose, ii) trehalose, or iii) sucrose and glucose as raw materials or carbon sources. Lactose is then produced by using i) sucrose, ii) trehalose, or iii) sucrose and glucose as carbon sources or raw materials, and the reaction is catalyzed by a combination of multiple enzymes. In one preferred embodiment, all enzymatic reactions are carried out in a single-tank manner. In another preferred embodiment, some enzymatic reactions are carried out separately from other reactions in the method.

[0053] Another process for producing lactose biotechnically can be found in WO2020048927, WO23121564, CN116496963, CN116064346 or CN117551713.

[0054] In a more preferred embodiment of the process for biotechnologically producing lactose, when the process is enzymatic, at least one of the enzymes is immobilized. Preferably, the at least one enzyme is immobilized on a support, or immobilized by encapsulation, adsorption, or covalent binding. The enzyme is immobilized by well-known techniques, such as by encapsulation (“encapsulated immobilized enzyme”), wherein the enzyme is encapsulated in, for example, collagen, gelatin, cellulose triacetate, polyacrylamide, and / or 1-carrageenan; by binding (“bound immobilized enzyme”); by adsorption (“adsorbed bound immobilized enzyme”), wherein the enzyme is adsorbed onto polysaccharide derivatives, synthetic polymers, and porous glass; and by covalent binding (“covalent immobilized enzyme”), wherein the enzyme is bound to glutaraldehyde, diazonium diisocyanate, and hexamethylene diisocyanate (see, for example, Rafiq Khan M, Bulletin of the National Research Centre 45, 207 (2021)).

[0055] In an alternative or more preferred embodiment of the process for biotechnologically producing lactose, when the process is an enzymatic process as described herein, at least one reaction is carried out between 50°C and 80°C, preferably between 55°C and 70°C.

[0056] In a more preferred embodiment of the process for biotechnologically producing lactose, when the process is a fermentation process or a biotransformation, the enzymatic conversion is carried out by at least one enzyme produced by the cell that synthesizes the enzyme. Preferably, such a process is carried out at a temperature of about 25°C to about 50°C, more preferably about 25°C to about 40°C.

[0057] In an alternative, more preferred embodiment of the process for producing lactose in a biotechnological manner, when the process is a fermentation process, the lactose is produced by cells that have been genetically modified to produce all the enzymes required for lactose production.

[0058] According to a preferred embodiment of the invention, preferably, the lactose crystallization step is followed by a drying step. The lactose crystals can then be dried using band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, vacuum drum drying, roller drying, vacuum roller drying, and other types of drying.

[0059] According to a preferred embodiment of the present invention, the method described herein includes the steps of drying and / or crystallizing the lactose, preferably the drying step including any one or more of the following: spray drying, freeze drying, evaporation, precipitation, spray freeze drying, freeze spray drying, strip drying, belt drying, vacuum strip drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying, vacuum roller drying, and stirred film drying.

[0060] According to a preferred embodiment of the present invention, the process for producing lactose in a biotechnological manner further includes a step of purifying the lactose.

[0061] In a preferred aspect, the present invention provides a method for producing crystalline lactose, wherein the lactose is produced via a biotechnological production process, wherein the lactose is purified by at least one of the following purification steps: microfiltration, centrifugation, ultrafiltration, nanofiltration, ion exchange, simulated moving bed (SMB), color removal, carbon source removal, removal of sucrose, glucose, or trehalose by adding yeast, removal of trehalose by adding trehalase, removal of sucrose by adding invertase; and preferably further comprising a concentration step, a drying step, and / or a crystallization step as described herein. Preferably, the ion exchange step is a cation and / or anion exchange step. In a preferred embodiment, the lactose is purified from a liquid produced by the biotechnological production process comprising sucrose, glucose, trehalose, fructose, and / or glycerol.

[0062] According to a preferred embodiment of the invention, the purity of lactose before purification is < 70%, < 60%, < 50%, < 40%, < 30%, < 20%, < 10% based on total solids, and / or the purity at the end of the method including purification is > 80% based on dry solids, preferably > 85% based on dry solids, more preferably > 90% based on dry solids, even more preferably > 95% based on dry solids, even more preferably > 97% based on dry solids, even more preferably > 98% based on dry solids, and most preferably > 99% based on dry solids.

[0063] In a further preferred embodiment, the purification of lactose produced by enzymatic means includes the steps of microfiltration or ultrafiltration to recover the used enzyme.

[0064] In a further preferred embodiment, the cells obtained from the fermentation or biotransformation after purification of the fermentation or biotransformation medium are reused in a further fermentation or biotransformation process.

[0065] In a further preferred embodiment of the invention, the biotechnology production process described herein may include: i) a step of nanofiltration to remove / reduce monosaccharides and / or salts; and / or ii) a step of ion exchange to remove charged materials.

[0066] According to the invention described herein, the purified lactose preferably has an ash content of less than 1% based on total solids, more preferably less than 0.5% based on total solids. According to an alternative or further preferred embodiment, the purified lactose has one or more of the following:

[0067] a) Lead content less than 0.1 mg / kg solids, preferably less than 0.02 mg / kg solids;

[0068] b) Arsenic content less than 0.2 mg / kg solids, preferably less than 0.02 mg / kg solids;

[0069] c) Cadmium content less than 0.1 mg / kg solids, preferably less than 0.01 mg / kg solids; or

[0070] d) Mercury content less than 0.5 mg / kg solids, preferably less than 0.1 mg / kg solids.

[0071] In another preferred embodiment of the invention, the purified lactose has at least one or more of the following: i) a protein content of less than 100 mg / kg dry solids, ii) a DNA content of less than 10 ng / g dry solids, and iii) an endotoxin content of less than 10,000 EU / g dry solids.

[0072] Therefore, the present invention provides a crystalline lactose product, wherein the lactose product is derived from a biotechnological production process. Preferably, the lactose is produced by any of the biotechnological production processes described herein. More preferably, the biotechnological production process is an enzymatic process, a biotransformation process, or a fermentation process.

[0073] In a preferred embodiment, the lactose product described herein is lactose monohydrate.

[0074] In a preferred embodiment, the lactose product described herein has a purity of at least 90%, preferably at least 94%, based on dry weight.

[0075] In another or alternative preferred embodiment, the lactose product described herein does not contain milk proteins. Such milk proteins may be derived from milk proteins from cattle, horses, sheep, goats, bison, etc.

[0076] Based on the description, implementation schemes and examples in this invention, all other implementation schemes that can be obtained by those skilled in the art without inventive effort fall within the protection scope of this invention.

[0077] In summary, the process for producing lactose according to the present invention is completely different from known processes for producing lactose, and the final product lactose can be used in industry, such as in food.

[0078] In one embodiment, the crystalline lactose product obtained as described herein can be used in medicine, preferably for the prevention or treatment of gastrointestinal disorders.

[0079] This invention provides the use of crystalline lactose obtained as described herein in food or feed preparations, in dietary supplements, in cosmetic ingredients, or in pharmaceutical ingredients.

[0080] Furthermore, the present invention provides the use of the crystalline lactose described herein in food or feed preparations, in dietary supplements, in cosmetic ingredients, or in pharmaceutical ingredients.

[0081] The present invention provides for the uses described herein, wherein the food is human food, preferably infant food and / or infant formula or infant supplement.

[0082] Furthermore, the present invention provides the use of crystalline lactose as an additive in food, preferably as an additive in human food and / or pet food, and more preferably as an additive in human infant food.

[0083] The present invention provides for the uses described herein, wherein the feed is pet food, animal milk substitute, veterinary product, post-weaning feed or creep feed.

[0084] Further advantages can be obtained from specific implementation schemes and embodiments. It goes without saying that the features mentioned above and those to be explained below can be used not only in their respective specified combinations, but also in other combinations or independently, without departing from the scope of the invention.

[0085] This invention relates to the following specific embodiments:

[0086] 1. A method for producing lactose, characterized in that the method comprises using i) a synthase and ii) UDP for the formation of UDP-glucose.

[0087] 2. The method according to embodiment 1, wherein UDP is provided in a catalytic amount, preferably UDP is provided at a concentration of 0.1 mM to 10 mM.

[0088] 3. The method according to embodiment 1, the method further comprising using UDP-glucose-4-epimerase to convert the UDP-glucose into UDP-galactose.

[0089] 4. The method according to any one of embodiments 1 or 2, wherein the UDP-galactose is a galactose donor for the formation of lactose by β1,4-galactosyltransferase.

[0090] 5. The method according to any one of embodiments 1 to 3, the method further comprising using β1,4-galactosyltransferase to convert glucose and UDP-galactose to form lactose and UDP.

[0091] 6. The method according to any one of embodiments 1 to 5, wherein the UDP released by β1,4-galactosyltransferase is recycled for use by the synthase to form UDP-glucose.

[0092] 7. The method according to any one of embodiments 1 to 6, wherein the synthase further forms glucose or fructose.

[0093] 8. The method according to any one of embodiments 1 to 7, wherein the synthase is sucrose synthase or trehalose synthase.

[0094] 9. The method according to any one of embodiments 1 to 8, wherein the β1,4-galactosyltransferase uses glucose as an acceptor and UDP-galactose as a donor.

[0095] 10. The method according to any one of embodiments 8 to 9, wherein the fructose is converted into glucose.

[0096] 11. The method according to any one of embodiments 1 to 10, wherein the method is a microbial fermentation process, a biotransformation process, or an enzymatic process.

[0097] 12. The method according to any one of embodiments 1 to 11, wherein the method is an enzymatic process, and wherein at least one of the enzymes is immobilized, preferably the enzyme is immobilized on a carrier, or immobilized by encapsulation, adsorption or covalent binding.

[0098] 13. The method according to any one of embodiments 1 to 12, wherein the method is an enzymatic process, and wherein the reaction is carried out between 50°C and 80°C, preferably between 55°C and 70°C.

[0099] 14. The method according to any one of embodiments 1 to 11, wherein the method is a microbial fermentation process or biotransformation, characterized in that the enzymatic transformation is carried out by at least one enzyme, said at least one enzyme being produced by a cell that synthesizes said enzyme.

[0100] 15. The method according to any one of embodiments 1 to 11, wherein the method is a microbial fermentation process, characterized in that the lactose is produced by cells that have been genetically modified to produce all the enzymes required for lactose production.

[0101] 16. The method according to any one of the foregoing embodiments, wherein the carbon source used for conversion by the synthase is selected from the list of: sucrose, trehalose, and a combination of sucrose and glucose.

[0102] 17. The method according to any one of embodiments 11 to 13, used for enzymatically producing lactose using i) sucrose, ii) trehalose or iii) sucrose and glucose as raw materials or carbon sources, characterized in that lactose is produced by using i) sucrose, ii) trehalose or iii) sucrose and glucose as raw materials or carbon sources, and the reaction is catalyzed by a combination of multiple enzymes.

[0103] 18. The method according to any one of embodiments 1 to 17, wherein the synthase is trehalose synthase, and wherein i) trehalose is converted into UDP-glucose and glucose using the trehalose synthase, and ii) the UDP-glucose is converted into UDP-galactose, and iii) glucose and UDP-galactose are converted into lactose.

[0104] 19. The method according to any one of embodiments 1 to 17, wherein the synthase is a sucrose synthase, and wherein i) sucrose is converted into UDP-glucose and fructose by the sucrose synthase, ii) the UDP-glucose is converted into UDP-galactose, and iii) the fructose is converted into glucose, and iv) the UDP-galactose from step ii) and the glucose from step iii) are converted into lactose.

[0105] 20. The method according to any one of embodiments 1 to 17, wherein the synthase is a sucrose synthase and the carbon source is sucrose and glucose, wherein i) sucrose is converted into UDP-glucose and fructose by the sucrose synthase, ii) the UDP-glucose is converted into UDP-galactose, and iii) the UDP-galactose from step ii) and the glucose provided together with sucrose are converted into lactose.

[0106] 21. The method according to any one of embodiments 4, 5 or 9, wherein the β1,4-galactosyltransferase is lactose synthase.

[0107] 22. The method according to any one of the foregoing embodiments, wherein the method further includes the step of separating the lactose.

[0108] 23. The method according to any one of the foregoing embodiments, wherein the method further includes the step of purifying the lactose.

[0109] 24. A method for purifying lactose synthesized enzymatically or biotechnically, wherein the lactose is purified by a purification step selected from the following: microfiltration, centrifugation, ultrafiltration, nanofiltration, ion exchange, preferably cation and / or anion exchange, simulated moving bed, color removal, removal of sucrose, glucose or trehalose by adding yeast, removal of trehalose by adding trehalase, removal of sucrose by adding invertase, and preferably further comprising a concentration step, a spray drying step and / or a crystallization step.

