Method of preparing a milk product containing hmos, kit and milk product prepared therefrom

Abstract

The present invention relates to a method for preparing a human milk oligosaccharide-containing milk product, a related kit, and a human milk oligosaccharide-containing milk product prepared thereby. The method or kit according to the present invention is capable of catalytically generating LNT and LNnT in a milk product with extremely high efficiency, thereby obtaining a milk product with a higher content of LNT and LNnT, which are the main components of HMOs, saving procedures, reducing packaging, transportation, and storage costs, and thus reducing the overall cost of HMO-containing milk products, compared to the conventional method of directly adding HMOs.

Description

Technical Field

[0001] This invention relates to the field of dairy technology, and more specifically to a method for preparing dairy products containing human milk oligosaccharides, related kits, and dairy products containing human milk oligosaccharides prepared therefrom. Background Technology

[0002] The literature "Scientific Consensus on Human Milk Oligosaccharides (HMOs)" (Chinese Journal of Food Science and Technology, https: / / kns.cnki.net / kcms2 / detail / 11.4528.TS.20230627.0936.004.html) discloses that human milk oligosaccharides (HMOs) play an important role in improving gut microbiota, maintaining the intestinal barrier, regulating immunity, resisting pathogenic bacterial infections, and promoting neural development. Furthermore, clinical studies have shown that HMOs can increase the proportion of Bifidobacteria in the gut of infants and young children, and decrease the proportion of Escherichia coli, Peptostreptococcus, and Clostridium difficile. The gut microbiota composition of infants and young children who consume foods containing HMOs is more similar to that of breastfed infants. Simultaneously, HMO intake also reduces the incidence of lower respiratory tract infections and otitis media, reduces the use of antipyretics and antibiotics, and decreases the occurrence of intestinal diseases.

[0003] Lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) have been found to be major components of HMOs, playing the role of bifidus factors.

[0004] LNT and LNnT have similar structures and both have a molecular weight of 707.63. The chemical structural formula of LNT is as follows:

[0005]

[0006] The chemical structural formula of LNnT is shown below:

[0007]

[0008] The European Union's EFSA and the United States' FDA have approved the marketing of lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT).

[0009] Currently, most infant formula milk powders use cow's or goat's milk as a base. However, the oligosaccharide content in cow's and goat's milk is only 1% to 10% of that in breast milk, and cow's and goat's milk does not contain LNT and LNnT, the main components of HMOs. Furthermore, the oligosaccharides in cow's and goat's milk are primarily acidic, while the oligosaccharides in breast milk are primarily neutral. Based on the nutritional and health benefits of HMOs, and the significant differences between animal milk-based oligosaccharides and those in breast milk, the addition of HMOs to infant formula to improve the nutritional and health status of this population is now widely accepted.

[0010] Studies have shown that adding HMOs to infant formula made primarily from cow's or goat's milk and feeding it to infants can achieve effects similar to breastfeeding in terms of nutrition and immunity. Therefore, infant formula containing added HMOs, as well as dietary supplements, are already available in markets in countries such as the United States, the European Union, Australia, and New Zealand.

[0011] Commercially available infant formula or formula milk is made by purchasing various HMOs or other components from manufacturers or commercial channels, adding them to milk powder or milk, and mixing them thoroughly. This process requires HMO manufacturers to synthesize HMOs using methods such as spray drying, freezing, or chemical crystallization, and then handling packaging, storage, and transportation before supplying them to dairy producers. The entire process involves multiple steps, resulting in high costs and placing a burden on businesses.

[0012] Therefore, there is an urgent need in the field for a simple and cost-effective method for preparing dairy products containing HMOs. Summary of the Invention

[0013] Purpose of the invention

[0014] The purpose of this invention is to provide a method for preparing HMO-containing dairy products that is simple in procedure, cost-effective, and highly efficient in production, along with related kits and HMO-containing dairy products prepared therefrom.

[0015] Solution

[0016] To achieve the objectives of this invention, the following technical solutions are provided:

[0017] In a first aspect, the present invention provides a method for preparing dairy products containing human milk oligosaccharides, characterized in that the method comprises: adding one or more polypeptides and lacto-N-trisaccharides or lacto-N-trisaccharide derivatives to a lactose-containing dairy product and reacting therewith;

[0018] The polypeptide has an amino acid sequence selected from the following: SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0019] The inventors of this application obtained amino acid sequences such as SEQ ID NO:1 (which is a β-galactosidase derivative) and the polypeptides shown in SEQ ID NO:6 to SEQ ID NO:13 through computer-aided design technology. All of them have the ability to catalyze the synthesis of LNT and LNnT. Moreover, compared with the original polypeptide (i.e., the polypeptide shown in SEQ ID NO:1), the derived polypeptides shown in SEQ ID NO:6 to SEQ ID NO:13 have a more superior ability to catalyze the synthesis of LNT and LNnT.

[0020] In the above method, preferably, the lacto-N-trisaccharide derivative is selected from lacto-N-trisaccharide-1-phosphate, UDP-lacto-N-trisaccharide, or GDP-lacto-N-trisaccharide.

[0021] In the above method, preferably, the lactose-containing dairy product is selected from: cow's milk, goat's milk, or dairy products containing cow's milk or goat's milk.

[0022] Preferably, the polypeptide is an immobilized polypeptide;

[0023] Preferably, the immobilization carrier of the immobilized polypeptide is a particulate carrier, preferably selected from: porous silica gel, activated carbon, cellulose, nonpolar macroporous adsorption resin, gold nanoparticles, and nanotubes.

[0024] More preferably, the non-polar macroporous adsorption resin is a styrene-based macroporous adsorption resin;

[0025] More preferably, the styrene-based macroporous adsorption resin is AB-8, X-5, HP20, H107 or S-8 macroporous adsorption resin.

[0026] All of the aforementioned carriers are materials that do not affect the safety of dairy products.

[0027] In a preferred embodiment, the preparation method includes the following steps:

[0028] (1) The polypeptide is immobilized on a fixation carrier to obtain an immobilized polypeptide;

[0029] (2) The immobilized polypeptide, lacto-N-trisaccharide or lacto-N-trisaccharide derivative obtained in step (1) are mixed with lactose-containing dairy products and reacted to generate dairy products containing human milk oligosaccharides.

[0030] And, optionally, (3) the immobilized peptide is separated from the dairy product containing human milk oligosaccharides.

[0031] In step (1) above, the polypeptide can be immobilized on a fixation carrier by traditional immobilization methods such as adsorption, embedding, covalent bonding and / or cross-linking.

[0032] As an example, peptides can be immobilized on a support via adsorption. This method is simple, operates under mild conditions, and the immobilized peptides can be reactivated to restore their activity after a decrease in activity, thanks to immobilized enzyme regeneration technology; the support can also be regenerated.

