A method for preparing a whey protein powder enriched in lpe components

Abstract

The application discloses a preparation method of whey protein powder rich in LPE components. The application provides a preparation method of whey protein powder rich in LPE components, which comprises the following steps: a centrifugal separation step, an enzyme coagulation separation step, an ultrafiltration step and a homogenization step. The preparation method of the whey protein powder rich in LPE components provided by the application realizes directional enrichment of LPE specific molecules from milk raw materials, and breaks through the limitation of only enriching total phospholipids.

Description

Technical Field

[0001] This invention relates to the field of deep processing technology of milk-derived functional lipids, and more specifically, to a method for preparing whey protein powder rich in LPE components. Background Technology

[0002] Milk fat globule membrane is a natural interfacial membrane structure that encapsulates milk fat globules. Phospholipids and sphingolipids are its important bioactive lipid components, mainly including phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine, and phosphatidylinositol. In recent years, milk fat globule membrane and its polar lipids have attracted attention in the food, nutrition, and pharmaceutical fields due to their emulsifying properties, nutritional value, and potential physiological activities.

[0003] Compared with common plant lecithin, milk-derived polar lipids have a higher proportion of sphingomyelin and phosphatidylethanolamine, and their fatty acid composition has milk-derived characteristics.

[0004] Lysophosphatidylethanolamine, or LPE for short, is a monoacylphosphatidylphosphatidylethanolamine formed by the removal of a fatty acyl group from phosphatidylethanolamine via a type A phospholipase reaction. Existing literature indicates... [1] LPE can be generated from phosphatidylethanolamine in the cell membrane via a type A phospholipase reaction and can be detected in human serum. At the same time, LPE has been reported to have biological effects such as inducing neural differentiation of PC12 cells, regulating intracellular calcium signaling, and promoting cell chemotaxis and migration, suggesting that LPE molecules have application value as functional lipids, lipidomics standards, and bioactivity research objects.

[0005] LPE 18:2 is a hemolytic phosphatidylethanolamine molecule with a fatty acyl group consisting of a linoleic acid chain, and can include 1-acyl-2-hydroxy and 2-acyl-1-hydroxy regioisomers. Lipidomics studies have already demonstrated... [2] The degree of unsaturation of the fatty acyl chain and the position of the acyl group in LPE molecules affect their chromatographic behavior, mass spectrometric fragmentation characteristics, and distribution in biological samples. (Furukawa) [2] They established a library of synthetic LPE standards and used it for liver LPE regional isomer analysis. They found that C18:2-LPE and C20:5-LPE were significantly reduced in the livers of mice with non-alcoholic steatohepatitis. They also pointed out that LPE research is limited by problems such as insufficient availability of standards, 1,2-O-acyl migration, and difficulty in distinguishing regional isomers.

[0006] Existing LPE preparation technologies mainly include natural source isolation, chemical synthesis, enzymatic modification, and the formulation of LPE-containing compositions. Natural source isolation primarily extracts LPE from raw materials such as soybean lecithin, egg yolk lecithin, and rapeseed lecithin. However, conventional lecithin contains low levels of LPE; EP1907401B1 records that ordinary soybean lecithin contains approximately 0.5%–2% LPE, and egg yolk lecithin contains approximately 1%–3%. Therefore, directly isolating LPE from natural lecithin is quite difficult. This patent also describes enriching LPE through phospholipase D and phospholipase A reactions, or by treating lecithin with phospholipase A2, pancreatic phospholipase A2, or neutral lipase. However, its technical focus is on obtaining LPE-containing lecithin or improving the stability of water-soluble compositions, and it does not address the preparation and enrichment of LPE, especially LPE 18:2, in dairy systems.

[0007] Current research on the industrialization of milk-derived phospholipids mainly focuses on enriching total phospholipids from buttermilk, whey buttermilk, or whey protein phospholipid concentrates. For example, some studies have used microfiltration combined with supercritical fluid extraction to concentrate polar lipids from milk fat globule membranes in buttermilk. [3] Other studies have used enzymatic deproteinization, ultrafiltration, and percolation to obtain phospholipid concentrates from whey buttermilk. [4] The final powder phospholipid content can reach about 14%, and the phospholipid recovery rate can reach 95% to 99%. These methods can increase the total phospholipid content of milk, but the target is usually total phospholipids, milk fat globule membrane materials or phospholipid concentrate powder, without further converting phosphatidylethanolamine into LPE, nor enriching LPE 18:2 at the molecular level.

[0008] In recent years, food-grade green extraction technology has been used for the enrichment of milk-derived phospholipids. For example, Ubeyitogullari and Rizvi used buttermilk powder as raw material and employed ethanol-modified supercritical carbon dioxide extraction to obtain a total lipid extract at 60°C, 30 MPa, and 15% ethanol, with a phospholipid content of approximately 49%. Further extraction using pure supercritical carbon dioxide to remove nonpolar lipids, followed by extraction of polar lipids using ethanol-modified supercritical carbon dioxide, yielded a milk-derived phospholipid concentrate with a phospholipid content of approximately 76%. This method provides a feasible path for the industrial-scale green enrichment of buttermilk phospholipids, but the product is still a mixture of total phospholipids, including phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and other categories, and does not solve the problems of targeted generation, selective retention, and enrichment purification of LPEs, especially LPE 18:2.

[0009] Regarding existing patents, US20210386083A1 discloses a method for preparing phospholipid concentrate from dairy product compositions. This method involves decalcification using a cation exchange resin, microfiltration with controlled transmembrane pressure gradient, and recovery of the microfiltration retentate to obtain the phospholipid concentrate. This method is applicable to raw materials such as buttermilk or concentrated buttermilk. US11425915B2 and its related subsequent patents disclose methods using buttermilk or butter whey as raw materials, involving a first-step ultrafiltration, carbon dioxide treatment, microfiltration, a second carbon dioxide treatment, and a second-step ultrafiltration to obtain a concentrate containing at least approximately 30 wt% phospholipids. All of the above methods pertain to the total enrichment of milk-derived phospholipids or milk fat globule membrane components. They do not disclose the selective conversion of phosphatidylethanolamine in phospholipids into LPE, especially LPE 18:2, nor do they address the separation, purification, and quality control of the LPE 18:2 concentrate.

[0010] Regarding the preparation of LPE 18:2 monomers, the closest academic technical solution is Furukawa. [2] This paper reports a tin-mediated rapid synthesis route for LPE. Using commercially available phosphatidylethanolamine derivatives as starting materials, various LPE standards were prepared through protection, deacylation, tin-mediated monoacylation, and deprotection. C18:2-LPE can be obtained by reacting with linoleic chloride. Literature tables show that the acylation yield of C18:2-LPE is 44%, the deprotection yield is 60%, and the 2-LPE:1-LPE ratio is approximately 3:1. This route is suitable for constructing analytical standard libraries, but it has drawbacks including the use of organotin reagents, the water sensitivity of the reaction system, the complexity of the deprotection process, the difficulty in completely controlling the proportion of regioisomers, and insufficient applicability for food-grade industrial scale-up.

[0011] In addition, existing research has shown that... [5] Phospholipids in milk and whey systems can have their surface properties and emulsifying functions altered by phospholipase treatment, indicating that phospholipase can be used for structural modification of milk-derived phospholipids; however, this type of research mainly focuses on improving the functional properties of dairy products rather than preparing specific LPE molecular species.

[0012] In summary, while existing technologies have disclosed methods for enriching total phospholipids in buttermilk, separating milk fat globule membrane materials, enriching LPEs from plant or egg yolk sources, developing water-soluble LPE compositions, and chemically synthesizing LPEs such as LPE 18:2 standards, a method is still lacking that utilizes dairy raw materials while considering food-grade raw material utilization, pre-enrichment of milk phospholipids, enrichment of LPEs, especially LPE 18:2 molecular species, hypoxia antioxidant protection, and feasibility for industrial scale-up. Therefore, developing a method for separating and preparing LPEs, especially LPE 18:2 enrichments, is of great significance for improving the high-value utilization of dairy raw materials, developing functional lipid raw materials from dairy sources, and establishing standardized preparation processes for LPEs, especially LPE 18:2 molecular species.