[0110] 25. The method according to any one of the foregoing embodiments, wherein the method includes the step of drying or crystallizing the lactose, preferably the drying step includes any one or more of the following: spray drying, freeze drying, evaporation, precipitation, spray freeze drying, freeze spray drying, strip drying, belt drying, vacuum strip drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying, vacuum roller drying, and stirred film drying.

[0111] 26. The method according to any one of embodiments 23 to 25, wherein the purity of lactose before purification is < 70%, < 60%, < 50%, < 40%, < 30%, < 20%, < 10% based on total dry solids, and / or the purity at the end of the method including purification is > 80% based on total dry solids, preferably > 85% based on total dry solids, more preferably > 90% based on total dry solids, even more preferably > 95% based on total dry solids, even more preferably > 97% based on total dry solids, even more preferably > 98% based on total dry solids, and most preferably > 99% based on total dry solids.

[0112] 27. The method according to any one of embodiments 23 to 26, wherein the purification of lactose synthesized by enzymatic means includes the steps of microfiltration or ultrafiltration to recover the used enzyme.

[0113] 28. The method according to any one of embodiments 23 to 27, wherein the purification of lactose synthesized by biotechnology includes the steps of centrifugation, microfiltration and / or ultrafiltration to remove biomass.

[0114] 29. The method according to any one of embodiments 23 to 28, comprising i) a step of nanofiltration to remove / reduce monosaccharides and / or salts; and / or ii) a step of ion exchange to remove charged materials.

[0115] 30. The method according to any one of embodiments 23 to 29, wherein the purified lactose has an ash content of less than 1% on total dry solids, preferably less than 0.5% on total dry solids, and preferably has one or more of the following:

[0116] a) Lead content less than 0.1 mg / kg dry solids, preferably less than 0.02 mg / kg dry solids;

[0117] b) Arsenic content less than 0.2 mg / kg dry solids, preferably less than 0.02 mg / kg dry solids;

[0118] c) A cadmium content of less than 0.1 mg / kg dry solids, preferably less than 0.01 mg / kg dry solids; or

[0119] d) Mercury content less than 0.5 mg / kg dry solids, preferably less than 0.1 mg / kg dry solids.

[0120] 31. The method according to any one of embodiments 23 to 30, wherein the purified lactose has at least one or more of the following: i) a protein content of less than 100 mg / kg dry solids, ii) a DNA content of less than 10 ng / g dry solids, and iii) an endotoxin content of less than 10,000 EU / g dry solids.

[0121] The present invention relates to the following particular preferred embodiments:

[0122] 1. A method for producing crystalline lactose, wherein the lactose is produced via a biotechnological production process, wherein the method includes steps of drying and / or crystallizing the lactose.

[0123] 2. The method according to preferred embodiment 1, wherein the lactose is purified by a purification step selected from the following: microfiltration, centrifugation, ultrafiltration, nanofiltration, ion exchange, simulated moving bed, color removal, removal of sucrose, glucose, or trehalose by adding yeast, removal of trehalose by adding trehalase, removal of sucrose by adding invertase; and preferably further comprising a concentration step; preferably, the ion exchange step is a cation and / or anion exchange step, and / or a mixed-bed ion exchange step.

[0124] 3. The method according to any one of the foregoing preferred embodiments, wherein the biotechnology production process is a fermentation process, a biotransformation process, or an enzymatic process.

[0125] 4. The method according to preferred embodiment 3, wherein the fermentation or bioconversion is followed by a biomass removal step.

[0126] 5. The method according to preferred embodiment 3, wherein the biotechnology production process is an enzymatic process, and wherein at least one enzyme is immobilized.

[0127] 6. The method according to any one of preferred embodiments 1 to 3 or 5, wherein the biotechnology production process is an enzymatic process, and wherein the reaction is carried out between 50°C and 80°C, preferably between 55°C and 70°C.

[0128] 7. The method according to any one of preferred embodiments 1 to 4, wherein the biotechnology production process is a fermentation process or a biotransformation process, wherein the fermentation process or biotransformation process is characterized in that the enzymatic transformation is carried out by at least one enzyme, said at least one enzyme being produced by the cell that synthesizes said enzyme.

[0129] 8. The method according to preferred embodiment 7, wherein the fermentation process or biotransformation process is carried out at a temperature of about 25°C to about 50°C, preferably about 25°C to about 40°C.

[0130] 9. The method according to any one of preferred embodiments 1 to 4, 7 or 8, wherein the biotechnological production process is a fermentation process, characterized in that the lactose is produced by cells that have been genetically modified to produce all the enzymes necessary for lactose production.

[0131] 10. The method according to any one of the foregoing preferred embodiments, wherein the lactose is purified from a liquid produced by the biotechnological production process comprising sucrose, glucose, trehalose, fructose and / or glycerol.

[0132] 11. The method according to any one of preferred embodiments 2 to 10, wherein the purity of lactose before purification is < 70%, < 60%, < 50%, < 40%, < 30%, < 20%, < 10% based on total solids, and / or the purity at the end of the method including purification is > 80% based on dry solids, preferably > 85% based on dry solids, more preferably > 90% based on dry solids, even more preferably > 95% based on dry solids, even more preferably > 97% based on dry solids, even more preferably > 98% based on dry solids, and most preferably > 99% based on dry solids.

[0133] 12. The method according to any one of preferred embodiments 2, 3, 5, 6, 10 or 11, wherein the purification of lactose produced by enzymatic means includes the step of microfiltration or ultrafiltration to recover the used enzyme.

[0134] 13. The method according to any one of preferred embodiments 2 to 4, 7 to 10, wherein the purification of the lactose produced by fermentation or bioconversion process includes the steps of centrifugation, microfiltration and / or ultrafiltration to remove biomass.

[0135] 14. The method according to any one of preferred embodiments 2 to 4, 7 to 11 or 13, wherein the cells removed from the fermentation or biotransformation are reused in a further fermentation or biotransformation process.

[0136] 15. The method according to any one of preferred embodiments 1 to 14, wherein the biotechnology production process comprises: i) a step of nanofiltration to remove / reduce monosaccharides and / or salts; and / or ii) a step of ion exchange to remove charged materials.

[0137] 16. The method according to any one of preferred embodiments 1 to 15, wherein the lactose has an ash content of less than 1% based on total solids, preferably less than 0.5% based on total solids, and preferably has one or more of the following:

[0138] a) Lead content less than 0.1 mg / kg solids, preferably less than 0.02 mg / kg solids;

[0139] b) Arsenic content less than 0.2 mg / kg solids, preferably less than 0.02 mg / kg solids;

[0140] c) Cadmium content less than 0.1 mg / kg solids, preferably less than 0.01 mg / kg solids; or

[0141] d) Mercury content less than 0.5 mg / kg solids, preferably less than 0.1 mg / kg solids.

[0142] 17. The method according to any one of preferred embodiments 1 to 16, wherein the lactose has at least one or more of the following: i) a protein content of less than 100 mg / kg dry solids, ii) a DNA content of less than 10 ng / g dry solids, and iii) an endotoxin content of less than 10,000 EU / g dry solids.

[0143] 18. The method according to any one of preferred embodiments 1 to 17, wherein the lactose crystals are dried by strip drying, strip drying, vacuum strip drying, vacuum strip drying, drum drying, vacuum drum drying, roller drying, vacuum roller drying and other types of drying.

[0144] 19. The method according to any one of preferred embodiments 1 to 17, wherein the drying step comprises any one or more of the following: spray drying, freeze drying, evaporation, precipitation, spray freeze drying, freeze spray drying, strip drying, belt drying, vacuum strip drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying, vacuum roller drying, and stirred film drying.

[0145] 20. A crystalline lactose product, wherein the lactose product is derived from a biotechnology production process, preferably produced by any one of preferred embodiments 1 to 19.

[0146] 21. The lactose product according to preferred embodiment 20, wherein the lactose is lactose monohydrate.

[0147] 22. The lactose product according to any one of preferred embodiments 20 or 21, wherein the lactose has a purity of at least 90% by dry weight, preferably at least 94% by dry weight.

[0148] 23. The lactose product according to any one of preferred embodiments 20 to 22, wherein the lactose contains no milk protein.

[0149] 24. The lactose product according to any one of preferred embodiments 20 to 23, wherein the lactose has at least one or more of the following: i) a protein content of less than 100 mg / kg dry solids, ii) a DNA content of less than 10 ng / g dry solids, and iii) an endotoxin content of less than 10,000 EU / g dry solids.

[0150] 25. Lactose according to any one of preferred embodiments 20 to 24, wherein the lactose has an ash content of less than 1% based on total solids, preferably less than 0.5% based on total solids, and preferably has one or more of the following:

[0151] a) Lead content less than 0.1 mg / kg solids, preferably less than 0.02 mg / kg solids;

[0152] b) Arsenic content less than 0.2 mg / kg solids, preferably less than 0.02 mg / kg solids;

[0153] c) Cadmium content less than 0.1 mg / kg solids, preferably less than 0.01 mg / kg solids; or

[0154] d) Mercury content less than 0.5 mg / kg solids, preferably less than 0.1 mg / kg solids.

[0155] 26. The crystalline lactose product according to any one of preferred embodiments 20 to 25, for use in medicine, preferably for use in the prevention or treatment of gastrointestinal disorders.

[0156] 27. Use of crystalline lactose obtained according to any one of the preferred embodiments 1 to 19 in food or feed preparations, in dietary supplements, in cosmetic ingredients or in pharmaceutical ingredients.

[0157] 28. Use of crystalline lactose according to any one of preferred embodiments 20 to 25 in food or feed preparations, in dietary supplements, in cosmetic ingredients or in pharmaceutical ingredients.

[0158] 29. The use according to any one of preferred embodiments 27 or 28, wherein the food is human food, preferably infant food and / or infant formula or infant supplement.

[0159] 30. The use of crystalline lactose according to any one of preferred embodiments 20 to 25 as an additive in food, preferably as an additive in human food and / or pet food, and more preferably as an additive in human infant food.

[0160] 31. The use according to any one of preferred embodiments 27 or 28, wherein the feed is pet food, animal milk substitute, veterinary product, post-weaning feed or pen supplement.

[0161] Example

[0162] Example 1: Materials and Methods

[0163] Analytical analysis

[0164] Standards, such as but not limited to sucrose, lactose, glucose, fructose, UDP-glucose, and UDP-galactose, were purchased from Sigma, Carbosynth (UK), Elicityl (France), and IsoSep (Sweden). Other compounds were analyzed using in-house prepared standards.

[0165] Carbohydrates were analyzed on a Waters Acquity H-class UPLC with either an evaporative light scattering detector (ELSD) or a refractive index (RI) detector. A 0.7 μL sample volume was injected onto a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm; 130 Å; 1.7 μm) equipped with an Acquity UPLC BEH Amide VanGuard column (130 Å, 2.1 x 5 mm). The column temperature was 50 °C. The mobile phase consisted of ¼ water and ¾ acetonitrile solution with 0.2% triethylamine added. The method was solvent-constant, with a flow rate of 0.130 mL / min. The ELS detector was set to a drift tube temperature of 50 °C, an N2 gas pressure of 50 psi, a gain of 200, and a data transfer rate of 10 pps. The RI detector temperature was set to 35 °C.

[0166] Ash content

[0167] Ash content is a measure of the total amount of minerals present in a food or ingredient, such as oligosaccharides, while mineral content is a measure of the amount of a specific inorganic component (e.g., Ca, Na, K, Mg, phosphates, sulfates, and Cl) present in a food. The determination of ash and mineral content in foods or oligosaccharides is important for many reasons. Nutrition labeling: The concentration and type of minerals present often must be specified on the label of a food or ingredient, such as oligosaccharides. The quality of many foods depends on the concentration and type of minerals they contain, including their taste, appearance, texture, and stability. Microbiological stability: High mineral content is sometimes used to delay the growth of certain microorganisms. Nutrition: Some minerals are essential for a healthy diet (e.g., calcium, phosphorus, potassium, and sodium), while others can be toxic (e.g., lead, mercury, cadmium, and aluminum). Processing: Knowing the mineral content of a food / product during processing is often important because it affects the physicochemical properties of the food or ingredient, such as oligosaccharides.