[0033] Preferably, in step (2) above, the reaction temperature is 18–60°C, more preferably 20–45°C. In specific implementations, the reaction time and temperature can be adjusted appropriately to produce the desired LNT and LNnT and ensure that the dairy products do not spoil.

[0034] In some specific embodiments, in step (2), the reaction of the immobilized polypeptide with lactotrisaccharide or lactotrisaccharide derivative and lactose-containing dairy products is carried out in a reaction column; preferably, the bottom end of the reaction column has a sieve hole, which prevents the immobilized polypeptide from passing through but allows the reaction liquid to flow out.

[0035] Preferably, step (2) includes:

[0036] 1) The immobilized polypeptide is loaded into a reaction column;

[0037] 2) Mix lacto-N-trisaccharide or lacto-N-trisaccharide derivative with lactose-containing dairy products, inject the mixture from the top of the reaction column, and collect the effluent from the bottom of the reaction column to obtain dairy products containing human milk oligosaccharides.

[0038] In specific implementation schemes, the flow rate can be adjusted according to the required content of LNT and LNnT in the HMOs-containing dairy products to be prepared, as well as parameters such as the length of the reaction column. For example, taking a reaction column with a column length of 20cm as an example, the flow rate can be adjusted to 0.5-3ml / min to synthesize HMOs-containing dairy products that meet the expected requirements.

[0039] As demonstrated by the embodiments of the present invention, in the above method, using the polypeptides shown in SEQ ID NO:6-SEQ ID NO:13, lactose in dairy products can be catalyzed to react with lactose-N-trisaccharide (LNT II) or lactose-N-trisaccharide derivatives at room temperature within 30 minutes to produce the desired concentrations of LNT and LNnT.

[0040] Secondly, the present invention provides a kit for producing dairy products containing human milk oligosaccharides, the kit comprising:

[0041] I) One or more polypeptides having an amino acid sequence selected from the following: SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13;

[0042] II) Lacto-N-trisaccharide or lacto-N-trisaccharide derivatives;

[0043] And, optionally, (III) dairy products containing lactose.

[0044] The one or more polypeptides, the lacto-N-trisaccharide or lacto-N-trisaccharide derivative, and optionally the lactose-containing dairy products are each packaged separately.

[0045] In the above kit, preferably, the lacto-N-trisaccharide derivative is selected from lacto-N-trisaccharide-1-phosphate, UDP-lacto-N-trisaccharide, or GDP-lacto-N-trisaccharide;

[0046] Preferably, the lactose-containing dairy product is selected from cow's milk, goat's milk, or dairy products containing cow's milk or goat's milk.

[0047] Preferably, the polypeptide is an immobilized polypeptide;

[0048] Preferably, the immobilization carrier of the immobilized polypeptide is a particulate carrier, preferably selected from: porous silica gel, activated carbon, cellulose, nonpolar macroporous adsorption resin, gold nanoparticles, and nanotubes.

[0049] More preferably, the non-polar macroporous adsorption resin is a styrene-based macroporous adsorption resin;

[0050] More preferably, the styrene-based macroporous adsorption resin is AB-8, X-5, HP20, H107 or S-8 macroporous adsorption resin.

[0051] Preferably, the immobilized polypeptide is loaded in a reaction column, the bottom of which has sieve holes that prevent the immobilized polypeptide from passing through while allowing the reaction solution to flow out.

[0052] Thirdly, the present invention provides a dairy product containing human milk oligosaccharides, which is prepared by the method described in the first aspect above, or by using the kit described in the second aspect above.

[0053] Beneficial effects

[0054] This invention provides a method for preparing dairy products containing HMOs, which utilizes polypeptides as shown in SEQ ID NO:6-SEQ ID NO:13, or polypeptides having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO:6-13 to catalyze the reaction of lacto-N-trisaccharide or lacto-N-trisaccharide derivatives with lactose present in the dairy product to generate LNT and LNnT.

[0055] The method for preparing HMO-containing dairy products of the present invention has the following beneficial effects:

[0056] (1) It can catalyze the generation of LNT and LNnT in dairy products with extremely high efficiency, thereby obtaining dairy products with high content of LNT and LNnT (which are the main components of HMOs), which is of great benefit to human growth and health maintenance;

[0057] (2) It can reduce the lactose content in dairy products such as milk, providing more options for people with lactose intolerance; at the same time, the byproduct galactose produced by the reaction can improve the flavor of dairy products such as milk, without the need to add other sweeteners.

[0058] (3) According to the preparation method of the present invention, dairy product manufacturers can directly add peptides or immobilized peptides provided by peptide manufacturers to lactose-containing dairy products to produce dairy products containing LNT and LNnT. Compared with the traditional method of directly adding HMOs, it not only saves energy and simplifies the process, but also reduces packaging costs, transportation costs and storage costs, thereby reducing the overall production cost of HMOs-containing dairy products, which will benefit the public. Detailed Implementation

[0059] The present invention provides a method for preparing dairy products containing human milk oligosaccharides, the method comprising: adding one or more polypeptides selected from those shown in SEQ ID NO:6 to 13 and lact-N-trisaccharide to a lactose-containing dairy product, and allowing it to react.

[0060] The present invention also provides a kit for producing dairy products containing human milk oligosaccharides, the kit comprising:

[0061] I) One or more polypeptides having an amino acid sequence selected from the following: SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13;

[0062] II) Lacto-N-trisaccharide or lacto-N-trisaccharide derivatives;

[0063] And, optionally, (III) dairy products containing lactose.

[0064] The one or more polypeptides, the lacto-N-trisaccharide or lacto-N-trisaccharide derivative, and optionally the lactose-containing dairy products are each packaged separately.

[0065] Furthermore, the present invention provides a dairy product containing human milk oligosaccharides, which is prepared by the method described above, or by using the kit described above.

[0066] To implement the above invention, the inventors used computer-aided design technology to screen for a polypeptide with the amino acid sequence shown in SEQ ID NO:1, which is a derivative of β-galactosidase and has the ability to catalyze the synthesis of HMOs, particularly the simultaneous synthesis of LNT and LNnT. Furthermore, the inventors modified the polypeptide shown in SEQ ID NO:1 in a series of ways, obtaining a series of derivative polypeptides with significantly enhanced ability to catalyze the synthesis of LNT and LNnT, namely, the polypeptides shown in SEQ ID NO:6 to SEQ ID NO:13, which exhibit superior performance in the simultaneous catalytic synthesis of LNT and LNnT compared to the original polypeptide (i.e., the polypeptide shown in SEQ ID NO:1).

[0067] The modifications made to the amino acid sequences shown in SEQ ID NO:6 to SEQ ID NO:13 compared to the amino acid sequence shown in SEQ ID NO:1 are as follows:

[0068] i) The glutamic acid residue at position 65 of the amino acid sequence shown in SEQ ID NO:1 is replaced with a leucine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:6.