[0013] References:

[0014] [1] Makide K, Kitamura H, Sato Y, et al. Emerging lysophospholipidmediators, lysophosphatidylserine, lysophosphatidylthreonine,lysophosphatidylethanolamine and lysophosphatidylglycerol[J]. Prostaglandins& other lipid mediators, 2009, 89(3-4): 135-139.

[0015] [2] Furukawa T, Fuda H, Miyanaga S, et al. Rapid tin-mediated accessto a lysophosphatidylethanolamine (LPE) library: Application to positionalLC / MS analysis for hepatic LPEs in non-alcoholic steatohepatitis model mice[J]. Chemistry and physics of lipids, 2016, 200: 133-138.

[0016] [3] Ubeyitogullari A, Rizvi S S H. Production of high-purityphospholipid concentrate from buttermilk powder using ethanol-modifiedsupercritical carbon dioxide[J]. Journal of dairy science, 2020, 103(10):8796-8807.

[0017] [4] Konrad G, Kleinschmidt T, Lorenz C. Ultrafiltration of wheybuttermilk to obtain a phospholipid concentrate[J]. International DairyJournal, 2013, 30(1): 39-44.

[0018] [5] Lilbæk HM, Fatum TM, Ipsen R, et al. Modification of milk and whey surface properties by enzymatic hydrolysis of milk phospholipids[J]. Journal of agricultural and food chemistry, 2007, 55(8): 2970-2978. Summary of the Invention

[0019] The problem the invention aims to solve

[0020] While existing technologies have disclosed methods for enriching total phospholipids in buttermilk, separating milk fat globule membrane materials, enriching LPE from plant or egg yolk sources, creating water-soluble LPE compositions, and chemically synthesizing LPE such as LPE 18:2 standards, a method is still lacking that utilizes milk-derived raw materials, takes into account the use of food-grade raw materials, pre-enrichment of milk-derived phospholipids, enrichment of LPE, especially LPE 18:2 molecular species, low-oxygen antioxidant protection, and feasibility for industrial scale-up.

[0021] Therefore, developing a method for separating and preparing LPE enrichment is of great significance for improving the high-value utilization of dairy raw materials, developing functional lipid raw materials from dairy sources, and establishing a standardized preparation process for LPE, especially LPE 18:2 molecular species.

[0022] Solution for solving the problem

[0023] [1]. A method for preparing whey protein powder rich in LPE components, wherein the preparation method includes: a centrifugal separation step, wherein the pretreated milk phospholipid-containing raw material is centrifuged to separate the fat-containing components and protein-containing components in the pretreated milk phospholipid-containing raw material to obtain a milk material rich in LPE milk phospholipid components and a milk protein-containing component; an enzymatic coagulation separation step, wherein the milk protein-containing component material obtained in the centrifugal separation step is treated with rennet and the whey is separated; an ultrafiltration step, wherein the whey obtained in the enzymatic coagulation separation step is ultrafiltered to obtain a concentrate; and a homogenization step, wherein the milk material rich in LPE milk phospholipid components obtained in the centrifugal separation step is mixed with the concentrate obtained in the ultrafiltration step and homogenized.

[0024] [2]. According to the preparation method described in [1], wherein in the enzymatic coagulation separation step, the amount of rennet added is 50-100 mL / ton of milk protein-containing material; and / or, in the enzymatic coagulation separation step, the coagulation time is 5-60 min.

[0025] [3]. According to the preparation method described in [1] or [2], wherein, in the step of enzyme coagulation separation, the enzyme activity of the rennet is above 600 U / mL; and / or, in the step of enzyme coagulation separation, the rennet is derived from Aspergillus niger and / or the rennet includes abomasin.

[0026] [4]. The preparation method according to any one of [1]-[3], wherein, in the ultrafiltration step, the amount of water added is 2-6 times the mass of the whey.

[0027] [5]. The preparation method according to any one of [1]-[4], wherein, in the ultrafiltration step, the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 1-50KD.

[0028] [6]. The preparation method according to any one of [1]-[5], wherein in the centrifugal separation step, a butterfly centrifuge is used with a rotation speed of 2000-7000 r / min; optionally, the processing capacity of the butterfly centrifuge is 10-45 t / h.

[0029] [7]. The preparation method according to any one of [1]-[6], wherein the milk phospholipid-containing milk raw material includes at least one of whey and whey powder.

[0030] [8]. The preparation method according to any one of [1]-[7], wherein the pretreatment includes standardization treatment of the milk phospholipid-containing milk raw material and / or removal of impurities.

[0031] [9]. According to the preparation method described in [8], wherein the standardization treatment adjusts the solid content in the milk phospholipid raw material to 5-25 kg / 100 L; and / or the impurity removal treatment is carried out by filtration.

[0032]

[10] . A whey protein powder rich in LPE components, which is prepared by any one of the preparation methods of whey protein powder rich in LPE components described in [1]-[9].

[0033]

[11] . Use of LPE-rich whey protein powder in food preparation, wherein the LPE-rich whey protein powder is prepared by any one of the preparation methods of LPE-rich whey protein powder described in [1]-[9].

[0034] The effects of the invention

[0035] The present invention has the following differences and advantages compared with the prior art.

[0036] (1) To achieve the targeted enrichment of specific LPE molecules in dairy raw materials, breaking through the limitation of only enriching total phospholipids.

[0037] Existing milk-derived phospholipid processes and related patents all target total phospholipids and milk fat globule membrane components for enrichment, achieving only the overall extraction of phospholipids and failing to selectively enrich LPEs, especially LPE 18:2, a single functional molecule. This invention uses whey / whey powder as raw material and relies on a complete coupled process to directionally increase the content of LPEs, especially LPE 18:2, upgrading from "crude extraction of total phospholipids" to precise enrichment of the target active lipid molecule. This solves the core problem of existing technologies' inability to achieve the specialized preparation of LPEs, especially LPE 18:2.

[0038] (2) Construct a coupling system of chyme enzymatic hydrolysis and multi-stage ultrafiltration to enhance the separation and retention of target components.

[0039] Existing processes often employ single separation methods such as centrifugation, microfiltration, and supercritical fluid extraction, resulting in incomplete removal of impurities such as protein, lactose, and inorganic salts, and significant loss of phospholipid components. This invention utilizes a directional enzymatic process derived from Aspergillus niger to coagulate casein, combined with a standardized ultrafiltration process using a 5-30 kDa ultrafiltration membrane and 2-4 times the required water volume. This process removes large-molecule casein and small-molecule water-soluble impurities stepwise, maximizing the retention of LPE, especially LPE 18:2, and associated milk phospholipids, significantly improving the recovery rate and purity of the target product. Experimental verification shows that omitting ultrafiltration or reducing enzyme addition / water volume causes a sharp decline in LPE content, especially LPE 18:2. This coupled separation system is the core innovative process of this invention.

[0040] (3) Adopting a combination of physical and biological processes of food grade, suitable for industrial production in the food industry.

[0041] LPE, especially LPE 18:2, requires organotin reagents and organic solvents for chemical synthesis, with harsh reaction conditions and the risk of reagent residue, making it completely unsuitable for food production. Traditional plant / egg yolk lecithin extraction methods result in low background LPE content and are difficult to purify. This invention employs common food-grade unit operations such as filtration, centrifugation, enzymatic coagulation, ultrafiltration, homogenization, low-temperature sterilization, triple-effect concentration, and spray drying. All rennet and separation equipment used are food-grade, with no toxic reagents or high-risk reaction conditions. The process route is safe and compliant, and can be directly applied to the industrial production of dairy products and functional foods, overcoming the shortcomings of chemical synthesis methods in food applications.

[0042] (4) Optimize the range of process parameters throughout the entire process, with strong process fault tolerance and adaptability to industrial scale-up.

[0043] Existing milk phospholipid enrichment processes have narrow parameter ranges, stringent production control requirements, and poor batch stability. This invention defines a wide controllable range for key parameters such as raw material solids, centrifugation temperature and speed, rennet dosage, coagulation time, ultrafiltration makeup water volume, homogenization pressure, sterilization, and drying temperature. Within a range of 8%–20% raw material solids, 3500–5000 r / min centrifugation speed, 75–85 mL / ton rennet dosage, and 2–4 times the ultrafiltration makeup water volume, high-content LPE, especially LPE 18:2, can be stably obtained. Product index fluctuations are minimal under different parameter combinations, making this process more suitable for large-scale continuous production compared to existing processes.