[0168] Ash is the inorganic residue remaining after water and organic matter have been removed by heating in the presence of an oxidizing agent, providing a measure of the total amount of minerals in food. Analytical techniques used to provide information about total mineral content are based on the fact that minerals (analytes) can be distinguished from all other components (matrix) in food in some measurable way. The most widely used methods are based on the fact that minerals are not destroyed by heating and that they have low volatility compared to other food components. The three main types of analytical procedures used to determine the ash content of food are based on this principle: dry ashing, wet ashing, and low-temperature plasma dry ashing. The method chosen for a particular analysis depends on the reason for performing the analysis, the type of food or ingredient being analyzed, and the available equipment. Ashing can also be used as the first step in preparing a sample for analyzing a specific mineral (by atomic spectrometry or the various conventional methods described below).

[0169] For sample preparation, samples whose composition is representative of the components are selected to ensure that their composition does not change significantly before analysis. For example, dried oligosaccharide samples are often hygroscopic, and the selected samples should be kept in dry conditions to avoid absorbing moisture. Typically, 1–10 g of sample is used in ash content analysis. The solid components are finely ground and then carefully mixed to facilitate the selection of a representative sample. Samples with high moisture or solution content are usually dried before ash analysis to prevent splashing during ashing. Other potential problems include sample contamination by minerals in the grinder, glassware, or crucible that come into contact with the sample during analysis. For the same reason, deionized water is used when preparing samples and in blank samples.

[0170] The dry ashing process uses a muffle furnace capable of maintaining temperatures between 500 and 600°C. Water and other volatile materials are vaporized, and organic matter is burned into CO2, H2O, and N2 in the presence of oxygen in the air. Most minerals are converted into oxides, sulfates, phosphates, chlorides, or silicates. While most minerals have relatively low volatility at these high temperatures, some are volatile and may be partially lost, such as iron, lead, and mercury. For these minerals, ICP-MS analysis of the products is more suitable for quantification.

[0171] Food samples are weighed before and after ashing to determine the concentration of ash present. Ash content can be expressed on a dry basis by dividing the mass of the ashed material, ingredient, or food by the mass of the dry material, ingredient, or food before ashing. Multiplying by 100 gives the percentage of ash in the material, ingredient, or food. In a similar manner, the percentage of wet ash can be determined for liquid products, where the mass of the liquid before and after ashing is used instead of the mass of the dry material, ingredient, or food.

[0172] Heavy metal determination

[0173] For each of the following elements, a robust and universal inductively coupled plasma mass spectrometry (ICP-MS)-based method was used for detection and quantification: arsenic (As), selenium (Se), cadmium (Cd), tin (Sn), lead (Pb), silver (Ag), palladium (Pd), platinum (Pt), mercury (Hg), molybdenum (Mo), sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), zinc (Zn), manganese (Mn), phosphorus (P), and selenium (Se).

[0174] Nitric acid (≥ 65%, Sigma-Aldrich) was used for microwave digestion and standard / sample preparation. All dilutions were performed using 18.2 MΩ·cm (Millipore, Bedford, MA, USA) deionized water (DIW). Approximately 0.2 g of each oligosaccharide, component, or sample was digested in 5 mL of HNO3 using the following microwave digestion (CEM, Mars 6) procedure: 15 min ramp time and 15 min hold time (at 100 W and 50 °C), followed by 15 min ramp time and 20 min hold time (at 1800 W and 210 °C). The samples were cooled for 30 min after digestion. 1. The fully digested samples were then diluted to 50 mL with DIW.

[0175] Analysis was performed using a standard Agilent 7800 ICP-MS, including the fourth-generation ORS cup system, which effectively controls polyatomic interferences using helium collision mode (He mode). The ORS uses He to control polyatomic interferences to reduce the propagation of all common matrix-based polyatomic interferences. Kinetic energy discrimination (KED) was used to separate smaller, faster analyte ions from larger, slower interfering ions. All elements, except Se, were measured in He mode at a flow rate of 5 mL / min. Se was measured in high-energy He (HEHe) mode using a cup gas flow rate of 10 mL / min. The 7800 ICP-MS was equipped with a standard sample introduction system consisting of a MicroMist glass concentric nebulizer, a quartz spray chamber, a quartz torch with a 2.5 mm inner diameter injector, and a nickel interface cone. The ICP-MS operating conditions are: 1550 W RF power, 8 mm sampling depth, 1.16 l / min nebulizer gas, automatically adjusted lens tuning, 5 or 10 ml / min helium gas flow, and 5 V KED.

[0176] Quantitative analysis of dry matter and moisture content

[0177] The dry matter content of oligosaccharides was determined using a Sartorius MA150 infrared moisture analyzer. 0.5 g of oligosaccharide was weighed on an analytical balance and dried in the infrared moisture analyzer until the sample weight stabilized. The dry matter content (in percentage) of the oligosaccharide, or a sample containing oligosaccharides, was given by dividing the mass of the dried sample by the mass of the sample before drying. Liquid samples were weighed in a similar manner, however, the amount of liquid weighed was adjusted to suit the expected amount of dry matter in the liquid so that the mass of the dry matter could be accurately measured on the analytical balance.

[0178] Moisture analyzers measure dry matter, but not water content. Karl Fischer titration is used to determine the amount of water present in powders, ingredients, or food products. The KF titration is performed using a Karl Fischer titrator from Mettler Toledo. The titration was performed using a Fischer DL31 titrator with a two-component technique, employing Hydra-Point Solvent G and Hydra-Point titrant (5 mg H2O / ml), both purchased from JT Baker (Deventer, Holland). The polarization current for endpoint determination using the dual-indicating electrode potentiometric titration was 20 μA, and the termination voltage was 100 mV. Endpoint criteria were drift stability (15 μg H2O / min) or the maximum titration time (10 min).

[0179] The moisture content (MC) of a sample is calculated using the following equation:

[0180] MC = V_KF W_eq 100 / W_sample; where V_KF is the titer consumed in mL, W_eq is the titer of the titer in mgH2O / mL, and W_sample is the weight of the sample in mg.

[0181] Protein quantification

[0182] For protein quantification, a method compatible with reducing agents (e.g., reducing sugars or oligosaccharides with reducing ends) is used. For this purpose, the Bradford assay (Thermo Scientific, Pierce) with a linear range of 1 to 1500 μg / ml is used. This assay is calibrated using a BSA standard curve. The protein content of the dried oligosaccharide product is quantified by dissolving a pre-weighed amount in 18.2 MΩ·cm (Millipore, Bedford, MA, USA) deionized water (DIW) down to 50% (m / v). The amount of protein is measured at 595 nm and converted to concentration using a BSA-based calibration curve.

[0183] DNA quantification

[0184] The production host-specific DNA residue was quantified by RT-qPCR, using host-specific primers designed to amplify the production host's residual DNA. The RT-qPCR was performed according to the standard operating protocol of a kit obtained from Sigma and based on SYBR Green detection.

[0185] Total DNA was measured using a threshold assay (Molecular Devices), based on an immunoassay that allows measurement of DNA down to 2 pg in a sample in solution. Double-stranded DNA was measured using SpectraMax, which has a linear range of 5 pg to 3 ng of dsDNA. ® Quant TM AccuBlue TM The Pico dsDNA Assay kit (MolecularDevices) is used for measurement.

[0186] Endotoxin measurement

[0187] Endotoxins in liquids are measured using the LAL test.

[0188] A. E. coli

[0189] Culture medium, culture and cell lysis

[0190] Luria Broth (LB) medium consists of 1% tryptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco), and 0.5% sodium chloride (VWR. Leuven, Belgium). The basal medium used in 96-well agar or shake flask culture experiments contains 2.00 g / L NH₄Cl, 5.00 g / L (NH₄)₂SO₄, 2.993 g / L KH₂PO₄, 7.315 g / L K₂HPO₄, 8.372 g / L MOPS, 0.5 g / L NaCl, 0.5 g / L MgSO₄·7H₂O, 30 g / L sucrose or 30 g / L glycerol, 1 mL / L vitamin solution, 100 μl / L molybdate solution, and 1 mL / L selenium solution. The basal medium is adjusted to pH 7 using 1M KOH. The vitamin solution consists of 3.6 g / L FeCl₂·4H₂O, 5.0 g / L CaCl₂·2H₂O, 1.3 g / L MnCl₂·2H₂O, 0.38 g / L CuCl₂·2H₂O, 0.5 g / L CoCl₂·6H₂O, 0.94 g / L ZnCl₂, 0.0311 g / L H₃BO₄, 0.4 g / L Na₂EDTA·2H₂O, and 1.01 g / L thiamine hydrochloride. The molybdate solution contains 0.967 g / L NaMoO₄·2H₂O. The selenium solution contains 42 g / L SeO₂. The basal medium used for fermentation contained 6.75 g / L NH₄Cl, 1.25 g / L (NH₄)₂SO₄, 2.93 g / L KH₂PO₄ and 7.31 g / L KH₂PO₄, 0.5 g / L NaCl, 0.5 g / L MgSO₄·7H₂O, 30 g / L sucrose or 30 g / L glycerol, 1 mL / L vitamin solution, 100 μL / L molybdate solution, and 1 mL / L selenium solution, having the same composition as described above. The composite medium was sterilized by autoclaving (121°C, 21 min), and the basal medium was sterilized by filtration (0.22 μm Sartorius). When necessary, antibiotics were added to make the medium selective: for example, chloramphenicol (20 mg / L), carbenicillin (100 mg / L), spectinomycin (40 mg / L), and / or kanamycin (50 mg / L). Inducers were added when necessary.

[0191] For fermentation-produced cultures, a pre-culture was started in 150 μL LB flasks for 96-well microtiter plate experiments and incubated overnight at 37°C on a fixed-track shaker at 800 rpm. This culture was then diluted 400x to serve as inoculum for 96-well square microtiter plates (with 400 μL of basal medium). These final 96-well plates were then incubated at 37°C on a fixed-track shaker at 800 rpm for 72 hours, or less, or longer. To measure sugar concentrations at the end of the culture experiment, a total culture sample (mean of intracellular and extracellular sugar concentrations) was taken from each well by boiling the culture solution at 60°C for 15 minutes before centrifuging the cells.

[0192] Pre-culture for the bioreactor begins with a whole 1 mL frozen vial of a specific strain, inoculated into 250 mL or 500 mL of basal medium in a 1 L or 2.5 L shake flask, and incubated at 37°C on a steady-state shaker at 200 rpm for 24 hours. Then, 250 mL of inoculum in 2 L batch medium is inoculated into a 5 L bioreactor; the process is controlled using MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Culture conditions are set to 37°C and maximum stirring; the pressurized gas flow rate depends on the strain and the bioreactor. The pH is maintained at 6.8 using 0.5 M H₂SO₄ and 20% NH₄OH. The exhaust gas is cooled. When foam rises during fermentation, a 10% silicone defoamer solution is added.

[0193] For heterologous expression of the enzyme, a pre-culture of the enzyme was started in 175 μL LB medium from a frozen vial for 96-well microtiter plate experiments and incubated overnight at 37°C on a fixed-track shaker at 800 rpm. This culture was diluted 100x and used as inoculum for 96-well deep-well microtiter plates (with 1 mL LB medium). These final 96-well plates were then incubated at 37°C on a fixed-track shaker at 800 rpm, induced if necessary, and further grown at 16–37°C for 5–24 hours. After expression, the DW plates were centrifuged at 4200 rpm for 30 minutes at 4°C, the supernatant was discarded, and the granular precipitate was frozen at -80°C for at least one hour. The granular precipitate was then resuspended in a buffer containing 1 mg / mL lysozyme, 10 U / mL DNase, and 1 mM protease inhibitor and incubated at 37°C for 30 minutes. Optionally, the cell lysate is clarified by centrifuging the plate (at 4200 rpm for 30 minutes at 4°C) and transferring the supernatant to a new plate.

[0194] Begin with a pre-culture of the culture in 5 mL LB medium in frozen vials and incubate overnight at 37°C on a steady-state shaker at 200 rpm. Use this culture as an inoculum for shake flask experiments (with 50–250 mL LB medium) by diluting it 50x. Then, incubate these final cultures at 37°C on a steady-state shaker at 200 rpm, inducing growth if necessary, and further growing at 16–37°C for 5–24 hours, or longer or shorter. After expression, harvest cells by centrifugation at 4°C for 30 minutes at 4200 rpm. Discard the supernatant and freeze the granular pellet at -20°C for at least one hour. Resuspend the granular pellet and lyse it by sonication. Optionally, clarify the cell lysate by centrifugation (4°C, 4200 rpm for 30 minutes) and transfer the supernatant to a new container.