[0069] ii) The tyrosine residue at position 414 of the amino acid sequence shown in SEQ ID NO:1 is replaced with a phenylalanine residue or an alanine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8.

[0070] iii) The tryptophan residue at position 535 of the amino acid sequence shown in SEQ ID NO:1 is replaced with a leucine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:9;

[0071] iv) The glutamic acid residue at position 65 of the amino acid sequence shown in SEQ ID NO:1 is replaced with a phenylalanine residue and the tyrosine residue at position 414 is replaced with a leucine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:10.

[0072] v) The glutamic acid residue at position 65 of the amino acid sequence shown in SEQ ID NO:1 is replaced with a phenylalanine residue and the tyrosine residue at position 414 is replaced with an alanine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:11.

[0073] vi) The glutamic acid residue at position 65 of the amino acid sequence shown in SEQ ID NO:1 is replaced with a phenylalanine residue and the tryptophan residue at position 535 is replaced with an alanine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:12.

[0074] vii) The glutamic acid residue at position 65 of the amino acid sequence shown in SEQ ID NO:1 is replaced with a phenylalanine residue, the tyrosine residue at position 414 is replaced with an alanine residue, and the glutamic acid residue at position 566 is replaced with a leucine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:13.

[0075] Furthermore, the inventors, using computer-aided design technology, screened out polypeptides with amino acid sequences as shown in SEQ ID NO:3, which are derivatives of 1,3-β-galactosyl-N-acetylhexosamine phosphorylase (also known as Lacto-N-biose phosphorylase, LNBP). Further, the inventors performed a series of modifications on the polypeptide shown in SEQ ID NO:3 to obtain a series of derived peptides, which are as follows:

[0076] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the tryptophan acid residue with a phenylalanine residue at position 233, thereby obtaining the amino acid sequence shown in SEQ ID NO:14.

[0077] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the tryptophan residue with an alanine residue at position 271, thereby obtaining the amino acid sequence shown in SEQ ID NO:15.

[0078] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the glycine residue with a leucine residue at position 312, thereby obtaining the amino acid sequence shown in SEQ ID NO:16.

[0079] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the threonine residue with a leucine residue at position 342, thereby obtaining the amino acid sequence shown in SEQ ID NO:17.

[0080] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the methionine residue with a phenylalanine residue at position 345, thereby obtaining the amino acid sequence shown in SEQ ID NO:18.

[0081] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the methionine residue at position 457 with a leucine residue or an alanine residue, thereby obtaining the amino acid sequences shown in SEQ ID NO:19 and SEQ ID NO:20.

[0082] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the glycine residue at position 458 with a phenylalanine residue or a leucine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:21 or SEQ ID NO:22.

[0083] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the 459th position of the glycine residue with a phenylalanine residue, or a leucine residue, or an alanine residue, thereby obtaining the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.

[0084] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the asparagine residue with a phenylalanine residue at position 460, thereby obtaining the amino acid sequence shown in SEQ ID NO:26.

[0085] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the tryptophan residue at position 233 with a phenylalanine residue and the phenylalanine residue at position 456 with a tyrosine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:27.

[0086] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the tryptophan residue at position 233 with a phenylalanine residue and the methionine residue at position 457 with an alanine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:28.

[0087] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the tryptophan residue at position 233 with a phenylalanine residue and the asparagine residue at position 460 with a phenylalanine residue, thereby obtaining the amino acid sequence shown in SEQ ID NO:29.

[0088] The amino acid sequence shown in SEQ ID NO:3 is obtained by replacing the tryptophan residue at position 233 with a phenylalanine residue, the threonine residue at position 342 with a leucine residue, and the asparagine residue at position 460 with a phenylalanine residue.

[0089] Furthermore, the inventors used computer-aided design technology to screen out polypeptides with amino acid sequences as shown in SEQ ID NO:5, which is β-galactosidase GenBank: CP022572.1 (derived from Neobacillus mesonae, referred to as D1 in the examples). Further, the inventors performed a series of modifications on the polypeptide shown in SEQ ID NO:5 to obtain a series of derived peptides, namely:

[0090] The amino acid residues at positions 182-185 of the amino acid sequence shown in SEQ ID NO:5 are replaced by QPSS with GMNC, the amino acid residue at position 189 is replaced by S with T, and the amino acid residues at positions 408-412 are replaced by QSKKP with YGHYQ, thereby obtaining the amino acid sequence shown in SEQ ID NO:31.

[0091] The amino acid residues at positions 408-413 of the amino acid sequence shown in SEQ ID NO:5 are replaced with PMNRLT by QSKKPY, the amino acid residues at positions 481-482 are replaced with DN by ED, and the amino acid residues at positions 484-492 are replaced with IKANWNESN by TRGDKVNVT, thereby obtaining the amino acid sequence shown in SEQ ID NO:32.

[0092] The amino acid residues at positions 182-184 of the amino acid sequence shown in SEQ ID NO:5 are replaced by QPS with RAD, the amino acid residue at position 189 is replaced by S with T, the amino acid residues at positions 408-413 are replaced by QSKKPY with PMNRLT, the amino acid residue at position 593 is replaced by I with V, the amino acid residue at position 600 is replaced by I with L, and the amino acid residue at position 606 is replaced by Y with W, thereby obtaining the amino acid sequence shown in SEQ ID NO:33;

[0093] The amino acid residues at positions 182-184 of the amino acid sequence shown in SEQ ID NO:5 are replaced by QPS with RAD, the amino acid residue at position 189 is replaced by S with T, the amino acid residues at positions 408-413 are replaced by QSKKPY with PMNRLT, the amino acid residues at positions 449-453 are replaced by IYDTT with SGHGA, the amino acid residue at position 593 is replaced by I with V, the amino acid residue at position 600 is replaced by I with L, and the amino acid residue at position 606 is replaced by Y with W, thereby obtaining the amino acid sequence shown in SEQ ID NO:34;

[0094] The amino acid residues at positions 182-185 of the amino acid sequence shown in SEQ ID NO:5 are replaced by QPSS with GMNC, the amino acid residue at position 189 is replaced by S with T, the amino acid residues at positions 408-412 are replaced by QSKKP with YGHYQ, the amino acid residues at positions 481-482 are replaced by ED with DN, the amino acid residues at positions 484-492 are replaced by TRGDKVNVT with IKANWNESN, the amino acid residues at positions 567-568 are replaced by DY with SA, the amino acid residue at position 573 is replaced by R with A, the amino acid residue at position 593 is replaced by I with V, the amino acid residues at positions 595-597 are replaced by TGF with DWV, and the amino acid residues at positions 599-606 are replaced by YIGEPTPY with QSLIKYDE, thereby obtaining the amino acid sequence shown in SEQ ID NO:35;