[0044] (5) The process product is a composite emulsion matrix system that takes into account both functional activity and product application attributes.

[0045] Existing technologies primarily produce pure phospholipid powders and concentrated phospholipid solutions, which are characterized by limited components and poor emulsification and processing compatibility. This invention homogenizes and mixes the phospholipid-rich lipid phase obtained by centrifugation with the whey protein liquid phase obtained by ultrafiltration, ultimately yielding a whey protein complex product rich in LPE, especially LPE 18:2. This product naturally retains beneficial components such as milk protein and various milk-derived phospholipids, resulting in superior physicochemical properties. It can be directly used as an ingredient in formula powders, liquid dairy products, and other end products, expanding its application scenarios. Attached Figure Description

[0046] Figure 1 A schematic flowchart of a method for preparing whey protein powder rich in LPE according to one embodiment of the present invention. Detailed Implementation

[0047] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0048] Terminology Definition

[0049] In this invention, "comprising," "having," "including," or "containing" can mean included or open-ended, and does not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0050] In this invention, the meaning of "may" includes both performing a certain process and not performing a certain process.

[0051] In this invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions, etc., are used or not used.

[0052] In this invention, the numerical ranges represented by "value A ~ value B", "value A - value B", and "value A above / below" refer to the ranges including the endpoint values ​​A and B.

[0053] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by the term "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1%, or ±0.5% of a specific value or range. Furthermore, the values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0054] In this invention, terms such as "some specific / preferred embodiments," "other specific / preferred embodiments," and "implementation" refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to a particular embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0055] In this invention, all unit names used are international standard unit names, and unless otherwise stated, the "%" used refers to weight or mass percentage content.

[0056] In this invention, "infants and toddlers" refers to the human group under the age of 3 years, including infants aged 0-6 months, older infants aged 6-12 months, and toddlers aged 12-36 months.

[0057] In this invention, "children" refers to the human group aged 3-6 years.

[0058] In this invention, "teenagers" refers to the human group aged 7-18.

[0059] In this invention, "pregnant women" includes women who are pregnant and women who are breastfeeding.

[0060] In this invention, "middle-aged and elderly" refers to the human group aged 41 and above, including middle-aged people aged 41-65 and elderly people aged 65 and above.

[0061] In this invention, "animal milk" refers to the liquid obtained from the mammary glands of a mammal in the process of lactation.

[0062] In this invention, "lysophosphatidylethanolamine (LysoPE, LPE)" is a glycerophospholipid composed of a glycerol backbone, a fatty acid chain, a phosphate group, and an ethanolamine head group. Compared to phosphatidylethanolamine, it lacks a fatty acid chain at the sn-1 or sn-2 position of its glycerol backbone.

[0063] In this invention, for ease of description of glycerophospholipids, the following characters are used to refer to different types of fatty acids: straight-chain fatty acids with a chain length of 18 carbon atoms and no carbon-carbon double bonds (18:0), such as stearic acid; straight-chain fatty acids with a chain length of 18 carbon atoms and one carbon-carbon double bond (18:1), such as oleic acid; straight-chain fatty acids with a chain length of 18 carbon atoms and two carbon-carbon double bonds (18:2), such as linoleic acid; and other fatty acid chain lengths are treated similarly. In this invention, for specific lysophosphatidylethanolamines, the format "LPE + the above characters" is used to indicate the type of lysophosphatidylethanolamine linked with different fatty acids, such as LPE 18:0, LPE 18:1, LPE 18:2, etc.

[0064] In some implementations, LPE 18:2 includes the 1-acyl-2-hydroxy type of LPE 18:2.

[0065] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0066] Preparation method of whey protein powder rich in LPE

[0067] In some aspects of the present invention, a method for preparing whey protein powder rich in LPE is provided, wherein the method includes: a centrifugal separation step, wherein a pretreated milk phospholipid-containing raw material is centrifuged to separate the fat-containing and protein-containing components in the pretreated milk phospholipid-containing raw material to obtain a milk material rich in LPE milk phospholipid components and a milk protein-containing material; an enzymatic coagulation separation step, wherein the milk protein-containing material obtained in the centrifugal separation step is treated with rennet and the whey is separated; an ultrafiltration step, wherein the whey obtained in the enzymatic coagulation separation step is ultrafiltered to obtain a concentrate; and a homogenization step, wherein the milk material rich in LPE milk phospholipid components obtained in the centrifugal separation step is mixed with the concentrate obtained in the ultrafiltration step and homogenized.

[0068] In some preferred embodiments, the LPE comprises LPE 18:2. In some more preferred embodiments, the LPE 18:2 comprises 1-acyl-2-hydroxy type LPE 18:2.

[0069] Milk raw materials containing milk phospholipids

[0070] In some embodiments of the present invention, there are no particular limitations on the milk raw material containing milk phospholipids; it can be any milk raw material containing milk phospholipids. In some preferred embodiments, the milk raw material containing milk phospholipids includes at least one of whey and whey powder.

[0071] Preprocessing

[0072] In some embodiments of the present invention, the milk raw material containing milk phospholipids may be pretreated before centrifugation.

[0073] In some embodiments, the pretreatment includes standardization of the milk phospholipid-containing raw material and / or removal of impurities.

[0074] In some specific implementations, the standardization process includes the step of adjusting the solids content in the milk phospholipid-containing raw material, specifically adjusting the solids content to 5-25 kg / 100L.

[0075] In some preferred embodiments, the solids content in the milk phospholipid-containing raw material is adjusted to 6-25 kg / 100L, more preferably 8-23 kg / 100L, even more preferably 10-23 kg / 100L, and even more preferably 10-18 kg / 100L, such as 10 kg / 100L, 12 kg / 100L, 16 kg / 100L, 17 kg / 100L or 18 kg / 100L.

[0076] In some implementations, the impurity removal process is carried out by filtration.

[0077] In some specific implementations, the standardized milk phospholipid-containing raw material can be passed through a bag filter to remove impurities. In some specific implementations, an 80-120 mesh bag filter can be used, preferably a 90-110 mesh bag filter, such as 90 mesh, 100 mesh, or 110 mesh.

[0078] Centrifugal separation steps

[0079] In some embodiments of the present invention, the centrifugal separation step is performed using a disc centrifuge with a rotation speed of 2000-7000 r / min, preferably 3500-5000 r / min, more preferably 3500-4800 r / min, even more preferably 3500-4500 r / min, and even more preferably 3800-4500 r / min, for example 3800 r / min, 3900 r / min, 4000 r / min, 4100 r / min, 4200 r / min or 4500 r / min.

[0080] In some embodiments, the throughput of the disc centrifuge is 10-45 t / h, preferably 20-40 t / h, for example 20 t / h, 30 t / h or 40 t / h.

[0081] In some embodiments, during the centrifugation step, the milk raw material containing milk phospholipids is separated into milk components rich in LPE (milk components rich in LPE milk phospholipid I) and milk protein components (milk protein and other milk components such as lactose).

[0082] In some specific implementation schemes, the milk component I containing LPE phospholipids obtained from the centrifugation step is cooled to 10-15°C and temporarily stored for later use, and the milk protein component obtained from the centrifugation step is cooled to 30-40°C and temporarily stored for later use.

[0083] Enzymatic coagulation separation steps

[0084] In some embodiments of the present invention, in the enzymatic coagulation separation step, the amount of rennet added is 50-100 mL / ton of milk protein-containing material (obtained by centrifugation separation).

[0085] In some preferred embodiments, the rennet comprises rennet derived from Aspergillus niger and / or the rennet comprises abomasin.

[0086] In some embodiments, the abomasal enzyme is a natural rennet extracted from the abomasum of young ruminant animals such as cattle and sheep. In some preferred embodiments, the abomasal enzyme is calf abomasal enzyme.

[0087] In some alternative embodiments, the amount of rennet or abomasalase derived from Aspergillus niger added is 60-90 mL / ton of milk protein-containing material.