[0195] Pre-cultures for the bioreactor are started with a whole 1 mL frozen vial of a specific strain, inoculated into 250 mL or 500 mL of LB medium in a 1 L or 2.5 L shake flask, and incubated at 37°C on a steady-state shaker at 200 rpm for 24 hours. Then, 250 mL of inoculum in 2 L batch medium is inoculated into a 5 L bioreactor; the process is controlled using MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Culture conditions are set at 25–37°C and maximum agitation; the pressurized gas flow rate depends on the strain and the bioreactor. The pH is maintained at 6.8 using 0.5 M H₂SO₄ and 20% NH₄OH. The exhaust gas is cooled. When foaming occurs during fermentation, a 10% silicone defoamer solution is added. A gentle release of the product is established by physically disrupting the cells using sonication. Other commonly used methods known in the art include methods such as freeze-thaw and / or by mixing shear stress, homogenizers and / or Freund's crushers.

[0196] Cells for whole-cell biotransformation were obtained by centrifuging the culture (as described above) overnight at 4200 rpm for 30 minutes at 4°C. The resulting granular precipitate was then resuspended in 0.9% NaCl solution and transferred to 50 mL tubes, and centrifuged at 4200 rpm for 30 minutes at 4°C. The supernatant was removed, and 1000 mg of each granular precipitate was taken and resuspended in 2 mL of 0.9% NaCl (cell concentration 0.5 g / mL). Optionally, the cells could be permeabilized (e.g., with xylene).

[0197] strains and mutations

[0198] Escherichia coli K12 MG1655 [λ] - , F - [rph-1] was obtained from Coli Genetic Stock Center (US) in March 2007, CGSC strain #: 7740, and Escherichia coli NiCo21 (DE3) was obtained from New England Biolabs in November 2017. Gene disruption, gene introduction, and gene replacement were performed using techniques published by Datsenko and Wanner (PNAS 97(2000), 6640-6645). All constitutive promoter, UTR, and terminator sequences were derived from libraries described by Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148), Dunn et al. (Nucleic Acids Res. 1980, 8, 2119-2132), Edens et al. (Nucleic Acids Res. 1975, 2, 1811-1820), Kim and Lee (FEBS Letters 1997, 407, 353-356), and Mutalik et al. (Nat. Methods 2013, No.10, 354-360). Genes were synthesized and ordered from Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com), and the vendor's tools were used to tune codon usage. All strains were stored in frozen vials at -80°C (overnight LB culture, which was mixed with 70% glycerol in a 1:1 ratio).

[0199] In cases where lactose is produced via whole-cell biotransformation, cell extracts, enzymatic synthesis using partially purified enzymes, or fermentation, *E. coli* K12 MG1655 or NiCo21 (DE3) uses an *E. coli* gene encoding β-galactosidase (e.g., ...). lacZ Modification is achieved by knocking out a specific enzyme. In the case of lactose enzymatic synthesis using purified enzymes, lacZ Knockout may not be necessary. For fermentation production, the strain is further modified with a suitable sugar input (e.g., cscB for sucrose). The strain may also be modified by, for example, knocking out proteases (e.g., lon, OmpT), nucleotide sugar degrading enzymes (e.g., ushA), or glucose-consuming enzymes (e.g., glk). Optionally, the strain may also be modified by overexpressing an export protein (e.g., setA) to secrete lactose from the cell.

[0200] B. brewing yeast

[0201] Culture medium, culture and cell lysis

[0202] The strains were grown on synthetically formulated yeast media (SD CSM) or CSM drop-out (SD CSM-Ura, SD CSM-Trp, SD CSM-His) with a complete complement mixture containing 6.7 g / L yeast nitrogen base (YNB w / o AA, Difco) without amino acids, 20 g / L agar (Difco) (solid culture), 22 g / L sucrose monohydrate or glucose or fructose, and 0.79 g / L CSM or 0.77 g / L CSM-Ura, 0.77 g / L CSM-Trp, or 0.77 g / L CSM-His (MP Biomedicals). Typically, the yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were incubated at 30°C for 2–3 days. Starting with a single colony, the pre-culture was incubated overnight at 30°C in 5 mL, with shaking at 200 rpm. Subsequently, 2% of this preculture was inoculated into 125 mL shake flasks in 25 mL of culture medium. These shake flasks were incubated at 30°C with steady-state shaking at 200 rpm. Slow release of the product was established by physically disrupting the cells using sonication. Other commonly used methods known in the art include methods such as freeze-thaw and / or by mixed shear stress, homogenizers, and / or Freund's crushers.

[0203] strains, plasmids and mutants

[0204] The *Saccharomyces cerevisiae* BY4742 strain, created by Brachmann et al. (Yeast (1998) 14:115-32), was used and is available at the Euroscarf Culture Collection. All mutant strains were created by homologous recombination or plasmid transformation, using the method of Gietz (Yeast 11:355-360, 1995). Gene expression is achieved using synthetic constitutive promoters, as described by, for example, Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012), Redden and Alper (Nat. Commun. 2015, 6, 7810), Liu et al. (Microb. Cell Fact. 2020, 19, 38), Xu et al. (Microb. Cell Fact. 2021, 20, 148), and Lee et al. (ACS Synth. Biol. 2015, 4(9), 975-986).

[0205] C. Bacillus subtilis

[0206] Culture medium, culture and cell lysis

[0207] Two media were used to culture Bacillus subtilis: a composite medium such as enriched Luria Broth (LB) and a basal medium for shake-flask cultures. LB medium consisted of 1% tryptone (Difco), 0.5% yeast extract (Difco), and 0.5% sodium chloride (VWR). Luria Broth agar (LBA) plates consisted of LB medium supplemented with 12 g / L agar (Difco). The basal medium contained 2.00 g / L (NH4)2SO4, 7.5 g / L KH2PO4, 17.5 g / L K2HPO4, 1.25 g / L sodium citrate, 0.25 g / L MgSO4·7H2O, 0.05 g / L tryptophan, glucose from 10 to 30 g / L (or another carbon source, including but not limited to fructose, maltose, sucrose, glycerol, and maltotriose), a 10 mL / L trace element mixture, and a 10 mL / L ferric citrate solution. The culture medium was set to pH 7 using 1 M KOH. The trace element mixture consisted of 0.735 g / L CaCl₂·2H₂O, 0.1 g / L MnCl₂·2H₂O, 0.033 g / L CuCl₂·2H₂O, 0.06 g / L CoCl₂·6H₂O, 0.17 g / L ZnCl₂, 0.0311 g / L H₃BO₄, 0.4 g / L Na₂EDTA·2H₂O, and 0.06 g / L Na₂MoO₄. The ferric citrate solution contained 0.135 g / L FeCl₃·6H₂O and 1 g / L sodium citrate (Hoch 1973 PMC1212887). The composite culture medium (e.g., LB) was sterilized by autoclaving (121 °C, 21 min), and the basic culture medium was sterilized by filtration (0.22 μm Sartorius). When necessary, antibiotics are added to make the culture medium selective. Initially, Bacillus subtilis strains are grown on LB agar to obtain single colonies. These plates are incubated overnight at 37°C. Starting with single colonies, the pre-culture is incubated overnight at 37°C in 5 mL agar, with shaking at 200 rpm. Subsequently, 2% of this pre-culture is inoculated into 125 mL shake flasks for experiments in 25 mL agar. These shake flasks are incubated at 37°C for 72 hours, or shorter or longer, with steady-state shaking at 200 rpm. At the end of the culture experiment, samples are taken to measure the supernatant concentration (extracellular sugar concentration, after centrifugation of cells for 5 minutes), or by boiling the culture solution at 90°C for 15 minutes or at 60°C for 60 minutes before centrifugation of cells (= total culture concentration, i.e., intracellular and extracellular sugar concentration)). A gentle release of the product is established by physically disrupting the cells using sonication.Other commonly used methods known in the art include methods such as freeze-thaw and / or by mixing shear stress, homogenizers and / or Freund's crushers.

[0208] strains, plasmids and mutants

[0209] Bacillus subtilis 168, which is available at the Bacillus Genetic Stock Center (Ohio, USA), was used. Plasmids for gene deletion via Cre / lox were constructed as described by Yan et al. (Appl & Environm. Microbial., Sep 2008, pp. 5556-5562). Gene disruption was performed by homologous recombination with linear DNA and transformation via electroporation, as described by Xue et al. (J. microb. Meth. 34 (1999) 183-191). The gene knockout method was described by Liu et al. (Metab. Engine. 24 (2014) 61-69). The integration vector described by Popp et al. (Sci. Rep., 2017, 7, 15158) was used as the expression vector and can be further used for genome integration if desired. Suitable promoters for expression can be derived from a partial repository (iGem): sequence id: BBa_K143012, BBa_K823000, BBa_K823002, or BBa_K823003. Cloning can be performed using Gibson Assembly, Golden Gate Assembly, Cliva Assembly, LCR, or restricted linking.

[0210] D. Corynebacterium glutamicum

[0211] Culture medium, culture and cell lysis

[0212] Two different culture media were used: a composite medium, such as tryptone yeast extract-rich (TY) medium, and a basal medium (MMsf) for shake flasks. The basal medium used a 1000x stock trace element mixture. The trace element mixture consisted of 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 10 g / L MnSO4·H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·6H2O, 0.2 g / L biotin (pH 7), and 0.03 g / L protocatechuic acid. The basic medium (MMsf) used in shake flask experiments contains 20 g / L (NH4)2SO4, 5 g / L urea, 1 g / L KH2PO4, 1 g / L K2HPO4, 0.25 g / L MgSO4·7H2O, 42 g / L MOPS, glucose or another carbon source from 10 to 30 g / L (including but not limited to fructose, maltose, sucrose, glycerol, and maltotriose) (when specifically specified in the examples), and a 1 mL / L trace element mixture. TY medium consists of 1.6% tryptone (Difco, Erembodegem, Belgium), 1% yeast extract (Difco), and 0.5% sodium chloride (VWR. Leuven, Belgium). TY agar (TYA) plates consist of TY medium supplemented with 12 g / L agar (Difco, Erembodegem, Belgium). Complex media (e.g., TY) are sterilized by autoclaving (121°C, 21 minutes), while basic media are sterilized by filtration (0.22 μm Sartorius). When necessary, antibiotics are added to make the media selective.

[0213] A preculture is started in 6 mL of TY medium from a frozen vial or a single colony from a TY plate and incubated overnight at 37°C on a stationary shaker at 200 rpm. Subsequently, 2% of this preculture is inoculated into 125 mL shake flasks in 25 mL of MMsf medium. These shake flasks are incubated at 37°C for 72 hours, or less, or longer, with stationary shaking at 200 rpm. At the end of the culture experiment, samples are taken to measure the supernatant concentration (extracellular sugar concentration, after centrifugation for 5 minutes), or by boiling the culture at 90°C for 15 minutes or at 60°C for 60 minutes before centrifugation (= total culture concentration, i.e., intracellular and extracellular sugar concentration)). The gentle release of the product is established by physically disrupting the cells using sonication. Other commonly used methods known in the art include methods such as freeze-thaw and / or by mixed shear stress, homogenizers, and / or Freund's crushers.

[0214] strains and mutations

[0215] Corynebacterium glutamicum, available at the American Type Culture Collection (ATCC 13032), was used. Integrative plasmid vectors were prepared using the Cre / loxP technique described by Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 Apr, 67(2):225-33), and temperature-sensitive shuttle vectors were constructed using the technique described by Okibe et al. (Journal of Microbiological Methods 85, 2011, 155-163) for gene deletion, mutation, and insertion. Suitable promoters for (heterologous) gene expression were derived from Yim et al. (Biotechnol. Bioeng., 2013 Nov, 110(11):2959-69). Cloning was performed using Gibson assembly, Golden Gate assembly, Cliva assembly, LCR, or restriction ligation.

[0216] E. Optical density

[0217] Cell density in cultures was frequently monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium, or Spark 10M microplate reader, Tecan, Switzerland). Maximum growth rate (mumax) was calculated based on the observed optical density at 600 nm using the R package grofit.

[0218] F. Growth rate / speed measurement

[0219] The maximum growth rate (μMax) was calculated based on the observed optical density at 600 nm using the R package grofit.