[0095] The amino acid residues at positions 182-185 of the amino acid sequence shown in SEQ ID NO:5 are replaced by QPSS with GMNC, the amino acid residue at position 189 is replaced by S with T, the amino acid residues at positions 408-413 are replaced by QSKKPY with PMNRLT, the amino acid residues at positions 450-454 are replaced by YDTTN with KNKET, the amino acid residues at positions 567-569 are replaced by DYV with SLG, and the amino acid residues at positions 571-573 are replaced by WGR with FAK, thereby obtaining the amino acid sequence shown in SEQ ID NO:36;

[0096] The amino acid residues at positions 450-454 of the amino acid sequence shown in SEQ ID NO:5 are replaced by YDTTN with KNKET, the amino acid residue at position 481 is replaced by E with A, the amino acid residue at position 484 is replaced by T with F, the amino acid residues at positions 486-492 are replaced by GDKVNVT with FGNGSGG, the amino acid residues at positions 567-569 are replaced by DYV with SLG, the amino acid residues at positions 571-573 are replaced by WGR with FAK, the amino acid residue at position 593 is replaced by I with V, the amino acid residues at positions 595-597 are replaced by TGF with DWV, and the amino acid residues at positions 599-606 are replaced by YIGEPTPY with QSLIKYDE, thereby obtaining the amino acid sequence shown in SEQ ID NO:37;

[0097] The amino acid residues at positions 182-184 of the amino acid sequence shown in SEQ ID NO:5 are replaced by QPS with RAD, the amino acid residue at position 189 is replaced by S with T, the amino acid residues at positions 449-453 are replaced by IYDTT with SGHGA, the amino acid residue at position 481 is replaced by E with A, the amino acid residue at position 484 is replaced by T with F, the amino acid residues at positions 486-492 are replaced by GDKVNVT with FGNGSGG, the amino acid residues at positions 567-568 are replaced by DY with SA, the amino acid residue at position 573 is replaced by R with A, the amino acid residue at position 593 is replaced by I with V, the amino acid residues at positions 595-597 are replaced by TGF with DWV, and the amino acid residues at positions 599-606 are replaced by YIGEPTPY with QSLIKYDE, thereby obtaining the amino acid sequence shown in SEQ ID NO:38;

[0098] The amino acid residues at positions 182-185 of the amino acid sequence shown in SEQ ID NO:5 are replaced by QPSS with GMNC, the amino acid residue at position 189 is replaced by S with T, the amino acid residues at positions 408-413 are replaced by QSKKPY with PMNRLT, the amino acid residues at positions 449-453 are replaced by IYDTT with SGHGA, the amino acid residue at position 481 is replaced by E with A, the amino acid residue at position 484 is replaced by T with F, the amino acid residues at positions 486-492 are replaced by GDKVNVT with FGNGSGG, the amino acid residues at positions 567-568 are replaced by DY with SA, the amino acid residue at position 573 is replaced by R with A, the amino acid residue at position 593 is replaced by I with V, the amino acid residues at positions 595-597 are replaced by TGF with DWV, and the amino acid residues at positions 599-606 are replaced by YIGEPTPY with QSLIKYDE, thereby obtaining the sequence shown in SEQ ID NO:5. The amino acid sequence shown in NO:39.

[0099] In the following examples, the polypeptide shown in SEQ ID NO:3 and its derivative polypeptides shown in SEQ ID NO:14-SEQ ID NO:30, as well as the polypeptide shown in SEQ ID NO:5 and its derivative polypeptides shown in SEQ ID NO:31-SEQ ID NO:39, were used as control polypeptides. They were added to lactose-containing dairy products together with LNT II or lacto-N-trisaccharide derivatives for catalytic reaction. It was found that none of them could catalyze the reaction between lactose in the dairy products and LNT II or lacto-N-trisaccharide derivatives within 40 minutes to produce the required concentration of LNT and LNnT. If the reaction time was extended, the dairy products may spoil and the desired concentration of LNT and LNnT could not be produced.

[0100] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; all materials and reagents are commercially available unless otherwise specified.

[0101] The present application will be further described in detail below through embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. Any modifications or substitutions to the details and form of the technical solution without departing from the structural concept and scope of use of the present application shall fall within the protection scope of the present application.

[0102] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art. Generally, the nomenclature used in this specification and the experimental methods described below are well-known and commonly used in the art.

[0103] In the examples below, the products obtained from the polypeptide catalytic reaction were analyzed or identified using HPLC, mass spectrometry, and ion chromatography.

[0104] The HPLC analysis conditions are as follows:

[0105] Column type: Cosmosil Sugar-D amino column (nacalaitesque, INC.), detector: UV detector (Hitachi Chromaster), detection wavelength: 210 nm, injection volume: 10 μL, flow rate: 1.0 mL / min, column temperature: 30 ℃, mobile phase: acetonitrile:water = 70:30.

[0106] The LC-MS analysis conditions are as follows:

[0107] Column type: Cosmosil Sugar-D amino column (nacalaitesque, INC.), detector: UV detector (Hitachi Chromaster), detection wavelength: 210 nm, injection volume: 10 μL, flow rate: 1.0 mL / min, column temperature: 30 ℃, mobile phase: acetonitrile:water = 70:30; H-ESI mode, molecular weight scan range 400–900.

[0108] The analytical conditions for ion chromatography are as follows:

[0109] Chromatographic column: MetroSep Carb2 (4.0 mm × 250 mm), eluent: 140 mM NaOH / 20 mM NaAc, isocratic elution, flow rate: 0.500 mL / min, amperometric detector, column temperature: 40 °C, injection volume: 20 μL, run time: 50 min.

[0110] The standards for lactose-N-trisaccharide (LNTII), lactose-N-tetrasaccharide (LNT), and lactose-N-neotetrasaccharide (LNnT) were produced by ELICITYL, France.

[0111] It should be noted that the terminology used herein is for descriptive purposes only and is not intended to limit the exemplary embodiments of the invention. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the invention is for descriptive purposes only and is not intended to limit the scope of protection of the invention. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, and such techniques and conditions are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0112] Example 1. Preparation of polypeptides

[0113] I. Through steps such as synthesizing polynucleotides encoding polypeptides, constructing recombinant plasmids, and expressing polypeptides in E. coli. Step by step, polypeptides as shown in SEQ ID NO:1 and SEQ ID NO:6-13 were obtained.

[0114] 1. By DNA synthesis, a polynucleotide encoding a mutant BgaD-D of natural β-galactosidase was obtained, the sequence of which is shown in SEQ ID NO:2; then, based on the polynucleotide shown in SEQ ID NO:2, PCR amplification was performed using the PCR amplification primers shown in Table 1 to obtain a polynucleotide encoding a polypeptide shown in SEQ ID NO:1, 6-13.