[0088] Preferably, the amount of rennet or abomasin derived from *Aspergillus niger* added is 65-90 mL / ton of milk protein-containing material. More preferably, the amount of rennet or abomasin derived from *Aspergillus niger* added is 70-90 mL / ton of milk protein-containing material. More preferably, the amount of rennet or abomasin derived from *Aspergillus niger* added is 72-88 mL / ton of milk protein-containing material. Even more preferably, the amount of rennet or abomasin derived from *Aspergillus niger* added is 75-85 mL / ton of milk protein-containing material. More preferably, the amount of rennet or abomasalase derived from Aspergillus niger added is 75-83 mL / ton of milk protein-containing material. Even more preferably, the amount of rennet or abomasalase derived from Aspergillus niger added is 78-83 mL / ton of milk protein-containing material, for example, 78 mL / ton, 80 mL / ton, or 82 mL / ton.

[0089] In some embodiments, the enzyme activity of the rennet or abomasin derived from Aspergillus niger is above 600 U / mL.

[0090] In some embodiments, the enzyme activity of the rennet or abomasin derived from Aspergillus niger is below 800 U / mL.

[0091] In this invention, the rennet can be food-grade rennet, which is commercially available.

[0092] In some embodiments, the rennet treatment time (i.e., curdling time) is 5-60 min, preferably 10-50 min, more preferably 15-45 min, further preferably 20-45 min or 25-45 min, even more preferably 25-40 min, and even more preferably 25-35 min, for example 25 min, 30 min or 35 min.

[0093] In this invention, there are no particular limitations on the method of separating whey from the material after rennet treatment. For example, demulsification stirring can be used, and a press (e.g., a fully automated press) can be used to separate and remove the curd solids to obtain whey and separate the casein.

[0094] In some embodiments, the demulsification stirring can be carried out in a curdling tank, with the stirring paddle speed at 5-60 rpm, preferably 10-60 rpm, more preferably 10-50 rpm, and even more preferably 10-30 rpm.

[0095] In some implementations, a fully automated press is used to separate and remove curd solids, with a pressing pressure of 0.1-0.7 MPa, preferably 0.2-0.7 MPa, more preferably 0.2-0.6 MPa, and even more preferably 0.3-0.6 MPa.

[0096] In this invention, the role of rennet is to specifically hydrolyze the phenylalanine-methionine peptide bonds of casein, disrupting the spatial structure of casein micelles and causing them to aggregate. This allows for the full release of phospholipid components bound to the milk fat globule membrane into the whey, achieving initial separation of protein and phospholipids. When the amount of rennet added is insufficient or the coagulation time is too short, casein hydrolysis is incomplete, micelle aggregation is incomplete, and a large amount of phospholipid components remain bound to casein, failing to be effectively released into the whey. This results in the inability to achieve effective enrichment of LPE 18:2 in the subsequent ultrafiltration stage. Simultaneously, the insufficiently hydrolyzed casein binds to non-target PS components and precipitates with the finished product, leading to a significant increase in PS content and further reducing the enrichment selectivity of the target product.

[0097] Ultrafiltration steps

[0098] In some embodiments of the present invention, in the ultrafiltration step, the whey obtained in the enzymatic coagulation separation step is subjected to ultrafiltration to further separate lactose, milk mineral salts and milk protein peptides and amino acids in the whey to obtain a concentrate (i.e. whey protein containing LPE milk phospholipids).

[0099] In some specific implementations, during the ultrafiltration step, the amount of water added is 2-6 times the mass of the whey (i.e., a concentration factor of 2-6), preferably 2-4 times, more preferably 2.5-4 times, and even more preferably 2.5-3.5 times, for example, 3 times.

[0100] Ultrafiltration is one of the core steps in the LPE 18:2 enrichment process of this invention. Through the sieving action of the ultrafiltration membrane, small molecule impurities, unhydrolyzed whey protein, lactose, inorganic salts, etc., in the whey can be filtered out, while larger molecular weight phospholipid components are retained, achieving phospholipid concentration and purification. The amount of water added is a core control parameter of the ultrafiltration process, directly determining the concentration factor and impurity removal efficiency: when the amount of water added is insufficient, the concentration factor of the ultrafiltration process is insufficient, the LPE 18:2 phospholipid component in the whey cannot be effectively retained, and a large amount of the target product is lost with the filtrate, resulting in a significant reduction in the LPE 18:2 content in the final product; at the same time, due to the insufficient concentration factor, non-target phospholipid components (such as PI, PE, PC, SM) are still largely retained in the product. The total phospholipid content is close to that of the optimized process, but the proportion of the target product is greatly reduced, and the enrichment selectivity is significantly decreased.

[0101] In some specific embodiments, the ultrafiltration step uses an ultrafiltration membrane with a molecular weight cutoff of 1-50 KD, preferably 3-50 KD, more preferably 3-40 KD, even more preferably 3-30 KD, even more preferably 3-20 KD, and even more preferably 3-15 KD, 3-10 KD, or 3-8 KD, such as a 3 KD ultrafiltration membrane, a 5 KD ultrafiltration membrane, or an 8 KD ultrafiltration membrane.

[0102] Enzymatic coagulation separation only achieves the initial release of phospholipid components. The whey still contains a large amount of small molecule impurities such as whey protein, lactose, and inorganic salts, resulting in extremely low concentrations of phospholipid components. Furthermore, these impurities bind to the phospholipids, making effective enrichment impossible. Without ultrafiltration concentration, the retention, concentration, and purification of phospholipid components are impossible. Almost all LPE 18:2 components are lost along with the small molecule impurities in the whey. Simultaneously, non-target PS components bind to unhydrolyzed proteins and remain largely in the final product, ultimately leading to a complete failure to achieve the desired enrichment of the target product. The results of the embodiments and comparative examples in this application fully demonstrate that ultrafiltration concentration is one of the core and indispensable steps for achieving efficient enrichment of LPE 18:2 in this invention.

[0103] Homogenization steps

[0104] In some embodiments of the present invention, in the homogenization step, the LPE-rich milk phospholipid milk component material (LPE-rich milk phospholipid milk component I) obtained in the centrifugation step is mixed with the concentrate (i.e., LPE-containing whey protein) obtained in the ultrafiltration step and homogenized.

[0105] In some specific implementations, the homogenization is carried out at a homogenization pressure of 120-240 bar.

[0106] Post-processing

[0107] In some embodiments of the present invention, after the homogenization step, it may be understood that a post-processing step may also be included.

[0108] In some optional embodiments, the post-processing steps include at least one of sterilization, concentration, drying, and packaging.

[0109] In some optional embodiments, the sterilization temperature is 70-85°C and the holding time is 12-24 seconds.

[0110] In some alternative implementations, the concentration is achieved through evaporation.

[0111] In some specific implementation schemes, a triple-effect evaporator is used to concentrate the material, with the first effect temperature at 75-80℃, the second effect temperature at 70-75℃, and the third effect temperature at 65-70℃, concentrating the material to a solid content of 26-32%.

[0112] In some alternative implementations, the drying is performed using spray drying.

[0113] In some specific implementations, the inlet air temperature of the spray dryer is 160-195°C, and the exhaust air temperature is 60-85°C.

[0114] In some alternative implementations, the packaging includes packaging, coding, boxing, and final warehousing.

[0115] whey protein powder rich in LPE

[0116] In some aspects of the present invention, a whey protein powder rich in LPE is provided, which is prepared by the method for preparing whey protein powder rich in LPE as described above.

[0117] Application of whey protein powder rich in LPE

[0118] In some aspects of the present invention, the use of LPE-rich whey protein powder in the preparation of food is provided. The LPE-rich whey protein powder is prepared by the method for preparing LPE-rich whey protein powder as described above.

[0119] In some embodiments, the food is infant food, children's food, adolescent food, maternal food, adult food, or food for the middle-aged and elderly. In some embodiments, the food of the present invention is candy, such as hard candy, gel candy, shortbread candy, compressed candy, and aerated candy.