[0220] G. Heterologous and homologous expression

[0221] Genes to be expressed, whether derived from plasmids or genomes, are synthesized synthetically by one of the following companies: IDT or Twist Bioscience. The proteins described in this disclosure are summarized in Table 1. Unless otherwise stated, the UniProt ID of the described proteins corresponds to their sequence version 01 present in the UniProt database release 2023_02 on May 3, 2023. Expression can be further advanced by optimizing codon usage to suit the codon usage of the expression host. The vendor's tools are used to optimize the genes.

[0222] Table 1. Overview of the genes and their UniProtIDs described in this invention.

[0223]

[0224] H. analyze

[0225] To determine carbohydrates, the HPAEC-PAD system (Dionex) was used, employing a Dionex CarboPac PA1 Guard (2 × 50 mm) and a Dionex CarboPac PA1 Separation (2 × 250 mm), with a gradient of sodium acetate (1 M) (A), sodium hydroxide (200 mM) (B), water (C), and sodium acetate (25 mM) (D), wherein the gradient was 5% B, 88% C, and 7% over 0–10 minutes. D, in 10-15 minutes, change B from 5% to 17% and C from 88% to 76%; in 15-25 minutes, change B from 17% to 93% and C from 76% to 0%; in 25-28.1 minutes, change A from 0% to 20% and B from 93% to 73%; in 28.1 to 32 minutes, maintain the percentages of A, B, C, and D; in 32 to 32.1 minutes, change A from 20% to 0%, B from 73% to 0%, and C from 0% to 88%; in 32.1 to 50 minutes, maintain the percentages of A, B, C, and D.

[0226] To determine the nucleotide sugars, the same system was used, albeit with different gradients: sodium hydroxide (1 mM) (A) and sodium acetate (1 M) in sodium hydroxide (1 mM) (B), wherein the gradients varied A from 80% to 45% and B from 20% to 55% over 0–10 minutes, 45% A and 55% B over 10–25 minutes, A from 45% to 20% and B from 55% to 80% over 25–35 minutes, A from 20% to 0% and B from 80% to 100% over 35–40 minutes, 0% A and 100% B over 40–50 minutes, A from 0% to 80% and B from 100% to 20% over 50–50.001 minutes, and 80% A and 20% B over 50.001–60 minutes.

[0227] I. Enzyme purification

[0228] Clarified lysates containing an enzyme with an N-terminal histidine tag, in 50 mM Tris pH 7.0, 250 mM NaCl, and 10 mM imidazole, were applied to an ÄKTA Pure Protein Purification system equipped with a Ni-NTA column. The bound protein was washed with five column volumes of wash buffer (50 mM Tris, pH 7.0, 250 mM NaCl, 20–50 mM imidazole) and eluted with elution buffer (50 mM Tris, pH 7.0, 250 mM NaCl, 250 mM imidazole), and the target protein was collected. Buffer exchange was then performed on an Amicon centrifugal filter with a suitable MW cutoff value. Other commonly used purification methods known in the art include salting out, size exclusion chromatography, affinity chromatography, ion exchange chromatography, and / or any combination of the methods mentioned above.

[0229] J. SDS-PAGE and Western blot

[0230] Add 3 μL of Laemmli 4x buffer containing 2-mercaptoethanol (1:10) to 9 μL of protein sample (5 mg / mL final protein concentration for cell extracts; or 0.05–0.5 mg / mL final protein concentration for purified proteins) and heat at 95 °C for 5 min. Load the protein sample with a protein ladder and run at 120–220 V. After electrophoresis, stain the gel (in Coomassie Brilliant Blue R-250 for 1 h) or transfer the gel to a blot membrane for Western blotting. Block the membrane with the transferred protein with 5% milk powder and 0.2% Tween 20 for at least 30 min before applying the primary antibody (anti-His). After 2 h of incubation, wash the membrane and apply HRP-linked secondary antibody for 1 h. Finally, wash the membrane again and visualize the His-tagged proteins by chemiluminescence.

[0231] Example 2: Expression of active sucrose synthase

[0232] The *Escherichia coli* strain NiCo21 (DE3) described in Example 1 was transformed with an inducible expression plasmid containing a gene encoding sucrose synthase (SuSy) from soybean (UniProt ID P13708), *Arabidopsis thaliana* (UniProt ID P49040), *Anabaena* species (UniProt ID Q9ZEV2), or *Thiobacillus thermophilus* (UniProt ID A0A059ZV61). Enzyme expression was performed in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis of the clarified cell lysates was performed as described in Example 1, confirming the expression of the target enzyme. The activity was verified by incubating 100 mM sucrose, 10 mM UDP and 50% (v / v) SuSy cell extract in 50 mM MOPS buffer (pH 7.0) at 37°C for 24 hours and analyzing the formation of UDP-glucose and fructose (as described in Example 1).

[0233] Example 3: Expression of active UDP-glucose-4-epimerase

[0234] The *E. coli* strain NiCo21 (DE3) described in Example 1 was transformed with an inducible expression plasmid containing a gene encoding a UDP-glucose-4-epimerase from *E. coli* (UniProt ID P09147), *Bifidobacterium longum* (UniProt IDE8MF10), *Thermophyton* species WP30 (UniProt ID A0A0K1E1Q5), or *Campylobacter jejuni* (UniProt ID Q0P9C3). Enzyme expression was performed in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis of the clarified cell lysates was performed as described in Example 1, confirming the expression of the target enzyme. The activity was verified by incubating 10 mM UDP-glucose and 50% (v / v) GalE cell extract in 50 mM MOPS buffer (pH 6.5) at 37°C for 24 hours and analyzing the formation of UDP-galactose (as described in Example 1).

[0235] Example 4: Expression of active β1,4-galactosyltransferase

[0236] The *Escherichia coli* strain NiCo21 (DE3) described in Example 1 was transformed with an inducible expression plasmid containing a gene encoding a β1,4-galactosyltransferase (GalT) from *Neisseria meningitidis* with UniProt ID Q51116. Enzyme expression was performed in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis of the clarified cell lysates was performed as described in Example 1, confirming the expression of the target enzyme. Activity was validated by incubating the cell extract at 37°C for 24 hours in 50 mM MOPS buffer (pH 7.0) with 10 mM UDP-galactose, 10 mM glucose, 10 mM MgCl2, and 50% (v / v) cell extract and analyzing lactose formation (as described in Example 1).

[0237] Example 5: Expression of active glucose isomerase

[0238] The *Escherichia coli* strain NiCo21 (DE3) described in Example 1 was transformed with an inducible expression plasmid containing a gene encoding a glucose isomerase from *Streptomyces griseus* (UniProt ID P37031), *Arthrobacter* (UniProt IDP12070), or *Anaerobes ethanolophilus* (UniProt ID D2DK62). Enzyme expression was performed in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis of the clarified cell lysates was performed as described in Example 1, confirming the expression of the target enzyme. Activity was verified by incubating at 37°C for 24 hours in 50 mM MOPS buffer (pH 6.5) with 100 mM fructose, 10 mM MgCl2, and 50% (v / v) cell extract and analyzing glucose formation (as described in Example 1).

[0239] Example 6: Expression of active trehalose synthase

[0240] The *E. coli* strain NiCo21 (DE3) described in Example 1 was transformed with an inducible expression plasmid containing a gene encoding trehalose synthase (treT) from either *Thermococcus Bergensis* (UniProt ID Q7LYW5) or *Fireflya horikosa* (UniProt ID O58762). Enzyme expression was performed in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis of the clarified cell lysates was performed as described in Example 1, confirming the expression of the target enzyme. Activity was validated by incubating the cells at 37°C for 24 hours in 50 mM MOPS buffer (pH 6.5) with 10 mM trehalose, 10 mM UDP, 10 mM MgCl2, and 50% (v / v) cell extract and analyzing glucose formation (as described in Example 1).

[0241] Example 7: Activity of sucrose synthase at elevated temperatures

[0242] The *E. coli* strain NiCo21 (DE3) described in Example 1 was transformed with an inducible expression plasmid containing a gene encoding sucrose synthase (SuSy) from *Thiobacillus thermophilus* with UniProt ID A0A059ZV61. Enzyme expression was performed in shake flasks as described in Example 1, and cells were harvested and lysed. Activity was verified by incubating 100 mM sucrose, 10 mM UDP, and 50% (v / v) SuSy cell extract in 50 mM MOPS buffer (pH 7.0) at 60°C for 1 hour and analyzing the formation of UDP-glucose and fructose (as described in Example 1).

[0243] Example 8: Activity of UDP-glucose-4-epomerase at elevated temperatures

[0244] The *E. coli* strain NiCo21 (DE3) described in Example 1 was transformed with an inducible expression plasmid containing a gene encoding a UDP-glucose-4-epimerase from *Thermophyton* species WP30 with UniProt ID A0A0K1E1Q5. Enzyme expression was performed in shake flasks as described in Example 1, and cells were harvested and lysed. Activity was verified by incubating 10 mM UDP-glucose and 50% (v / v) GalE cell extract in 50 mM MOPS buffer (pH 6.5) at 60°C for 1 hour and analyzing UDP-galactose formation (as described in Example 1).

[0245] Example 9: Activity of glucose isomerase at elevated temperatures

[0246] The *Escherichia coli* strain NiCo21 (DE3) described in Example 1 was transformed with an inducible expression plasmid containing a gene encoding a glucose isomerase from an ethanol-producing anaerobic bacterium with UniProt ID D2DK62. Enzyme expression was performed in shake flasks as described in Example 1, and cells were harvested and lysed. Activity was verified by incubating at 60°C for 1 hour in 50 mM MOPS buffer (pH 6.5) with 100 mM fructose, 10 mM MgCl2, and 50% (v / v) cell extract and analyzing glucose formation (as described in Example 1).

[0247] Example 10: Trehalose synthase activity at elevated temperatures

[0248] The *Escherichia coli* strain NiCo21 (DE3) described in Example 1 was transformed with an inducible expression plasmid containing a gene encoding trehalose synthase (treT) from either *Thermococcus Bergensis* (UniProt ID Q7LYW5) or *Fireflya horikoshi* (UniProt ID O58762). Enzyme expression was performed in shake flasks as described in Example 1, followed by cell harvesting and lysis. Activity was verified by incubating at 60°C for 1 hour in 50 mM MOPS buffer (pH 6.5) with 10 mM trehalose, 10 mM UDP, 10 mM MgCl2, and 50% (v / v) cell extract and analyzing glucose formation (as described in Example 1).

[0249] Example 11: Co-expression of sucrose synthase, UDP-glucose-4-epimerase and β1,4-galactosyltransferase

[0250] The *Escherichia coli* strain NiCo21 (DE3) described in Example 1 was transformed with an expression plasmid containing a constitutive transcription unit for sucrose synthase (SuSy) from *Thiobacillus thermophilus* with UniProt ID A0A059ZV61, a constitutive transcription unit for UDP-glucose-4-epimerase (GalE) from *Thermophyton* species WP30 with UniProt ID A0A0K1E1Q5, and a constitutive transcription unit for β-1,4-galactosyltransferase (GalT) from *Neisseria meningitidis* with UniProt ID Q51116. Enzyme expression was performed in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis of the clarified cell lysates was performed as described in Example 1, and the expression of the target enzymes was confirmed.

[0251] Example 12: Co-expression of sucrose synthase, UDP-glucose-4-epimerase, β1,4-galactosyltransferase and glucose isomerase

[0252] The *Escherichia coli* NiCo21 (DE3) strain described in Example 1 was transformed with an expression plasmid containing a constitutive transcription unit for sucrose synthase (SuSy) from *Thiobacillus thermophilus* with UniProt ID A0A059ZV61, a constitutive transcription unit for UDP-glucose-4-epimerase (GalE) from *Thermotrophicella* species WP30 with UniProt ID A0A0K1E1Q5, a constitutive transcription unit for β-1,4-galactosyltransferase (GalT) from *Neisseria meningitidis* with UniProt ID Q51116, and a constitutive transcription unit for glucose isomerase (XylA) from *Anaerobes ethanolophilus* with UniProt ID D2DK62. Enzyme expression was performed in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis was performed on the clarified cell lysate as described in Example 1, and the expression of the target enzyme was confirmed.

[0253] Example 13: Co-expression of trehalose synthase, UDP-glucose-4-epimerase and β1,4-galactosyltransferase

[0254] The *E. coli* strain NiCo21 (DE3) described in Example 1 was transformed with an expression plasmid containing a constitutive transcription unit for trehalose synthase (treT) from *Thermococcus Bergensis* with UniProt ID Q7LYW5, a constitutive transcription unit for UDP-glucose-4-epimerase (GalE) from *Thermoplasia* species WP30 with UniProt ID A0A0K1E1Q5, and a constitutive transcription unit for β-1,4-galactosyltransferase (GalT) from *Neisseria meningitidis* with UniProt ID Q51116. Enzyme expression was performed in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis of the clarified cell lysates was performed as described in Example 1, and the expression of the target enzymes was confirmed.