[0115] 2. Following the methods described in Molecular Cloning: A Laboratory Manual, a series of recombinant plasmids pET32a-BgaD-MX (pET32a-BgaD-M1, M3, M6, M8, M11, M14, M15, M16, M17) were constructed using the Fast Mutagenesis Kit; the empty plasmid pET32a was purchased from Shanghai Sangon Biotech.

[0116] 3. Transform the above recombinant plasmids into Escherichia coli BL21(DE3) according to the following steps:

[0117] Prepared Escherichia coli BL21(DE3) competent cells were thawed on ice for 30 min. 100 μL of competent cells were mixed with 10 μL of pET32a-BgaD-MX recombinant plasmid (concentration 50 ng / μL), and heat-shocked in a 42℃ water bath for 45 s. Immediately afterward, the mixture was cooled on ice for 2 min. 1 mL of fresh LB medium (LB medium: peptone 1.0%, yeast extract 0.5%, NaCl 1.0%, plate with 1.5% agar powder) was added, and the cells were revived and cultured at 37℃ and 100 rpm for 1 h. Then, 100 μL of the bacterial solution was spread on an LB plate containing ampicillin (100 μg / mL), and cultured at 37℃ for 12 h. Single colonies were picked for colony PCR (PCR amplification primers are shown in Table 1), and positive transformants were screened.

[0118] 4. Cultivate positive transformants, then extract their plasmids, and use double enzyme digestion and gene sequencing to verify whether the pET32a-BgaD-MX recombinant plasmid has been successfully introduced into E. coli.

[0119] 5. Inoculate the correct transformants into LB liquid medium and culture on a shaker at 37°C and 200 rpm for 12 h to obtain the seed culture. Then, inoculate the seed culture into fresh LB medium at an inoculation rate of 1% (v / v) and culture at 37°C with shaking until the OD 600 reaches 0.8. Then, induce expression with isopropyl-β-D-thiopyranoside (IPTG) at a final concentration of 0.1 mmol / L at 16°C for 12 h at 200 rpm. After induction, collect the cells by centrifuging the fermentation broth at 5000 r / min for 30 min at 4°C. Resuspend the cells in 20 mM pH 7.4 PBS buffer and sonicate at a frequency of plus on 5 s / off 5 s for 30 min to lyse the cells. Centrifuge the lysed liquid at 13000 × g at 4°C for 30 min to remove cell debris and collect the supernatant.

[0120] 6. Soluble peptide sequences were purified using nickel column affinity chromatography as follows: Deionized water was added to the top of the nickel column, and after natural elution, elution was performed with 5 volumes of binding buffer. Crude enzyme solution filtered through a 0.45 μm membrane was then loaded onto the column, and the sample was allowed to fully bind to the nickel column at a flow rate of 1.5 mL / min. After the sample dried, impurities were removed by a continuous gradient elution with 5 column volumes of washing buffer. Finally, the target protein was eluted with 5 volumes of elution buffer, and the eluent was collected. The expression of the target protein was then analyzed using SDS-PAGE.

[0121] SDS-PAGE results showed that the genetically engineered bacteria had obvious specific expression bands after induction, and the molecular weight of the bands was basically consistent with the expected molecular weight of 110 kDa. Therefore, the polypeptides shown in SEQ ID NO:1 and 6-13 were obtained. For details, please refer to Table 2.

[0122] Table 1. Primers for PCR amplification of nucleic acids encoding the polypeptides shown in SEQ ID NO: 6-13

[0123]

[0124]

[0125] Table 2. Correspondence between recombinant plasmids, peptides and their amino acid sequence numbers

[0126] plasmid Peptide nomenclature peptide sequence number pET32a-BgaD-M1 M1 SEQ ID NO:1 pET32a-BgaD-M3 M3 SEQ ID NO:6 pET32a-BgaD-M6 M6 SEQ ID NO:7 pET32a-BgaD-M8 M8 SEQ ID NO:8 pET32a-BgaD-M11 M11 SEQ ID NO:9 pET32a-BgaD-M14 M14 SEQ ID NO:10 pET32a-BgaD-M15 M15 SEQ ID NO:11 pET32a-BgaD-M16 M16 SEQ ID NO:12 pET32a-BgaD-M17 M17 SEQ ID NO:13

[0127] II. Referring to the method described in Part I above, obtain the polypeptides shown in SEQ ID NO:3 and SEQ ID NO:14-30. And the polypeptides shown in SEQ ID NO:5 and SEQ ID NO:31-39 (in the following examples, they are used as comparative polypeptides).

[0128] Specifically, a polynucleotide encoding the polypeptide shown in SEQ ID NO:3 is synthesized, and its sequence is shown in SEQ ID NO:4. Then, based on the polynucleotide shown in SEQ ID NO:4, PCR amplification is performed using the PCR amplification primers shown in Tables 3 and 4 to obtain polynucleotides encoding the polypeptides shown in SEQ ID NO:14-30. The correspondence between the recombinant plasmid, the polypeptide, and its amino acid sequence number is shown in Table 5.

[0129] Table 3. Primers for PCR amplification of the polypeptide and its derivative peptides as shown in SEQ ID NO:3 (single mutation)

[0130]

[0131]

[0132] Table 4. Primers for PCR amplification of peptides derived from the polypeptide shown in SEQ ID NO:3 (double and triple mutations)

[0133]

[0134]

[0135] Table 5. Correspondence between recombinant plasmids, peptides, and their amino acid sequence numbers

[0136] plasmid Peptide nomenclature peptide sequence number pET32a-lnbp1 lnbp1 SEQ ID NO:3 pET32a-lnbp2 lnbp2 SEQ ID NO:14 pET32a-lnbp3 lnbp3 SEQ ID NO:15 pET32a-lnbp5 lnbp5 SEQ ID NO:16 pET32a-lnbp10 lnbp10 SEQ ID NO:17 pET32a-lnbp11 lnbp11 SEQ ID NO:18 pET32a-lnbp17 lnbp17 SEQ ID NO:19 pET32a-lnbp18 lnbp18 SEQ ID NO:20 pET32a-lnbp19 lnbp19 SEQ ID NO:21 pET32a-lnbp20 lnbp20 SEQ ID NO:22 pET32a-lnbp22 lnbp22 SEQ ID NO:23 pET32a-lnbp23 lnbp23 SEQ ID NO:24 pET32a-lnbp24 lnbp24 SEQ ID NO:25 pET32a-lnbp25 lnbp25 SEQ ID NO:26 pET32a-lnbp26 lnbp26 SEQ ID NO:27 pET32a-lnbp27 lnbp27 SEQ ID NO:28 pET32a-lnbp28 lnbp28 SEQ ID NO:29 pET32a-lnbp29 lnbp29 SEQ ID NO:30

[0137] In addition, a polynucleotide encoding the polypeptide shown in SEQ ID NO:5 was synthesized (its sequence can be found in GenBank:CP022572.1); then, based on the polynucleotide encoding the polypeptide shown in SEQ ID NO:5, PCR amplification was performed using the PCR amplification primers shown in Table 6 to obtain polynucleotides encoding the polypeptides shown in SEQ ID NO:31-39; the correspondence between the recombinant plasmid, polypeptide and its amino acid sequence number is shown in Table 7.