[0120] In some embodiments, the food described in this invention is a beverage, such as carbonated beverages, tea beverages, coffee beverages, fruit and vegetable juice beverages, and lactic acid bacteria beverages. In some embodiments, the food described in this invention is a dairy product, such as milk powder, cheese, yogurt, and liquid milk. In some embodiments, the food described in this invention is a baked product, such as bread, cakes, and biscuits. In some embodiments, the food described in this invention is a dietary supplement, such as hard capsules, soft capsules, tablets, oral liquids, pills, granules, and powders.

[0121] In some embodiments, the food described in this invention is in the form of a liquid or a solid under normal temperature conditions.

[0122] In addition to the whey protein powder containing LPE of the present invention, the food may also contain other ingredients, such as common food ingredients such as protein / amino acids, carbohydrates, fats, vitamins, and minerals.

[0123] Furthermore, depending on the type of food and the end needs of the target audience, in some embodiments, the food contains any one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, functional additives, and any acceptable excipients.

[0124] Examples of plant-based ingredients include fruits such as figs, pomegranates, kiwis, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, amla, and bilberries, or their extracts; fruits and vegetables such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, and oregano, or their extracts; grains such as rice (indica, japonica, glutinous rice), cereals (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow millet, buckwheat, soybeans, broad beans, peas, mung beans, red beans, and kidney beans, or their extracts; nuts such as walnuts, pistachios, cashews, hazelnuts, almonds, apricot kernels, pine nuts, peanuts, sunflower seeds, chestnuts, macadamia nuts, and ginkgo nuts, or their extracts; coffee or its extracts; and some medicinal and edible herbal medicines or their extracts.

[0125] Animal dairy product ingredients can include fresh milk from cows and sheep, as well as reprocessed dairy products such as whole milk powder, skim milk powder, whey protein concentrate, desalted whey powder, whey protein powder, hydrolyzed whey protein powder, and casein powder.

[0126] Examples of animal meat product ingredients include pork, beef, mutton, seafood, and poultry.

[0127] Examples of functional additives include vitamin supplements (such as vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, biotin, etc.), mineral supplements (such as iron, copper, manganese, zinc, cobalt, molybdenum, chromium, nickel, vanadium, fluorine, selenium, iodine, silicon, tin, etc.), nucleotide supplements (e.g.), dietary fiber (e.g., inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, soybean fiber, etc.), and functional polyunsaturated fatty acid supplements (e.g., arachidonic acid oil powder, docosahexaenoic acid oil powder, etc.).

[0128] Any acceptable excipients may include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, and food colorings.

[0129] like Figure 1 As shown, an exemplary embodiment of the present invention includes:

[0130] 1. Standardization: Standardize the milk raw material containing milk phospholipids and adjust its solid content to 8-20%, that is, standardize the milk raw material containing milk phospholipids; wherein the milk raw material containing milk phospholipids can be one or more of whey and whey powder.

[0131] 2. Filtration and impurity removal: The standardized milk phospholipid emulsion from step 1 is passed through a 100-mesh bag filter to remove impurities.

[0132] 3. Centrifugal Separation I: The standardized milk phospholipid-containing emulsion, after filtration and impurity removal, is heated to 50-55℃ for milk fat separation. Specifically, an industrial disc separator is used at a speed of 3500-5000 r / min to separate the fat containing LPE milk phospholipids (milk component I rich in LPE milk phospholipids) from milk protein and other milk components such as lactose (material containing milk protein components). The milk component I rich in LPE milk phospholipids is obtained and cooled to 10-15℃ for temporary storage. The material containing milk protein components is cooled to 30-40℃ for temporary storage.

[0133] 4. Enzymatic Coagulation Separation II: Add rennet to the milk protein-containing material obtained in step 3. The amount of rennet added per ton of milk protein-containing material is 75-85 mL. The coagulation time is 15-45 min. After demulsification and stirring, use a fully automated press to separate and remove the coagulated solids to obtain whey. Pour the whey into the ultrafiltration system. The ratio of water added to the protein side material in the ultrafiltration system is 2-4. Obtain the ultrafiltration system concentrate (i.e., whey protein solution containing LPE milk phospholipids) and store it for later use.

[0134] The rennet described in step 4 has an enzyme activity of 600-800 U / mL, is derived from Aspergillus niger, and is produced by Fujian Fuda Baite Biotechnology Co., Ltd.

[0135] The ultrafiltration system described in step 4 uses a 5-30KD ultrafiltration membrane, manufactured by Tongzhou Zongheng (Xiamen) Fluid Technology Co., Ltd.

[0136] 5. Homogenization: Mix and homogenize the LPE-rich milk phospholipid-rich milk component I obtained in step 3 with the LPE-containing whey protein solution obtained in step 4. The homogenization pressure is 120-240 bar.

[0137] 6. Sterilization: Sterilization temperature 70-85℃, heat preservation time 12-24s.

[0138] 7. Evaporation and Concentration: The material is concentrated using a triple-effect evaporator. The temperature of the first effect is 75-80℃, the temperature of the second effect is 70-75℃, and the temperature of the third effect is 65-70℃, concentrating to 26-32% solids.

[0139] 8. Spray drying: Inlet air temperature 160-195℃, outlet air temperature 60-85℃.

[0140] 9. Packaging: Packaging, coding, boxing, and finally warehousing.

[0141] Example

[0142] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0143] Example 1:

[0144] 1. Standardization: The milk raw material containing milk phospholipids is standardized and its solid content is adjusted to 8% (i.e., 8kg / 100L, the meaning of the solid content % unit in the subsequent examples and comparative examples is the same), that is, the standardized milk phospholipid emulsion; wherein the milk raw material containing milk phospholipids can be one or more of whey and whey powder.

[0145] 2. Filtration and impurity removal: The standardized milk phospholipid emulsion from step 1 is passed through a 100-mesh bag filter to remove impurities.

[0146] 3. Centrifugal Separation I: The standardized milk phospholipid-containing emulsion obtained in step 2 after filtration and impurity removal is heated to 50°C for milk fat separation. Specifically, an industrial disc separator (processing capacity 30t / h, the same in subsequent examples and comparative examples) is used. The separator speed is 5000r / min. The fat containing LPE milk phospholipids (milk component I rich in LPE milk phospholipids) is separated from milk protein and other milk components such as lactose (material containing milk protein components). The milk component I rich in LPE milk phospholipids is obtained and cooled to 10°C for temporary storage. The material containing milk protein components is obtained and cooled to 30°C for temporary storage.

[0147] 4. Enzymatic Coagulation Separation II: Add rennet to the milk protein-containing material obtained in step 3. The amount of rennet added per ton of milk protein-containing material is 85 mL. The coagulation time is 15 min. The mixture is broken up and stirred (stirring speed: 5 rpm; using a CV-5000DO coagulation tank, purchased from Changchun Jizhi Intelligent Manufacturing Technology Co., Ltd., model CV-5000DO). Use a fully automated press to separate and remove the coagulated solids (pressing pressure: 0.1 MPa; CP-66A fully automated press, purchased from Changchun Jizhi Intelligent Manufacturing Technology Co., Ltd., model CP-66A) to obtain whey. The whey is then fed into an ultrafiltration system. The ratio of water added to the protein side material in the ultrafiltration system is 2. The concentrated solution obtained from the ultrafiltration system (i.e., whey protein solution containing LPE milk phospholipids) is temporarily stored for later use.

[0148] The rennet described in step 4 has an enzyme activity of 600 U / mL, is derived from Aspergillus niger, and is produced by Fujian Fuda Baite Biotechnology Co., Ltd.

[0149] The ultrafiltration system described in step 4 uses a 30KD ultrafiltration membrane, manufactured by Tongzhou Zongheng (Xiamen) Fluid Technology Co., Ltd.

[0150] 5. Homogenization: The LPE-rich milk phospholipid component I obtained in step 3 is mixed and homogenized with the LPE-containing whey protein solution obtained in step 4. The homogenization pressure is 120 bar.

[0151] 6. Sterilization: Sterilization temperature 70℃, heat preservation time 24s.

[0152] 7. Evaporation and Concentration: The material is concentrated using a triple-effect evaporator. The temperature of the first effect is 75℃, the temperature of the second effect is 70℃, and the temperature of the third effect is 65℃, concentrating to 26% solids.

[0153] 8. Spray drying: Inlet air temperature 195℃, outlet air temperature 85℃.