[0255] Example 14: In vitro synthesis of lactose via cell extracts of an enzyme expressed exclusively

[0256] Cell lysates from strains expressing sucrose synthase (described in Example 2), UDP-glucose-4-epimerase (described in Example 3), and β1,4-galactosyltransferase (described in Example 4) were mixed with the substrate phase in a single-tank multi-enzyme reaction containing 100 mM sucrose, 100 mM glucose, 2 mM UDP, 10 mM MgCl2, and three times the volume of 16.6% (v / v) cell extract in 50 mM MOPS buffer (pH 7.0) and incubated at 37°C for 24 h. Lactose formation was analyzed as described in Example 1.

[0257] Example 15: In vitro synthesis of lactose via cell extracts of an enzyme expressed exclusively.

[0258] Cell lysates from strains expressing sucrose synthase (described in Example 2), UDP-glucose-4-epimerase (described in Example 3), β1,4-galactosyltransferase (described in Example 4), and glucose isomerase (described in Example 5) were mixed with the substrate phase in a single-tank multi-enzyme reaction containing 100 mM sucrose, 2 mM UDP, 10 mM MgCl2, and four times the volume of 12.5% ​​(v / v) cell extract in 50 mM MOPS buffer (pH 7.0) and incubated at 37°C for 24 h. Lactose formation was analyzed as described in Example 1.

[0259] Example 16: In vitro synthesis of lactose via cell extracts of an enzyme expressed exclusively

[0260] Cell lysates from strains expressing trehalose synthase (described in Example 6), UDP-glucose-4-epimerase (described in Example 3), and β1,4-galactosyltransferase (described in Example 4) were mixed with the substrate phase in a single-tank multi-enzyme reaction containing 100 mM trehalose, 2 mM UDP, 10 mM MgCl2, and three times the volume of 16.6% (v / v) cell extract in 50 mM MOPS buffer (pH 7.0) and incubated at 37°C for 24 h. Lactose formation was analyzed as described in Example 1.

[0261] Example 17: In vitro synthesis of lactose via cell extracts of co-expressed enzymes

[0262] Cell lysates from strains co-expressing the three enzymes described in Example 11 were mixed with the substrate phase in a single-tank multi-enzyme reaction containing 100 mM sucrose, 100 mM glucose, 2 mM UDP, 10 mM MgCl2, and 50% (v / v) cell extract in 50 mM MOPS buffer (pH 7.0) and incubated at 37°C for 24 hours. Lactose formation was analyzed as described in Example 1.

[0263] Example 18: In vitro synthesis of lactose via cell extracts of co-expressed enzymes

[0264] Cell lysates from strains co-expressing the four enzymes described in Example 12 were mixed with the substrate phase in a single-tank multi-enzyme reaction containing 100 mM sucrose, 2 mM UDP, 10 mM MgCl2, and 50% (v / v) cell extract in 50 mM MOPS buffer (pH 7.0) and incubated at 37°C for 24 hours. Lactose formation was analyzed as described in Example 1.

[0265] Example 19: In vitro synthesis of lactose via cell extracts of co-expressed enzymes

[0266] Cell lysates from strains co-expressing the three enzymes described in Example 13 were mixed with the substrate phase in a single-tank multi-enzyme reaction containing 100 mM trehalose, 2 mM UDP, 10 mM MgCl2, and 50% (v / v) cell extract in 50 mM MOPS buffer (pH 7.0) and incubated at 37°C for 24 hours. Lactose formation was analyzed as described in Example 1.

[0267] Example 20: In vitro synthesis of lactose via purified enzymes

[0268] As described in Example 1, cell lysates from strains expressing sucrose synthase (described in Example 2), glucose isomerase (described in Example 3), β1,4-galactosyltransferase (described in Example 4), and glucose isomerase (described in Example 5) were purified. The purified enzyme was then mixed in a single-flask multi-enzyme reaction containing 100 mM sucrose, 2 mM UDP, 10 mM MgCl2, and 0.1 mg / ml of the purified enzyme in 50 mM MOPS buffer (pH 7.0) and incubated at 37°C for 24 hours. Lactose formation was analyzed as described in Example 1.

[0269] Example 21: In vivo synthesis of lactose

[0270] The expression plasmid was transformed into a modified *E. coli* strain K-12 MG1655 (described in Example 1) that grows on sucrose and is defective in lactose digestion. The expression plasmid contains constitutive transcription units concerning sucrose synthase (SuSy) from soybean (UniProt ID P13708), *Arabidopsis thaliana* (UniProt ID P49040), *Anabaena* species (UniProt ID Q9ZEV2), or *Thiobacillus thermophilus* (UniProt ID A0A059ZV61); constitutive transcription units concerning UDP-glucose-4-epimerase (GalE) from *Escherichia coli* (UniProt ID P09147), *Bifidobacterium longum* (UniProt ID E8MF10), *Thermophyton* species WP30 (UniProt ID A0A0K1E1Q5), or *Campylobacter jejuni* (UniProt ID Q0P9C3); and constitutive transcription units concerning UniProt ID... Constituent transcription units of β1,4-galactosyltransferase (GalT) from Neisseria meningitidis and constitutive transcription units of glucose isomerase (XylA) from Streptomyces griseus (UniProt IDP37031), Arthrobacter (UniProt ID P12070), or ethanol-thermogenic anaerobic bacteria (UniProt ID D2DK62) were described in Example 6. The novel strain was evaluated for lactose production in 96-well plates according to the culture conditions provided in Example 1, wherein the strain was cultured in a basal medium containing 30 g / L sucrose. After 72 hours of incubation, the culture was harvested, and lactose formation was analyzed as described in Example 1.

[0271] Example 22: Whole-cell biotransformation synthesis of lactose

[0272] The *Escherichia coli* strain K-12 MG1655 described in Example 1 was transformed with an expression plasmid containing a constitutive transcription unit for sucrose synthase (SuSy) from *Thiobacillus thermophilus* with UniProt ID A0A059ZV61, a constitutive transcription unit for UDP-glucose-4-epimerase (GalE) from *Thermotrophicella* species WP30 with UniProt ID A0A0K1E1Q5, a constitutive transcription unit for β-1,4-galactosyltransferase (GalT) from *Neisseria meningitidis* with UniProt ID Q51116, and a constitutive transcription unit for glucose isomerase (XylA) from *Anaerobes ethanolophilus* with UniProt ID D2DK62. The strain was cultured in shake flasks as described in Example 1, and cells were harvested and prepared for whole-cell biotransformation. The reaction vessel containing 100 mM sucrose, 50 mM UDP, 100 mM MOPS, 15 mM MgSO4, and 50 mg / mL cells (pH 7) was incubated at 21°C and 175 rpm for 48 hours. Lactose formation was analyzed as described in Example 1.

[0273] Example 23: Recovery of enzymes used in synthesis

[0274] The enzyme can be recovered from solution by ultrafiltration, where the molecular cutoff value of the ultrafiltration membrane is less than the molecular weight of the enzyme (i.e., between 80 and 440 kDa for sucrose synthase; between 30 and 80 kDa for UPD-glucose-4-epimerase; between 30 and 45 kDa for β1,4-galactosyltransferase; between 40 and 200 kDa for glucose isomerase; and between 45 and 200 kDa for trehalose synthase). The retained enzyme is recycled to the reaction vessel of the column, while the filtrate, containing residual substrate, byproducts, and salts, is further purified to form highly purified lactose.

[0275] Typical ultrafiltration membranes are PES (Synder), PAN (Synder), PVDF (Synder), SPES (Synder), or ceramic (Tami) membranes, which have molecular weight cutoff values ​​that vary between 1 kDa and 100 kDa.

[0276] In a preferred production setup, the ultrafiltration and recycling of the enzyme is carried out continuously, thereby enabling continuous lactose synthesis.

[0277] Example 24: Removal of proteins, cells, other carbohydrates and salts from lactose solution

[0278] After lactose is synthesized as described in the preceding examples, typical impurities include sucrose, glucose, fructose, galactose, UDP-glucose, UDP-galactose, and buffer components (phosphates and cations such as magnesium).

[0279] These impurities need to be removed from the solution; however, separating the sugars from each other in solution is not trivial. These sugars have similar properties and molecular weights, making them difficult to separate.

[0280] Using selective yeast to purify lactose

[0281] A solution containing lactose, sucrose, glucose, fructose, galactose, UDP-glucose, UDP-galactose, and a buffer solution, along with an enzyme for synthesis, is inoculated with baker's yeast (e.g., Bruggeman's yeast, Saccharomyces cerevisiae). This yeast converts sucrose, fructose, galactose, UDP-glucose, and UDP-galactose into biomass and ethanol, but leaves lactose in solution.

[0282] Following this transformation, the yeast is centrifuged, leaving lactose, buffer solutions, and metabolites formed by Saccharomyces cerevisiae (e.g., ethanol, acetate, glycerol), as well as cell debris, in the supernatant. The formation of these metabolites and debris presents new challenges that require removal. An alternative method for yeast removal is flocculation and decanting or microfiltration with pore sizes from 0.1 to 10 μm (ceramic, PES, PVDF membranes).

[0283] Therefore, ultrafiltration was used to remove cell debris, resulting in a solution with less than 100 mg protein / kg dry solids, less than 10 ng / g dry solids DNA content, and less than 10,000 EU / g dry solids endotoxin. Ultrafiltration was followed by a nanofiltration step, in which buffer salts and yeast metabolites were separated from lactose. For nanofiltration, a molecular weight cutoff of 300 Da was used at 50°C. The NFX membrane (Synder) exhibits >99% repulsion for lactose. The membrane also has <40% salt repulsion, which facilitates the removal of salts and yeast metabolites during water percolation. The lactose yield after this step is higher than 95%. During nanofiltration, the solution is further concentrated to a concentration of >15% dry matter.

[0284] An alternative nanofiltration membrane is NFW (Synder, with a molecular weight cutoff of <600 Da). This membrane exhibits >98.5% lactose rejection and <20.0% salt rejection, which facilitates the removal of salts and yeast metabolites during the percolation step. The ash content, based on total solids, is reduced to <5% after percolation.

[0285] Using invertase to selectively hydrolyze sucrose

[0286] A solution containing lactose, sucrose, glucose, fructose, galactose, UDP-glucose, UDP-galactose, and a buffer solution, along with the enzyme for synthesis, was treated with invertase (β-fructofuranosidase, Novozymes, approximately 1 g / L, pH 5, 50°C). During this treatment, residual sucrose was converted to glucose and fructose, while lactose remained intact.

[0287] Following sucrose conversion, enzymes are removed by ultrafiltration, resulting in a solution with less than 100 mg protein / kg dry solids, less than 10 ng / g dry solids DNA, and less than 10,000 EU / g dry solids endotoxin. Ultrafiltration is followed by a nanofiltration step, in which buffer salts and monosaccharides are separated from lactose. For nanofiltration, a DL-series membrane (Suez) with a molecular weight cutoff of 300 Da is used at 50°C, exhibiting >99% repulsion for lactose but low repulsion (5-10%) for monosaccharides. Monosaccharides and salts are removed in this step by percolation. During nanofiltration, the solution is further concentrated to a solution with >15% dry matter. After percolation, the ash content based on total solids is reduced to <5% based on total solids.

[0288] Example 25: Removal of proteins, cells, other carbohydrates, and salts from lactose solution

[0289] After lactose is synthesized as described in the preceding examples, typical impurities include trehalose, glucose, galactose, UDP-glucose, UDP-galactose, and buffer components (phosphates and cations such as magnesium).

[0290] These impurities need to be removed from the solution; however, separating the sugars from each other in solution is not trivial. These sugars have similar properties and molecular weights, making them difficult to separate.

[0291] Using selective yeast to purify lactose

[0292] A solution containing lactose, trehalose, glucose, galactose, UDP-glucose, UDP-galactose, a buffer solution, and an enzyme for synthesis is inoculated with baker's yeast (e.g., Bruggeman's yeast, Saccharomyces cerevisiae). This yeast converts trehalose, glucose, galactose, UDP-glucose, and UDP-galactose into biomass and ethanol, but leaves lactose in solution.