[0138] Table 6. Primers for PCR amplification of the polypeptide and its derivative peptides as shown in SEQ ID NO:5

[0139]

[0140]

[0141]

[0142]

[0143]

[0144] Table 7. Correspondence between recombinant plasmids, peptides, and their amino acid sequence numbers

[0145] plasmid Polypeptides and Nomenclature peptide sequence number pET32a-BgaD-D1 D1 SEQ ID NO:5 pET32a-BgaD-D2 D2 SEQ ID NO:31 pET32a-BgaD-D3 D3 SEQ ID NO:32 pET32a-BgaD-D5 D5 SEQ ID NO:33 pET32a-BgaD-D6 D6 SEQ ID NO:34 pET32a-BgaD-D7 D7 SEQ ID NO:35 pET32a-BgaD-D8 D8 SEQ ID NO:36 pET32a-BgaD-D9 D9 SEQ ID NO:37 pET32a-BgaD-D10 D10 SEQ ID NO:38 pET32a-BgaD-D11 D11 SEQ ID NO:39

[0146] Example 2. Preparation of immobilized peptides

[0147] Take the polypeptides obtained in Example 1 and prepare immobilized polypeptides according to the following method.

[0148] Step 1. Vector pretreatment

[0149] 1000g of nonpolar macroporous adsorption resin HP20 was soaked in 95% ethanol for 10 hours with occasional stirring. After filtering off the ethanol, the resin was washed until the eluent evaporated to dryness and no residue remained. The HP20 macroporous adsorption resin was then dried and stored for later use.

[0150] Step 2. Immobilization

[0151] 1) Take 2g of each of the peptides shown in SEQ ID NO:1 and SEQ ID NO:6-13, or 8g of each of the peptides shown in SEQ ID NO:3 and SEQ ID NO:14-30, or 5g of each of the peptides shown in SEQ ID NO:5 and SEQ ID NO:31-39, and add 100g of nonpolar macroporous adsorption resin HP20 and 150ml of acetate-sodium acetate buffer (pH 6-8) obtained in step 1 respectively. Maintain the temperature at 35℃ and shake for adsorption for 8h.

[0152] 2) Add 2 ml of 3.0% glutaraldehyde solution, shake and crosslink for 5 h, vacuum filter to obtain immobilized peptide, and store the immobilized peptide in a 4℃ refrigerator for later use.

[0153] Example 3. Catalytic synthesis of LNT and LNnT in milk using immobilized peptides as catalysts.

[0154] In this embodiment, the immobilized peptides prepared in Example 2 were used as catalysts to mix and react with LNTⅡ and commercially available pure milk (with a lactose content of 43.91 mg / mL) to prepare milk containing LNT and LNnT (i.e., the main components of HMOs). The specific steps are as follows:

[0155] Add 40 mL of commercially available pure milk, 15 g / L of LNTⅡ, and 7 mg / mL of the immobilized polypeptide prepared in Example 2 to the Erlenmeyer flasks, respectively, and mix them. The resulting mixture is reacted at 25 °C and 150 rpm for 30 min (polypeptide of the present invention) or 40 min (comparative polypeptide). After the reaction, filter to separate the immobilized polypeptide and retain the filtrate. Dilute the filtrate 20 times and detect the contents of LNT, LNnT, lactose, and galactose, respectively. The results are recorded in Tables 3-5.

[0156] Identification of catalytically synthesized products:

[0157] High performance liquid chromatography (HPLC), LC-MS, and ion chromatography were used to analyze lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT) standards, and the reaction products obtained by the above procedures.

[0158] HPLC analysis

[0159] The HPLC analysis conditions were as described above. The analytical results show:

[0160] (1) The peak elution time rt of both LNT and LNnT standards was 23.800 min;

[0161] (2) The catalytic reaction solutions of the immobilized peptides shown in SEQ ID NO:6-SEQ ID NO:13 all showed strong absorption peaks around 23.800 min at a wavelength of 210 nm, which is consistent with the peak times of LNT and LNnT standards, indicating that the reaction solution contains LNT and / or LNnT. This shows that LNT and / or LNnT were indeed generated by the catalysis of the immobilized peptides shown in SEQ ID NO:6-SEQ ID NO:13.

[0162] (3) For the immobilized peptide groups of peptides such as SEQ ID NO:3, SEQ ID NO:14-30 and peptides such as SEQ ID NO:5, SEQ ID NO:31-39, no absorption peaks or only weak absorption peaks are shown at the corresponding positions in the HPLC spectrum of their catalytic reaction solution. This indicates that the catalytic reaction solution of these peptides does not contain or contains only trace amounts of LNT and / or LNnT.

[0163] LC-MS analysis

[0164] Then, the substance in the HPLC chromatogram at rt = 23.800 min was analyzed by LC-MS under the conditions shown above.

[0165] The LNnT standard has the same quality spectrum as the LNT standard.

[0166] LC-MS analysis results show that the molecular weight of 708.2567 should be LNnT / LNT+H; and the molecular weight of 730.2393 should be LNnT / LNT+Na.

[0167] The mass spectrometry results were retrieved as follows: Compound CID:440993, Chemical Formula:C26H45NO21, Extract Mass:707.63.

[0168] In this embodiment, the mass spectrometry results of the catalytic reaction solution of the immobilized peptides shown in SEQ ID NO:6-SEQ ID NO:13 at rt=23.800 min are all the same as those of LNT or LNnT. Therefore, it is speculated that the immobilized peptides shown in SEQ ID NO:6-SEQ ID NO:13 catalyze the reaction of LNT II with lactose in milk to synthesize a substance with a molecular weight of about 707, which may be LNT and / or LNnT.

[0169] As for the immobilized peptide groups such as those shown in SEQ ID NO:3, SEQ ID NO:14-30 and SEQ ID NO:5, SEQ ID NO:31-39, as mentioned above, the substance at rt=23.800 min was almost absent in the HPLC spectrum of their catalytic reaction solution. Therefore, no LC-MS analysis results were obtained.

[0170] Ion chromatography analysis

[0171] The analysis conditions are as shown above. The results show:

[0172] (1) The peak elution time of LNnT standard was 26.99 min; the peak elution time of LNT standard was 30.92 min.