[0154] 9. Packaging: Packaging, coding, boxing, and finally warehousing.

[0155] Example 2:

[0156] 1. Standardization: Standardize the milk raw material containing milk phospholipids and adjust its solid content to 20%, that is, standardize the milk raw material containing milk phospholipids; wherein the milk raw material containing milk phospholipids can be one or more of whey and whey powder.

[0157] 2. Filtration and impurity removal: The standardized milk phospholipid emulsion from step 1 is passed through a 100-mesh bag filter to remove impurities.

[0158] 3. Centrifugal Separation I: The standardized milk phospholipid-containing emulsion obtained in step 2 after filtration and impurity removal is heated to 55°C for milk fat separation. Specifically, an industrial disc separator is used at a speed of 3500 r / min to separate the fat containing LPE milk phospholipids (milk component I rich in LPE milk phospholipids) from milk protein and other milk components such as lactose (material containing milk protein components). The milk component I rich in LPE milk phospholipids is obtained and cooled to 15°C for temporary storage. The material containing milk protein components is cooled to 40°C for temporary storage.

[0159] 4. Enzymatic Coagulation Separation II: Add rennet to the milk protein-containing material obtained in step 3. The amount of rennet added per ton of milk protein-containing material is 75 mL. The coagulation time is 45 min. The mixture is broken up and stirred (stirring speed: 60 rpm; using a CV-5000DO coagulation tank, purchased from Changchun Jizhi Intelligent Manufacturing Technology Co., Ltd., model CV-5000DO). Use a fully automated press to separate and remove the coagulated solids (pressing pressure: 0.7 MPa; CP-66A fully automated press, purchased from Changchun Jizhi Intelligent Manufacturing Technology Co., Ltd., model CP-66A) to obtain whey. The whey is then fed into an ultrafiltration system. The water replenishment volume of the ultrafiltration system is 4 times the mass of the protein-side material. The ultrafiltration system concentrate (i.e., whey protein solution containing LPE milk phospholipids) is obtained and temporarily stored for later use.

[0160] The rennet described in step 4 has an enzyme activity of 600 U / mL, is derived from Aspergillus niger, and is produced by Fujian Fuda Baite Biotechnology Co., Ltd.

[0161] The ultrafiltration system described in step 4 uses a 10KD ultrafiltration membrane, manufactured by Tongzhou Zongheng (Xiamen) Fluid Technology Co., Ltd.

[0162] 5. Homogenization: The LPE-rich milk phospholipid component I obtained in step 3 is mixed and homogenized with the LPE-containing whey protein solution obtained in step 4. The homogenization pressure is 240 bar.

[0163] 6. Sterilization: Sterilization temperature 85℃, heat preservation time 12s.

[0164] 7. Evaporation and Concentration: The material is concentrated using a triple-effect evaporator. The temperature of the first effect is 80℃, the temperature of the second effect is 75℃, and the temperature of the third effect is 70℃, concentrating to 32% solids.

[0165] 8. Spray drying: Inlet air temperature 160℃, outlet air temperature 60℃.

[0166] 9. Packaging: Packaging, coding, boxing, and finally warehousing.

[0167] Example 3:

[0168] 1. Standardization: Standardize the milk raw material containing milk phospholipids and adjust its solid content to 16%, that is, standardize the milk raw material containing milk phospholipids; wherein the milk raw material containing milk phospholipids can be one or more of whey and whey powder.

[0169] 2. Filtration and impurity removal: The standardized milk phospholipid emulsion from step 1 is passed through a 100-mesh bag filter to remove impurities.

[0170] 3. Centrifugal Separation I: The standardized milk phospholipid-containing emulsion obtained in step 2 after filtration and impurity removal is heated to 53°C for milk fat separation. Specifically, an industrial disc separator is used at a speed of 4500 r / min to separate the fat containing LPE milk phospholipids (milk component I rich in LPE milk phospholipids) from milk protein and other milk components such as lactose (material containing milk protein components). The milk component I rich in LPE milk phospholipids is obtained and cooled to 12°C for temporary storage. The material containing milk protein components is cooled to 35°C for temporary storage.

[0171] 4. Enzymatic Coagulation Separation II: Add rennet to the milk protein-containing material obtained in step 3. The amount of rennet added per ton of milk protein-containing material is 80 mL. The coagulation time is 30 min. Demulsification and stirring are performed (stirring speed: 15 rpm; using a CV-5000DO coagulation tank, purchased from Changchun Jizhi Intelligent Manufacturing Technology Co., Ltd., model CV-5000DO). Use a fully automated press to separate and remove the coagulated solids (pressing pressure: 0.5 MPa; CP-66A fully automated press, purchased from Changchun Jizhi Intelligent Manufacturing Technology Co., Ltd., model CP-66A). Whey liquid is obtained and pumped into the ultrafiltration system. The ratio of water added to the protein side material in the ultrafiltration system is 3. The ultrafiltration system concentrate (i.e., whey protein liquid containing LPE milk phospholipids) is temporarily stored for later use.

[0172] The rennet described in step 4 has an enzyme activity of 600 U / mL, is derived from Aspergillus niger, and is produced by Fujian Fuda Baite Biotechnology Co., Ltd.

[0173] The ultrafiltration system described in step 4 uses a 5KD ultrafiltration membrane, manufactured by Tongzhou Zongheng (Xiamen) Fluid Technology Co., Ltd.

[0174] 5. Homogenization: The LPE-rich milk phospholipid component I obtained in step 3 is mixed and homogenized with the LPE-containing whey protein solution obtained in step 4. The homogenization pressure is 180 bar.

[0175] 6. Sterilization: Sterilization temperature 80℃, heat preservation time 18s.

[0176] 7. Evaporation and Concentration: The material is concentrated using a triple-effect evaporator. The temperature of the first effect is 78℃, the temperature of the second effect is 73℃, and the temperature of the third effect is 68℃, concentrating to 26-32% solids.

[0177] 8. Spray drying: Inlet air temperature 180℃, outlet air temperature 70℃.

[0178] 9. Packaging: Packaging, coding, boxing, and finally warehousing.

[0179] Comparative Example

[0180] Comparative Example 1:

[0181] 1. Standardization: Standardize the milk raw material containing milk phospholipids and adjust its solid content to 16%, that is, standardize the milk raw material containing milk phospholipids; wherein the milk raw material containing milk phospholipids can be one or more of whey and whey powder.

[0182] 2. Filtration and impurity removal: The standardized milk phospholipid emulsion from step 1 is passed through a 100-mesh bag filter to remove impurities.

[0183] 3. Centrifugal Separation I: The standardized milk phospholipid-containing emulsion obtained in step 2 after filtration and impurity removal is heated to 53°C for milk fat separation. Specifically, an industrial disc separator is used at a speed of 4500 r / min to separate the fat containing LPE milk phospholipids (milk component I rich in LPE milk phospholipids) from milk protein and other milk components such as lactose (material containing milk protein components). The milk component I rich in LPE milk phospholipids is obtained and cooled to 12°C for temporary storage. The material containing milk protein components is cooled to 35°C for temporary storage.

[0184] 4. Enzymatic Coagulation Separation II: Add rennet to the milk protein-containing material obtained in step 3. The amount of rennet added per ton of milk protein-containing material is 20 mL. The coagulation time is 10 min. Demulsification and stirring are performed (stirring speed: 15 rpm; using a CV-5000DO coagulation tank, purchased from Changchun Jizhi Intelligent Manufacturing Technology Co., Ltd., model CV-5000DO). Use a fully automated press to separate and remove the coagulated solids (pressing pressure: 0.5 MPa; CP-66A fully automated press, purchased from Changchun Jizhi Intelligent Manufacturing Technology Co., Ltd., model CP-66A). Whey liquid is obtained and pumped into the ultrafiltration system. The ratio of water added to the protein side material in the ultrafiltration system is 3. The ultrafiltration system concentrate (i.e., whey protein liquid containing LPE milk phospholipids) is temporarily stored for later use.

[0185] The rennet described in step 4 has an enzyme activity of 600 U / mL, is derived from Aspergillus niger, and is produced by Fujian Fuda Baite Biotechnology Co., Ltd.