[0293] Following this transformation, the yeast is centrifuged, leaving lactose, buffer solutions, and metabolites formed by Saccharomyces cerevisiae (e.g., ethanol, acetate, glycerol), as well as cell debris, in the supernatant. The formation of these metabolites and debris presents new challenges that require removal. An alternative method for yeast removal is flocculation and decanting or microfiltration with pore sizes from 0.1 to 10 μm (ceramic, PES, PVDF membranes).

[0294] Therefore, ultrafiltration was used to remove cell debris, resulting in a solution with less than 100 mg protein / kg dry solids, less than 10 ng / g dry solids DNA content, and less than 10,000 EU / g dry solids endotoxin. Ultrafiltration was followed by a nanofiltration step, in which buffer salts and yeast metabolites were separated from lactose. For nanofiltration, a molecular weight cutoff of 300 Da was used at 50°C. The NFX membrane (Synder) exhibits >99% repulsion for lactose. The membrane also has <40% salt repulsion, which facilitates the removal of salts and yeast metabolites during water percolation. The lactose yield after this step is higher than 95%. During nanofiltration, the solution is further concentrated to a concentration of >15% dry matter.

[0295] An alternative nanofiltration membrane is NFW (Synder, with a molecular weight cutoff of <600 Da). This membrane exhibits >98.5% lactose rejection and <20.0% salt rejection, which facilitates the removal of salts and yeast metabolites during the percolation step. The ash content, based on total solids, is reduced to <5% after percolation.

[0296] Using trehalase to selectively hydrolyze trehalose

[0297] A solution containing lactose, trehalose, glucose, fructose, galactose, UDP-glucose, UDP-galactose, and a buffer solution, along with the enzyme for synthesis, was treated with trehalase (α,α-trehalase, Megazymes, approximately 100 mg / L, 40°C, pH 5.5). During this treatment, residual trehalose was converted, while lactose remained intact.

[0298] Following trehalose hydrolysis, the enzymes were removed by ultrafiltration, resulting in a solution with less than 100 mg protein / kg dry solids, less than 10 ng / g dry solids DNA, and less than 10,000 EU / g dry solids endotoxin. Ultrafiltration was followed by a nanofiltration step, in which buffer salts and monosaccharides were separated from lactose. For nanofiltration, a DL-series membrane (Suez) with a molecular weight cutoff of 300 Da was used at 50°C, exhibiting >99% repulsion for lactose but low repulsion (5-10%) for monosaccharides. Monosaccharides and salts were removed in this step by percolation. During nanofiltration, the solution was further concentrated to a solution with >15% dry matter. After percolation, the ash content (based on total solids) was reduced to <5% (based on total solids).

[0299] Example 26: Demineralization of lactose solution

[0300] Solutions from any of Examples 14 to 22 contained residual salts and nucleotide sugars after treatment. To completely remove the salts from the solutions, the solutions were treated with cation and anion exchange resins.

[0301] First, the solution is passed through a column containing a strong acid cation exchange resin (1 L Amberlite IR120) in proton form at 10°C, resulting in the exchange of all cations with protons in the liquid. The liquid resulting from the cation exchange step is then passed through a column containing a weak base anion exchange resin (1 L Amberlite IR400) in hydroxide form at 10°C, thereby exchanging the anions in the liquid for hydroxide ions. After both cation and anion exchange, the pH is set to between 6 and 7. Lactose recovery is 95% to 98%. Ash content, based on total solids, is reduced to <1% after ion exchange.

[0302] The alternative cation and anion exchange resins are Amberlite IR100, Amberlite IR120, Amberlite FPC22, Dowex 50WX, Finex CS16GC, Finex CS13GC, Finex CS12GC, Finex CS11GC, Lewatit S, Diaion SK, Diaion UBK, Amberjet 1000, Amberjet 1200, Amberjet 4200, Amberjet 4600, Amberlite IR400, Amberlite IR410, Amberlite IR458, Diaion SA, Diaion UBA120, Lewatit MonoPlus M, and Lewatit S7468.

[0303] Example 27: Color Removal

[0304] To achieve decolorization, several samples throughout the process underwent activated carbon treatment with Norit SX PLUS activated carbon (0.5% m / v). Color removal was measured at 420 nm using a spectrophotometer. In all samples, the color intensity at 420 nm decreased by 50 to 100 times. The activated carbon was filtered out using a plate filter or a box filter press, preferably at elevated temperatures.

[0305] Example 28: Concentration

[0306] Nanofiltration was performed using an NF-2540 membrane (DOW) with a cutoff of 200 Da to concentrate the deionized solution after ion exchange and nanofiltration up to 25 Brix, or the original solution after ultrafiltration. During the filtration process, a transmembrane pressure in the range of 20-25 bar and a process temperature of 45°C were used. The solution was continuously recirculated on the membrane for concentration, resulting in a concentrate with a dry matter content up to 25% Brix.

[0307] An alternative concentration method is falling film evaporation or wiped film evaporation, in which the lactose concentration can be increased up to 80% before drying or crystallization at high temperatures. Typical temperatures are 50-100°C.

[0308] Example 29: Spray drying of lactose produced by enzymatic means

[0309] The lactose-containing solutions from Examples 14-22 were spray-dried using an experimental spray drying apparatus. The apparatus had an evaporation capacity of 2 kg / h.

[0310] For spray drying, the liquid is heated to a temperature of 50 to 100°C to reduce viscosity. The pH of the liquid is set to a pH of 4 to 6. More preferably, the pH is set to 4 to 5, and the temperature is maintained between 50 and 70°C.

[0311] Depending on whether the solution is concentrated (Example 14) or treated with nanofiltration (Example 11), the lactose concentration in the feed is between 5% and 80% Brix. These concentrations are obtained by rotary evaporation or wipe-membrane evaporation (Example 14). The concentrated liquid is fed into the spray dryer at a rate of 50% to 90%. The higher the Brix percentage, the faster the feed rate.

[0312] The inlet temperature was varied between 120°C and 280°C. The outlet temperature was varied between 100°C and 180°C. The atomizer wheel speed was set between 10,000 and 28,000 rpm. In one particular test, the inlet temperature was set to 184°C, the outlet temperature to 110°C, and the atomizer speed to 21,500 rpm.

[0313] The obtained spray-dried powder, after ultrafiltration, nanofiltration, and activated carbon treatment, is white to off-white in color and has a pH of 4 to 6 after being dissolved in water at a concentration of 10%. The lactose purity is an oligosaccharide exceeding 80% on a dry solids basis. The spray-dried oligosaccharide mixture has a water content of approximately 3 to 10%, a protein content of less than 100 mg / kg dry solids, a DNA content of less than 10 ng / g dry solids, and an endotoxin content of less than 10,000 EU / g dry solids. GMO DNA was undetectable in the powder. The powder treated with ion exchange resin has an ash content of less than 1% (on a total dry solids basis), a lead content of less than 0.1 mg / kg dry solids, an arsenic content of less than 0.2 mg / kg dry solids, a cadmium content of less than 0.1 mg / kg dry solids, and a mercury content of less than 0.5 mg / kg dry solids.

[0314] Example 30: Crystallization of lactose

[0315] The crystallization process was carried out in a stirred-tandem crystallizer. The concentrated lactose solution from Example 28 was heated above room temperature and cooled to 20°C to 25°C to maximize the yield of lactose crystals over a period of 12 to 48 hours. As the solution was cooled, the lactose crystals crystallized due to increased supersaturation. The yield of crystallized lactose in one cycle was approximately 80%. The mother liquor could be further concentrated by passing it through a nanofiltration step (to remove concentrated impurities) or by concentrating it again to approximately 50% dry matter content.

[0316] In another experiment, a biotechnologically produced lactose solution containing sucrose, glucose, and fructose was crystallized. The initial lactose concentration was 42% by mass / total sugar, and the solution contained 42% by mass / total sugar of sucrose, 8% by mass / total sugar of fructose, and 8% by mass / total sugar of glucose. The solution was concentrated to 70% m / m in water by evaporation in a rotary evaporator at 60°C and 70 mbar, but common alternative evaporation systems such as falling film evaporators, vacuum container evaporators, and wipe-off film evaporators could also be used. After evaporation, the solution was maintained at 60°C and brought to atmospheric pressure, after which it was slowly cooled to 45°C. At 45°C, one fraction of the solution was inoculated with lactose crystals; the other fraction was not inoculated. Both were slowly cooled until turbidity appeared, indicating crystallization in both the inoculated and uninoculated solutions. 40% of the lactose was recovered from the inoculated solution, which was centrifuged, washed once with cold water (5°C), and dried. The final purity of the separated lactose was 99% on a dry weight basis.

[0317] In another experiment, a biotechnologically produced lactose solution containing glucose and salts was crystallized. The solution contained 20% lactose (dry solids by mass), 15% glucose (dry solids by mass), and 10% salts (phosphate, sulfate, ammonium, and sodium salts) (dry solids by mass). The solution was concentrated to 70% by mass (dry matter by mass) at 60°C in the same manner described above and brought to atmospheric pressure, after which the solution was cooled to 45°C. The solution was inoculated with lactose crystals at 45°C and allowed to cool further to room temperature, causing the solution to become turbid. The crystals were separated by centrifugation and washed once with cold water. The lactose recovery rate from the solution was 30%, and the lactose purity was 95% (dry matter by mass).

[0318] Example 31: Separating lactose and disaccharides using SMB

[0319] For SMB chromatography, a closed-loop SMB system equipped with 12 glass columns of the stated dimensions can be used. Each glass column contains a strong cation exchange resin, typically Dowex Monosphere in Ca2+ form. SMB can be operated at ambient temperature, but preferably above 50°C. The flow rate is adjusted in different regions of the SMB system to maintain a constant feed rate to the SMB. Water is typically used as the eluent, but food-grade ethanol can be used up to approximately 10% (v / v) for better separation.

[0320] Example 32: Purification of lactose produced by biotechnology

[0321] The first step purifies the lactose produced during the fermentation process (in which cells and cell debris are present). This first step removes cell debris and cells from the fermentation broth through microfiltration (45 μm ceramic membrane, Tami) at temperatures exceeding 50°C. This step results in a clear solution, which is then further purified. This step can be replaced by centrifugation or direct ultrafiltration, which yield similar results.

[0322] The microfiltration step is followed by an ultrafiltration step (step 2), which removes proteins and other high molecular weight molecules such as DNA. This ultrafiltration step is performed using a PES spiral-wound membrane with a MWCO of 3 kDa. This step can be replaced with other types of ultrafiltration or nanofiltration membranes of different sizes, such as membranes with MWCOs varying from 500 Daltons to higher.

[0323] The filtrate from the ultrafiltration or nanofiltration step (step 2) is further treated in a nanofiltration step (step 3) to remove salts and monosaccharides from the liquid. The membrane used is a PA TFC membrane with an MWCO of 150 to 300 Da, at temperatures exceeding 40°C and pressures of approximately 20 bar. Its residue is percolated once in a percolation step, resulting in a 50% reduction in monosaccharides and a 20% reduction in salts. The total lactose yield from steps 1 to 3 is 90%. Further, the removal of monosaccharides and salts is enhanced by lowering the pH of the liquid with an acid (sulfuric acid, for example here, but other acids such as phosphoric acid, citric acid, acetic acid, and lactic acid will have the same effect), resulting in a 33% salt removal after one percolation, a 50% salt removal after two percolations, and a 75% salt removal after three percolations.

[0324] The residue from step 3 (obtained at neutral pH) is concentrated to 70% dry matter concentration in a rotary evaporator (step 4, 60°C and 70 mbar). Alternative evaporation methods include falling film evaporators, vacuum vessel evaporators, and wipe-off membrane evaporators, all of which will result in similar outputs.

[0325] Then, the concentrate from step 4 was further crystallized according to Example 30, wherein a similar yield was obtained in a single crystallization cycle.

[0326] Example 33: Purification of lactose produced by biotechnology

[0327] The first step purifies the lactose produced during the fermentation process (in which cells and cell debris are present). This first step removes cell debris and cells from the fermentation broth through microfiltration (45 μm ceramic membrane, Tami) at temperatures exceeding 50°C. This step results in a clear solution, which is then further purified. This step can be replaced by centrifugation or direct ultrafiltration, which yield similar results.

[0328] The microfiltration step is followed by an ultrafiltration step (step 2), which removes proteins and other high molecular weight molecules such as DNA. This ultrafiltration step is performed using a PES spiral-wound membrane with a MWCO of 3 kDa. This step can be replaced with other types of ultrafiltration or nanofiltration membranes of different sizes, such as membranes with MWCOs varying from 500 Daltons to higher.