[0173] (2) In this embodiment, substances with rt=23.800 min in the catalytic reaction solution were extracted and analyzed by ion chromatography. The following results were obtained: The catalytic reaction solutions of the immobilized peptides based on the peptides shown in SEQ ID NO:6-SEQ ID NO:13 all had strong absorption peaks near 26.99 min and 30.92 min, which were consistent with the peak times of LNnT and LNT standards, indicating that the catalytic reaction synthesized LNnT and LNT.

[0174] The yields of LNT and / or LNnT in each catalytic reaction solution were also determined by ion chromatography, and the results are recorded in Table 8-10.

[0175] In addition, the lactose and galactose content in the catalytic reaction solution were tested according to GOST 34304-2017 "Milk and dairy products - Determination of lactose and galactose content". The results showed that after the catalytic reaction of the present invention, the lactose content in the dairy products decreased while the galactose content increased. This indicates that the reaction process may be accompanied by the conversion of lactose to galactose, that is, the byproduct galactose is generated.

[0176] Table 8. Identification results of the catalytic reaction solution for immobilized peptides shown in SEQ ID NO:1, 6-13 (reaction time 30 min).

[0177]

[0178] Table 9. Identification results of the catalytic reaction solution for immobilized peptides shown in SEQ ID NO:3, 14-30 (reaction time 40 min).

[0179]

[0180] Table 10. Identification results of the catalytic reaction solution for immobilized peptides shown in SEQ ID NO:5, 31-39 (reaction time 40 min).

[0181]

[0182]

[0183] Table 8-10 shows the following:

[0184] 1) The peptide shown in SEQ ID NO:6-13 can synthesize high yields of LNT and LNnT within 30 min using LNT II and lactose contained in dairy products as substrates, and its activity is significantly improved compared with the peptide shown in SEQ ID NO:1.

[0185] 2) In contrast, the peptides shown in SEQ ID NO:3, 14-30 and SEQ ID NO:5, 31-39 have poor efficiency in catalyzing the synthesis of LNT and LNnT using LNTII and lactose contained in dairy products as substrates. Even if the reaction time is extended to 40 min, no LNT and LNnT are synthesized or only trace amounts are synthesized. If the reaction time is further extended or the reaction temperature is increased to about 37°C, the expected results cannot be obtained due to the spoilage of the milk.

[0186] in conclusion

[0187] The above results indicate that using immobilized peptides such as those shown in SEQ ID NO:6-SEQ ID NO:13 as catalysts, and using LNTⅡ and lactose naturally present in dairy products as substrates, LNnT and LNT are generated in a short time and with high efficiency, thereby preparing dairy products containing HMOs.

[0188] Example 4. Using the polypeptide shown in SEQ ID NO:7 as a catalyst, LNT and LNnT were synthesized in milk.

[0189] In this embodiment, the polypeptide (i.e., in free form) prepared in Example 1 as shown in SEQ ID NO:7 was used as a catalyst. It was mixed and reacted with LNTⅡ and commercially available pure milk (with a lactose content of 43.91 mg / mL) to prepare milk containing LNT and LNnT (i.e., the main components of HMOs). The specific steps are as follows:

[0190] Commercially available pure milk was used as the reaction solvent, and LNTⅡ and lactose from milk were used as substrates.

[0191] Add 40 mL of commercially available pure milk, 15 g / L of LNTⅡ, and 0.13 g / L of the polypeptide prepared in Example 1 as shown in SEQ ID NO:7 to the Erlenmeyer flasks, respectively, and mix them. The resulting mixture is reacted at 35 °C and 150 rpm for 30 min with stirring. The reaction solution is heated to 90 °C for 10 min to inactivate enzyme activity, and then cooled to room temperature. The reaction solution is diluted 20 times, and the contents of LNT, LNnT, lactose, and galactose are detected (the detection method is the same as in Example 3 above). The results are shown in Table 11 below.

[0192] Table 11. Identification results of the catalytic reaction solution for the polypeptide shown in SEQ ID NO:7

[0193]

[0194] Table 11 shows that the free form of the polypeptide shown in SEQ ID NO:7 can catalyze the reaction between LNTⅡ and lactose present in the dairy product in milk, and generate LNnT and LNT in a short time and with high efficiency, thereby preparing dairy products containing HMOs.

[0195] Example 5. Immobilized peptides were packed into a reaction column and used to catalytically synthesize LNT and LNnT in milk.

[0196] In this embodiment, the immobilized polypeptide of the polypeptide shown in SEQ ID NO:7 prepared in Example 2 was packed into a reaction column, and then a catalytic reaction of LNTⅡ with commercially available pure milk (with a lactose content of 43.91 mg / mL) was carried out in the reaction column to prepare milk containing LNT and LNnT (i.e., the main components of HMOs). The specific steps are as follows:

[0197] 1) At 20℃, add 40mL of commercially available pure milk to 15g / L LNT II and mix well to obtain a mixture;

[0198] 2) Take 7g of the immobilized polypeptide of the polypeptide shown in SEQ ID NO:7 obtained in Example 2 and fill it into a reaction column (30cm long and 2cm in diameter). The bottom end of the reaction column has a sieve hole, which prevents the immobilized polypeptide particles from passing through, but the reaction solution can flow out through the sieve hole.

[0199] 3) Inject the mixture obtained in step 1) from the top of the reaction column, control the outflow rate to about 2 ml / min and control the reaction temperature to 20°C, collect the outflowing material, and obtain dairy products containing HMOs.

[0200] The received effluent was diluted 20 times, and the contents of LNT, LNnT, lactose, and galactose were detected by the method described in Example 3. The results are recorded in Table 7.

[0201] Table 12. Identification results of the catalytic reaction solution for immobilized peptides loaded in the reaction column

[0202]

[0203] Table 12 shows that the immobilized peptide, as shown in SEQ ID NO:7, can catalyze the reaction between LNTⅡ and lactose naturally present in milk in the reaction column, and generate LNnT and LNT with high efficiency, thereby preparing dairy products containing HMOs.

[0204] Compare with Example 1

[0205] Take 40 mL of pure milk and add 7 g / L of the polypeptide shown in SEQ ID NO:6-13 to it at room temperature (25°C). React at 35°C and 150 rpm for 30 min, then heat to 90°C and cool to room temperature. Dilute the reaction solution 20 times and detect the contents of LNT, LNnT, lactose and galactose according to the method described in Example 3.

[0206] The results showed that no galactose, LNT, or LNnT were produced in the reaction solution, and the lactose content in the reaction solution remained unchanged.

[0207] This result indicates that mixing and reacting milk with immobilized peptides without the addition of lactose-N-trisaccharide does not produce galactose, LNT, or LNnT.