[0186] The ultrafiltration system described in step 4 uses a 5KD ultrafiltration membrane, manufactured by Tongzhou Zongheng (Xiamen) Fluid Technology Co., Ltd.

[0187] 5. Homogenization: The LPE-rich milk phospholipid component I obtained in step 3 is mixed and homogenized with the LPE-containing whey protein solution obtained in step 4. The homogenization pressure is 180 bar.

[0188] 6. Sterilization: Sterilization temperature 80℃, heat preservation time 18s.

[0189] 7. Evaporation and Concentration: The material is concentrated using a triple-effect evaporator. The temperature of the first effect is 78℃, the temperature of the second effect is 73℃, and the temperature of the third effect is 68℃, concentrating to 26-32% solids.

[0190] 8. Spray drying: Inlet air temperature 180℃, outlet air temperature 70℃.

[0191] 9. Packaging: Packaging, coding, boxing, and finally warehousing.

[0192] Comparative Example 2:

[0193] 1. Standardization: Standardize the milk raw material containing milk phospholipids and adjust its solid content to 16%, that is, standardize the milk raw material containing milk phospholipids; wherein the milk raw material containing milk phospholipids can be one or more of whey and whey powder.

[0194] 2. Filtration and impurity removal: The standardized milk phospholipid emulsion from step 1 is passed through a 100-mesh bag filter to remove impurities.

[0195] 3. Centrifugal Separation I: The standardized milk phospholipid-containing emulsion obtained in step 2 after filtration and impurity removal is heated to 53°C for milk fat separation. Specifically, an industrial disc separator is used at a speed of 4500 r / min to separate the fat containing LPE milk phospholipids (milk component I rich in LPE milk phospholipids) from milk protein and other milk components such as lactose (material containing milk protein components). The milk component I rich in LPE milk phospholipids is obtained and cooled to 12°C for temporary storage. The material containing milk protein components is cooled to 35°C for temporary storage.

[0196] 4. Enzymatic Coagulation Separation II: Add rennet to the milk protein-containing material obtained in step 3. The amount of rennet added per ton of milk protein-containing material is 80 mL. The coagulation time is 30 min. Demulsification and stirring are performed (stirring speed: 15 rpm; using a CV-5000DO coagulation tank, purchased from Changchun Jizhi Intelligent Manufacturing Technology Co., Ltd., model CV-5000DO). Use a fully automated press to separate and remove the coagulated solids (pressing pressure: 0.5 MPa; CP-66A fully automated press, purchased from Changchun Jizhi Intelligent Manufacturing Technology Co., Ltd., model CP-66A). Whey liquid is obtained and pumped into the ultrafiltration system. The water replenishment volume of the ultrafiltration system is equal to the mass of the protein-side material. The ultrafiltration system concentrate (i.e., whey protein liquid containing LPE milk phospholipids) is obtained and temporarily stored for later use.

[0197] The rennet described in step 4 has an enzyme activity of 600 U / mL, is derived from Aspergillus niger, and is produced by Fujian Fuda Baite Biotechnology Co., Ltd.

[0198] The ultrafiltration system described in step 4 uses a 5KD ultrafiltration membrane, manufactured by Tongzhou Zongheng (Xiamen) Fluid Technology Co., Ltd.

[0199] 5. Homogenization: The LPE-rich milk phospholipid component I obtained in step 3 is mixed and homogenized with the LPE-containing whey protein solution obtained in step 4. The homogenization pressure is 180 bar.

[0200] 6. Sterilization: Sterilization temperature 80℃, heat preservation time 18s.

[0201] 7. Evaporation and Concentration: The material is concentrated using a triple-effect evaporator. The temperature of the first effect is 78℃, the temperature of the second effect is 73℃, and the temperature of the third effect is 68℃, concentrating to 26-32% solids.

[0202] 8. Spray drying: Inlet air temperature 180℃, outlet air temperature 70℃.

[0203] 9. Packaging: Packaging, coding, boxing, and finally warehousing.

[0204] Comparative Example 3:

[0205] 1. Standardization: Standardize the milk raw material containing milk phospholipids and adjust its solid content to 16%, that is, standardize the milk raw material containing milk phospholipids; wherein the milk raw material containing milk phospholipids can be one or more of whey and whey powder.

[0206] 2. Filtration and impurity removal: The standardized milk phospholipid emulsion from step 1 is passed through a 100-mesh bag filter to remove impurities.

[0207] 3. Centrifugal Separation I: The standardized milk phospholipid-containing emulsion obtained in step 2 after filtration and impurity removal is heated to 53°C for milk fat separation. Specifically, an industrial disc separator is used at a speed of 4500 r / min to separate the fat containing LPE milk phospholipids (milk component I rich in LPE milk phospholipids) from milk protein and other milk components such as lactose (material containing milk protein components). The milk component I rich in LPE milk phospholipids is obtained and cooled to 12°C for temporary storage. The material containing milk protein components is cooled to 35°C for temporary storage.

[0208] 4. Enzymatic Coagulation Separation II: Add rennet to the milk protein-containing material obtained in step 3. The amount of rennet added per ton of milk protein-containing material is 80 mL. The coagulation time is 30 min. The mixture is broken up and stirred (stirring speed: 15 rpm; using a CV-5000DO coagulation tank, purchased from Changchun Jizhi Intelligent Manufacturing Technology Co., Ltd., model CV-5000DO). The coagulated solids are separated and removed using a fully automated press (pressing pressure: 0.5 MPa; CP-66A fully automated press, purchased from Changchun Jizhi Intelligent Manufacturing Technology Co., Ltd., model CP-66A). The resulting whey is temporarily stored for later use.

[0209] The rennet described in step 4 has an enzyme activity of 600 U / mL, is derived from Aspergillus niger, and is produced by Fujian Fuda Baite Biotechnology Co., Ltd.

[0210] 5. Homogenization: The LPE-rich milk phospholipid component I obtained in step 3 is mixed and homogenized with the LPE-containing whey protein solution obtained in step 4. The homogenization pressure is 180 bar.

[0211] 6. Sterilization: Sterilization temperature 80℃, heat preservation time 18s.

[0212] 7. Evaporation and Concentration: The material is concentrated using a triple-effect evaporator. The temperature of the first effect is 78℃, the temperature of the second effect is 73℃, and the temperature of the third effect is 68℃, concentrating to 26-32% solids.

[0213] 8. Spray drying: Inlet air temperature 180℃, outlet air temperature 70℃.

[0214] 9. Packaging: Packaging, coding, boxing, and finally warehousing.

[0215] Test case

[0216] Test Example 1: Content Verification

[0217] The samples from each embodiment and comparative example before evaporation and concentration were tested. LPE18:2 (1-acyl-2-hydroxy type) was detected using high-performance liquid chromatography (HPLC). 1 mL of the test sample was transferred to a 50 mL centrifuge tube, mixed, and then 1 mL of water was added. The mixture was vortexed to mix. 10 mL of chloroform-methanol (2+1, i.e., 2 volumes of chloroform + 1 volume of methanol (similar expression below), v / v) solution was added, and the mixture was extracted by vortexing for 30 s. The mixture was then centrifuged at 8000 r / min for 10 min at 4 °C. The lower layer was transferred to another centrifuge tube. 1 mL of water was added to the upper layer, and the mixture was mixed. 10 mL of chloroform-methanol (2+1, v / v) solution was added, and the mixture was vortexed for 30 s. The mixture was then centrifuged at 8000 r / min for 10 min at 4 °C. The lower layer liquid was transferred again and the lower layer supernatants from the two extractions were combined. 1 mL of potassium chloride solution was added and mixed well. The mixture was then centrifuged at 8000 r / min, 10 min, 4℃. The entire lower layer liquid was transferred to a 50 mL centrifuge tube and the solvent was evaporated to dryness under a nitrogen stream at 55℃. The solution was then reconstituted with 2 mL of chloroform-methanol (9+1, v / v) solution and filtered through a 0.22 μm organic membrane filter into a sample vial for analysis. Detailed results are shown in Table 1.