[0329] The filtrate from the ultrafiltration or nanofiltration step (step 2) is further treated in a nanofiltration step (step 3) to remove salts and monosaccharides from the liquid. The membrane used is a PA TFC membrane with an MWCO of 150 to 300 Da, at temperatures exceeding 40°C and pressures of approximately 20 bar. Its residue is percolated once in a percolation step, resulting in a 50% reduction in monosaccharides and a 20% reduction in salts. The total lactose yield from steps 1 to 3 is 90%. Further, the removal of monosaccharides and salts is enhanced by lowering the pH of the liquid with an acid (sulfuric acid, for example here, but other acids such as phosphoric acid, citric acid, acetic acid, and lactic acid will have the same effect), resulting in a 33% salt removal after one percolation, a 50% salt removal after two percolations, and a 75% salt removal after three percolations.

[0330] The residue from step 3 (obtained at neutral pH) was further treated in step 4 with activated carbon to remove color from the solution. 4% (w / w) activated carbon was added to the solution and incubated in a stirred tank for 1 hour. The activated carbon was removed through a plate filter (0.45 μm).

[0331] The second part of step 3 is treated with activated carbon in the column, using at least 5 bed volumes / soup volume and at a flow rate of 3 bed volumes / hour.

[0332] The liquid obtained after activated carbon treatment is concentrated to a dry matter concentration of 70% in a rotary evaporator (step 4, 60°C and 70 mbar). Alternative evaporation methods include falling film evaporators, evacuated container evaporators, and wipe-off film evaporators, all of which will result in similar outputs.

[0333] Then, the concentrate from step 4 was further crystallized according to Example 30, wherein a similar yield was obtained in a single crystallization cycle.

[0334] Example 34: Whole-cell biotransformation synthesis of lactose

[0335] The *Escherichia coli* strain K-12 MG1655, as described in Example 1, was transformed with an expression plasmid that naturally expressed the UDP-glucose-4-epimerase (GalE) with UniProt ID P09147. The expression plasmid comprised a combination of constitutive transcriptional units relating to sucrose synthase (SuSy) and constitutive transcriptional units relating to β-1,4-galactosyltransferase (GalT). The SuSy was derived either from *Thiobacillus thermophilus* (with UniProt ID A0A059ZV61) or soybean (with UniProt ID P13708), combined with GalT from *Neisseria meningitidis* with UniProt ID Q51116 or GalT from *Pasteurella multocida* with UniProt ID D0EAD4. For the SuSy and GalT enzymes from soybean, different transcription units (TUs) were evaluated, differing in their promoter, 5'-untranslated region, and / or terminator sequences as described in Example 1. Co-expression of the enzymes was performed in 24-well plates, and cells were harvested and permeabilized via a freeze-thaw cycle as described in Example 1. Whole-cell biotransformation was performed by incubating 10 g / L cells, 100 mM sucrose, 25 mM glucose, 10 mM UDP, and 5 mM MgCl2 in 25 mM phosphate-buffered saline (pH 7) and then incubating at 30°C for 48 h. Lactose formation was analyzed as described in Example 1. Various combinations of these three co-expressed enzymes resulted in lactose production in the whole-cell biotransformation setting (Table 2).

[0336] Table 2. Lactose production in whole-cell biotransformation using strains expressing different combinations of sucrose synthase, β-1,4-galactosyltransferase and UDP-glucose-4-epimerase (normalized to production of the soybean SuSy (TU variant 2) / Pasteurella multocida GalT (TU variant 1) combination).

[0337]

[0338] Example 35: Purification of biotechnologically produced lactose from biotransformation process

[0339] The first step purifies lactose produced through biotransformation or whole-cell biotransformation (in which cells and cell debris are present). This first step removes cell debris and cells from the biotransformation reaction mixture via microfiltration (45 μm ceramic membrane, Tami) at temperatures exceeding 50°C. This step results in a clear solution, which is then further purified. This step can be replaced by centrifugation or direct ultrafiltration, which yields similar results.

[0340] The microfiltration step is followed by an ultrafiltration step (step 2), which removes proteins and other high molecular weight molecules such as DNA. This ultrafiltration step is performed using a PES spiral-wound membrane with a MWCO of 3 kDa. This step can be replaced with other types of ultrafiltration or nanofiltration membranes of different sizes, such as membranes with MWCOs varying from 500 Daltons to higher.

[0341] The filtrate from the ultrafiltration or nanofiltration step (step 2) is further treated in a nanofiltration step (step 3) to remove salts and monosaccharides from the liquid. The membrane used is a PA TFC membrane with an MWCO of 150 to 300 Da, at temperatures exceeding 40°C and pressures of approximately 20 bar. Its residue is percolated once in a percolation step, resulting in a 50% reduction in monosaccharides and a 20% reduction in salts. The total lactose yield from steps 1 to 3 is 90%. Further, the removal of monosaccharides and salts is enhanced by lowering the pH of the liquid with an acid (sulfuric acid, for example here, but other acids such as phosphoric acid, citric acid, acetic acid, and lactic acid will have the same effect), resulting in a 33% salt removal after one percolation, a 50% salt removal after two percolations, and a 75% salt removal after three percolations.

[0342] The residue from step 3 (obtained at neutral pH) was further treated in step 4 with activated carbon to remove color from the solution. 4% (w / w) activated carbon was added to the solution and incubated in a stirred tank for 1 hour. The activated carbon was removed through a plate filter (0.45 μm).

[0343] The second part of step 3 is treated with activated carbon in the column, using at least 5 bed volumes / soup volume and at a flow rate of 3 bed volumes / hour.

[0344] The liquid obtained after activated carbon treatment is concentrated to a dry matter concentration of 70% in a rotary evaporator (step 4, 60°C and 70 mbar). Alternative evaporation methods include falling film evaporators, evacuated container evaporators, and wipe-off film evaporators, all of which will result in similar outputs.

[0345] Then, the concentrate from step 4 was further crystallized according to Example 30, wherein a similar yield was obtained in a single crystallization cycle.

Claims

1. A method for producing crystalline lactose, wherein the lactose is produced via a biotechnological production process, wherein the method includes steps of drying and / or crystallizing the lactose.

2. The method according to claim 1, wherein the lactose is purified by a purification step selected from the following: microfiltration, centrifugation, ultrafiltration, nanofiltration, ion exchange, simulated moving bed, color removal, removal of sucrose, glucose, or trehalose by adding yeast, removal of trehalose by adding trehalase, removal of sucrose by adding invertase; and preferably further comprising a concentration step; preferably, the ion exchange step is a cation and / or anion exchange step, and / or a mixed-bed ion exchange step.

3. The method according to any one of the preceding claims, wherein the biotechnology production process is a fermentation process, a biotransformation process, or an enzymatic process.

4. The method according to claim 3, wherein the fermentation or bioconversion is followed by a biomass removal step, preferably the biomass removal step includes centrifugation, microfiltration and / or ultrafiltration.

5. The method according to claim 3, wherein the biotechnology production process is an enzymatic process, and wherein at least one enzyme is immobilized.

6. The method according to any one of claims 1 to 3 or 5, wherein the biotechnology production process is an enzymatic process, and wherein the reaction is carried out between 50°C and 80°C, preferably between 55°C and 70°C.

7. The method according to any one of claims 1 to 4, wherein the biotechnology production process is a fermentation process or a biotransformation process, wherein the fermentation process or biotransformation process is characterized in that the enzymatic transformation is carried out by at least one enzyme, said at least one enzyme being produced by a cell that synthesizes said enzyme.

8. The method according to claim 7, wherein the fermentation process or biotransformation process is carried out at a temperature of about 25°C to about 50°C, preferably about 25°C to about 40°C.

9. The method according to any one of claims 1 to 4, 7 or 8, wherein the biotechnological production process is a fermentation process, characterized in that the lactose is produced by cells that have been genetically modified to produce all the enzymes necessary for lactose production.

10. The method according to any one of the preceding claims, wherein the lactose is purified from a liquid produced by the biotechnological production process comprising sucrose, glucose, trehalose, fructose and / or glycerol.

11. The method according to any one of claims 2 to 10, wherein the purity of lactose before purification is < 70%, < 60%, < 50%, < 40%, < 30%, < 20%, < 10% based on total solids, and / or the purity at the end of the method including purification is > 80% based on dry solids, preferably > 85% based on dry solids, more preferably > 90% based on dry solids, even more preferably > 95% based on dry solids, even more preferably > 97% based on dry solids, even more preferably > 98% based on dry solids, and most preferably > 99% based on dry solids.

12. The method according to any one of claims 2, 3, 5, 6, 10 or 11, wherein the purification of lactose produced by enzymatic means includes the step of microfiltration or ultrafiltration to recover the used enzyme.

13. The method according to any one of claims 2 to 4, 7 to 11, wherein the cells removed from the fermentation or biotransformation are reused in a further fermentation or biotransformation process.

14. The method according to any one of claims 1 to 13, wherein the biotechnology production process comprises: i) A nanofiltration step to remove / reduce monosaccharides and / or salts; and / or ii) the step of ion exchange to remove charged materials.

15. The method according to any one of claims 1 to 14, wherein the lactose has an ash content of less than 1% based on total solids, preferably less than 0.5% based on total solids, and preferably has one or more of the following: a) Lead content less than 0.1 mg / kg solids, preferably less than 0.02 mg / kg solids; b) Arsenic content less than 0.2 mg / kg solids, preferably less than 0.02 mg / kg solids; c) Cadmium content less than 0.1 mg / kg solids, preferably less than 0.01 mg / kg solids; or d) Mercury content less than 0.5 mg / kg solids, preferably less than 0.1 mg / kg solids.

16. The method according to any one of claims 1 to 15, wherein the lactose has at least one or more of the following: i) a protein content of less than 100 mg / kg dry solids, ii) a DNA content of less than 10 ng / g dry solids, and iii) an endotoxin content of less than 10,000 EU / g dry solids.

17. The method according to any one of claims 1 to 16, wherein the lactose crystals are dried by strip drying, strip drying, vacuum strip drying, vacuum strip drying, drum drying, vacuum drum drying, roller drying, vacuum roller drying and other types of drying.

18. The method according to any one of claims 1 to 16, wherein the drying step comprises any one or more of the following: spray drying, freeze drying, evaporation, precipitation, spray freeze drying, freeze spray drying, strip drying, belt drying, vacuum strip drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying, vacuum roller drying, and stirred film drying.

19. A crystalline lactose product, wherein the lactose product is derived from a biotechnological production process, preferably produced by the method described in any one of claims 1 to 18.

20. The lactose product according to claim 19, wherein the lactose is lactose monohydrate.

21. The lactose product according to any one of claims 19 or 20, wherein the lactose has a purity of at least 90% by dry weight, preferably at least 94% by dry weight.

22. The lactose product according to any one of claims 19 to 21, wherein the lactose is free of milk protein.

23. The lactose product according to any one of claims 19 to 22, wherein the lactose has at least one or more of the following: i) a protein content of less than 100 mg / kg dry solids, ii) a DNA content of less than 10 ng / g dry solids, and iii) an endotoxin content of less than 10,000 EU / g dry solids.

24. The lactose according to any one of claims 19 to 23, wherein the lactose has an ash content of less than 1% based on total solids, preferably less than 0.5% based on total solids, and preferably has one or more of the following: a) Lead content less than 0.1 mg / kg solids, preferably less than 0.02 mg / kg solids; b) Arsenic content less than 0.2 mg / kg solids, preferably less than 0.02 mg / kg solids; c) Cadmium content less than 0.1 mg / kg solids, preferably less than 0.01 mg / kg solids; or d) Mercury content less than 0.5 mg / kg solids, preferably less than 0.1 mg / kg solids.

25. The crystalline lactose product according to any one of claims 19 to 24, for use in medicine, preferably for use in the prevention or treatment of gastrointestinal disorders.

26. Use of crystalline lactose obtained according to any one of claims 1 to 18 in food or feed preparations, in dietary supplements, in cosmetic ingredients or in pharmaceutical ingredients.

27. Use of crystalline lactose according to any one of claims 19 to 24, wherein the use is: i) in food or feed preparations, in dietary supplements, in cosmetic ingredients or in pharmaceutical ingredients, or ii) as an additive in food, preferably as an additive in human food and / or pet food, more preferably as an additive in human infant food.

28. The use according to any one of claims 26 or 27, wherein i) the food is human food, preferably infant food and / or infant formula or infant supplement, or ii) the feed is pet food, animal milk substitute, veterinary product, post-weaning feed or pen feed.

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