[0208] Compare with Example 2

[0209] Take 40 mL of pure milk, add 15 g / L LNT II at room temperature (25°C), and react for 30 min at 35°C and 150 rpm. Then dilute the reaction solution 20 times and detect the LNT, LNnT, lactose and galactose content using the method described in Example 3.

[0210] The results showed that no galactose, LNT, or LNnT were produced in the reaction solution, and the lactose content in the reaction solution remained essentially unchanged.

[0211] The results indicate that, without the addition of the polypeptides described in this invention, mixing and reacting milk with lactose-N-trisaccharide does not produce galactose, LNT, and LNnT.

[0212] Compare with Example 3

[0213] Take 40 mL of pure milk, add 15 g / L LNT II and 200 U of lactase at room temperature (25°C), mix well, and react at 35°C and 150 rpm for 30 min. After the reaction is complete, heat the reaction solution to 90°C for 10 min, and then cool it to room temperature. Then, dilute the reaction solution 20 times and detect the content of LNT, LNnT, lactose and galactose using the method described in Example 3.

[0214] The results showed that no LNT and LNnT were produced in the reaction solution, and the galactose content in the reaction solution was about 5.97 mg / mL and the lactose content was about 31.08 mg / mL.

[0215] The results indicate that after the addition of lactase, milk mixes and reacts with lactose-N-trisaccharide to produce galactose, but no LNT or LNnT is generated.

[0216] Compare with Example 4

[0217] Take 40 mL of pure milk and add 15 g / L LNT II and 20 U / mL galactokinase (Galk; EC 2.7.1.6) at room temperature (25°C). Mix well and react at 35°C and 150 rpm for 30 min. After the reaction is complete, heat the reaction solution to 90°C for 10 min and then cool it to room temperature. Then, dilute the reaction solution 20 times and detect the LNT, LNnT, lactose and galactose content using the method described in Example 3.

[0218] The results showed that no galactose, LNT, or LNnT were produced in the reaction solution, and the lactose content in the reaction solution remained essentially unchanged.

[0219] This result indicates that when milk is mixed with lactose-N-trisaccharide and galactokinase is added, galactose, LNT, and LNnT cannot be synthesized.

[0220] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a milk product containing human milk oligosaccharides, characterized in that, The method includes: adding one or more polypeptides and lacto-N-trisaccharides or lacto-N-trisaccharide derivatives to a lactose-containing dairy product; The polypeptide is selected from the following amino acid sequences: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:

13.

2. The method of claim 1, wherein, The lacto-N-trisaccharide derivative is selected from lacto-N-trisaccharide-1-phosphate, UDP-lacto-N-trisaccharide, or GDP-lacto-N-trisaccharide; And / or, the lactose-containing dairy product is selected from: cow's milk, goat's milk, or dairy products containing cow's milk or goat's milk.

3. The method of claim 1, wherein, The polypeptide is an immobilized polypeptide.

4. The method of claim 3, wherein, The immobilization carrier for the immobilized polypeptide is a particulate carrier.

5. The method of claim 4, wherein, The immobilization carrier is selected from: porous silica gel, activated carbon, cellulose, non-polar macroporous adsorption resin, gold nanoparticles, and nanotubes.

6. The method of claim 5, wherein, The non-polar macroporous adsorption resin is a styrene-based macroporous adsorption resin.

7. The method of claim 6, wherein, The styrene-based macroporous adsorption resin is AB-8, X-5, HP20, H107, or S-8 macroporous adsorption resin.

8. The method according to any one of claims 3-7, characterized in that, The preparation method includes the following steps: (1) The polypeptide is immobilized on a fixation carrier to obtain an immobilized polypeptide; (2) The immobilized polypeptide, lacto-N-trisaccharide or lacto-N-trisaccharide derivative obtained in step (1) are mixed with lactose-containing dairy products and reacted to generate dairy products containing human milk oligosaccharides.

9. The method of claim 8, wherein, The preparation method further includes the following steps: (3) separating the immobilized polypeptide from the dairy product containing human milk oligosaccharides.

10. The method of claim 8, wherein, In step (1), the polypeptide is immobilized on a fixation carrier by adsorption, encapsulation, covalent bonding and / or cross-linking. And / or, in step (2), the temperature of the reaction is 18 to 60 °C.

11. The method of claim 10, wherein, In step (2), the reaction temperature is 20–45°C.

12. The method of claim 8, wherein, In step (2), the reaction of the immobilized polypeptide with lactotrisaccharide or lactotrisaccharide derivative and lactose-containing dairy products is carried out in a reaction column.

13. The method of claim 12, wherein, The bottom end of the reaction column has sieve holes that prevent the immobilized polypeptide from passing through while allowing the reaction solution to flow out.

14. The method of claim 12, wherein, Step (2) includes: 1) The immobilized polypeptide is loaded into a reaction column; 2) Mix lacto-N-trisaccharide or lacto-N-trisaccharide derivatives with lactose-containing dairy products, inject the mixture from the top of the reaction column, and collect the effluent from the bottom of the reaction column to obtain dairy products containing human milk oligosaccharides.

15. A kit for producing a milk product containing human milk oligosaccharides, characterized in that, The kit includes: I) One or more polypeptides, said polypeptides being selected from the following amino acid sequences: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13; II) Lacto-N-trisaccharide or lacto-N-trisaccharide derivatives; The one or more polypeptides are packaged separately from the lacto-N-trisaccharide or lacto-N-trisaccharide derivative.

16. The kit of claim 15, wherein The kit also includes: III) lactose-containing dairy products.

17. The kit of claim 16, wherein The one or more polypeptides, the lacto-N-trisaccharide or lacto-N-trisaccharide derivatives, and the lactose-containing dairy products are each packaged separately.

18. The kit of any one of claims 15-17, wherein, The lacto-N-trisaccharide derivative is selected from lacto-N-trisaccharide-1-phosphate, UDP-lacto-N-trisaccharide, or GDP-lacto-N-trisaccharide; And / or, the lactose-containing dairy product is selected from cow's milk, goat's milk, or dairy products containing cow's milk or goat's milk; And / or, the polypeptide is an immobilized polypeptide.

19. The kit of claim 18, wherein The immobilization carrier for the immobilized polypeptide is a particulate carrier.

20. The kit of claim 19, wherein The immobilization carrier is selected from: porous silica gel, activated carbon, cellulose, non-polar macroporous adsorption resin, gold nanoparticles, and nanotubes.

21. The kit of claim 20, wherein, The non-polar macroporous adsorption resin is a styrene-based macroporous adsorption resin.

22. The kit of claim 21, wherein The styrene-based macroporous adsorption resin is AB-8, X-5, HP20, H107, or S-8 macroporous adsorption resin.

23. The kit of claim 18, wherein The immobilized polypeptide is loaded in a reaction column, the bottom of which has sieve holes that prevent the immobilized polypeptide from passing through while allowing the reaction solution to flow out.

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