[0218] Table 1:

[0219]

[0220] As shown in Table 1, the LPE 18:2 content in the three sets of embodiments of the present invention is significantly higher than that in all comparative examples, achieving efficient enrichment of the target product. Among them, the optimal process group (Example 3) had an LPE 18:2 content of 44.11 mg / 100g, which is 2.13 times the best value in the comparative examples (Comparative Example 2, 20.70 mg / 100g) and 7.64 times the lowest value in the comparative examples (Comparative Example 3, 5.77 mg / 100g). The second-best process group (Example 2) had an LPE 18:2 content of 43.64 mg / 100g, with a relative deviation of only 1.08% from Example 3, which is also 2.11 times that of Comparative Example 2 and 7.56 times that of Comparative Example 3. Even in Example 1 with the most conservative parameters, the LPE 18:2 content reached 27.96 mg / 100g, which is 35.07% higher than Comparative Example 2, 45.78% higher than Comparative Example 1, and 4.85 times that of Comparative Example 3.

[0221] Test Example 2: Effect of different process conditions on the enrichment selectivity of LPE 18:2

[0222] To further evaluate the process's ability to selectively enrich LPE 18:2 molecules, this study calculated the total phospholipid content (PI+PE+PS+PC+SM) of different samples, as well as the mass percentage of LPE 18:2 in the total phospholipids. The results are shown in Table 2.

[0223] Table 2: Total phospholipid content and LPE 18:2 ratio under different process conditions

[0224]

[0225] As shown in Table 2, this invention not only increases the absolute content of LPE 18:2, but also significantly increases its proportion in total phospholipids, achieving highly selective enrichment of LPE 18:2 molecules. Specifically, the LPE 18:2 proportion in Example 1 reached 1.83%, the highest among all samples, being 3.51 times that of Comparative Example 1, 6.10 times that of Comparative Example 2, and 12.8 times that of Comparative Example 3. The LPE 18:2 proportion in Example 3 was 0.913%, also significantly higher than all comparative examples, being 1.75 times that of Comparative Example 1, 3.04 times that of Comparative Example 2, and 6.38 times that of Comparative Example 3.

[0226] It is worth noting that this invention breaks the limitation of the prior art that "total phospholipid content is positively correlated with LPE 18:2 content," and achieves targeted enrichment of the target molecular species. For example, the total phospholipid content of Comparative Example 1 is 3.677 g / 100 g, which is 2.4 times that of Example 1 (1.526 g / 100 g), but its LPE 18:2 content is only 68.6% of that of Example 1, and its LPE 18:2 percentage is only 28.5% of that of Example 1; the total phospholipid content of Comparative Example 2 is 6.906 g / 100 g, slightly higher than that of Example 2 (6.693 g / 100 g), but its LPE 18:2 content is only 47.4% of that of Example 2, and its LPE 18:2 percentage is only 46.0% of that of Example 2.

[0227] The above results fully demonstrate that the present invention achieves highly selective enrichment of LPE18:2 molecules through optimization of core parameters, significantly reduces the proportion of non-target phospholipid components, solves the core technical bottleneck of existing technologies that cannot achieve targeted enrichment of milk-derived LPE18:2 molecules, can significantly reduce the cost of subsequent separation and purification, and enhance the core competitiveness of the product.

[0228] The mechanism by which core process parameters affect the enrichment effect of LPE 18:2:

[0229] (1) The regulatory effect of rennet addition amount on coagulation time

[0230] Comparative Example 1 used a rennet addition of 20 mL / ton and a coagulation time of 10 min, which is much lower than the 75-85 mL / ton and 15-45 min of the present invention. The results showed that the LPE 18:2 content of Comparative Example 1 was only 19.18 mg / 100 g, which was 56.52% lower than that of Example 3. The proportion of LPE 18:2 in the total phospholipids was only 0.522%, which was 71.5% lower than that of Example 1. At the same time, the content of non-target PS components reached 0.682 g / 100 g, which was one of the highest values ​​among all samples.

[0231] (2) The regulating effect of ultrafiltration water supply

[0232] Comparative Example 2 used an ultrafiltration makeup water volume of 1 volume, which is much lower than the 2 to 4 times of that in the present invention. The results showed that the LPE 18:2 content of Comparative Example 2 was only 20.70 mg / 100g, which was 53.07% lower than that of Example 3. The proportion of LPE 18:2 in the total phospholipids was only 0.300%, which was one of the lowest values ​​among all comparative examples. Although its total phospholipid content was close to that of Example 2, the proportion of the target product was significantly reduced.

[0233] (3) The decisive role of the ultrafiltration concentration step

[0234] Comparative Example 3 completely omitted the core ultrafiltration concentration step of this process, and only performed enzymatic coagulation separation before proceeding to subsequent processing. The results showed that the LPE 18:2 content of Comparative Example 3 was only 5.77 mg / 100g, which was the lowest value among all samples, only 13.08% of that of Example 3. The proportion of LPE 18:2 in total phospholipids was only 0.143%, which was the lowest value among all samples. At the same time, the content of non-target PS components reached 0.707 g / 100g, which was the highest value among all samples.

[0235] (4) Analysis of process stability and industrial applicability

[0236] The core parameters of this process were all set within a wide optimization range, including solids content of 8%–20%, centrifugation speed of 3500–5000 r / min, rennet addition of 75–85 mL / ton, coagulation time of 15–45 min, water replenishment of 2–4 times, and homogenization pressure of 120–240 bar. Within this parameter range, the LPE 18:2 content in Examples 2 and 3 reached 43.64 mg / 100g and 44.11 mg / 100g, respectively, with a relative deviation of only 1.08%, both achieving highly efficient enrichment of LPE 18:2.

[0237] This result demonstrates that the core parameters of this invention possess strong fault tolerance. Even with minor fluctuations in parameters during industrial production, the stability of the product's core indicators can be guaranteed, significantly reducing the control difficulty of industrial production and making it fully feasible for industrial-scale production. Furthermore, this process employs food-grade physical separation and enzymatic hydrolysis techniques throughout, without organic reagents, heavy metal catalysts, or harsh reaction conditions involving high temperature and pressure. Unlike existing chemical synthesis processes for LPE 18:2, this process meets raw material safety standards and possesses extremely high commercial application value.

[0238] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0239] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing whey protein powder rich in LPE 18:2, wherein, The preparation method includes: The centrifugation separation step involves centrifuging the pretreated milk phospholipid-containing raw material to separate the fat-containing and protein-containing components in the pretreated milk phospholipid-containing raw material, thereby obtaining a material rich in LPE milk phospholipid components and a material containing milk protein components. The enzymatic coagulation separation step involves treating the milk protein-containing material obtained in the centrifugal separation step with rennet and separating the whey. The ultrafiltration step involves subjecting the whey obtained in the enzyme coagulation separation step to ultrafiltration to obtain a concentrate. The homogenization step involves mixing the LPE-rich milk phospholipid-rich milk component obtained in the centrifugation step with the concentrate obtained in the ultrafiltration step and homogenizing it. In the enzymatic coagulation separation step, the amount of rennet added is 50-100 mL / ton of milk protein-containing material, and the coagulation time is 15-45 min. In the ultrafiltration step, the amount of water added is 2-6 times the mass of the whey solution. In the enzymatic coagulation and separation step, the rennet activity is above 600 U / mL, and the rennet is derived from Aspergillus niger (…). Aspergillus niger ); In the ultrafiltration step, the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 5-30KD. The milk raw material containing milk phospholipids is at least one of whey and whey powder; The pretreatment includes a standardization process for the milk phospholipid-containing raw material, wherein the standardization process adjusts the solids content in the milk phospholipid-containing raw material to 5-25 kg / 100 L.

2. The preparation method according to claim 1, wherein, In the centrifugal separation step, a disc centrifuge is used with a rotation speed of 2000-7000 r / min and a processing capacity of 10-45 t / h.

3. The preparation method according to claim 1, wherein, The pretreatment includes removing impurities from the milk raw material containing milk phospholipids.

4. The preparation method according to claim 3, wherein, The impurity removal process is implemented through filtration.

5. Uses of whey protein powder rich in LPE 18:2 in food preparation, among which, The whey protein powder rich in LPE 18:2 is prepared by the preparation method of the whey protein powder rich in LPE 18:2 as described in any one of claims 1-4.

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