Preparation method of lactoferrin standard substance in formula milk powder and finished product thereof

Abstract

The present application belongs to the technical field of standard substance preparation, and in particular relates to a preparation method of lactoferrin standard substance in formula milk powder, which comprises the steps of milk cleaning, lactoferrin preparation, ingredient mixing and drying. The preparation method provided by the present application solves the contradiction between the uniformity of lactoferrin standard substance in formula milk powder and the long-term storage requirement of the sample by adopting innovative processes such as low-temperature mixing, membrane filtration cold sterilization and low-temperature spray drying, and the prepared standard substance sample meets the requirements of the national certified standard substance regulations in all aspects. Meanwhile, the preparation method provided by the present application does not involve external purchase of lactoferrin standard sample, the process starts from the preparation of lactoferrin, and the preparation of standard samples with different gradients of lactoferrin content under the conditions of various complex formula milk powders can be realized, which greatly reduces the cost of lactoferrin detection and has a very broad market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of standard substance preparation technology, specifically relating to the preparation method of lactoferrin standard substance in formula milk powder and its finished product. Background Technology

[0002] Lactoferrin, an iron-binding glycoprotein, not only participates in iron transport but also possesses powerful biological functions such as broad-spectrum antibacterial, antioxidant, anticancer, and immune system regulation. In recent years, lactoferrin has been widely used in infant formula. However, in actual testing, two lactoferrin standards (CAS No.: 936541-36-5 and CAS No.: 146897-68-9) produced by GE Healthcare (USA) are primarily used as quality control samples, which are extremely expensive, costing as much as 150,000 RMB per gram. Because a more economical and practical lactoferrin standard substance for infant formula has not yet been developed, infant formula manufacturers and testing institutions can only purchase expensive lactoferrin standards.

[0003] Meanwhile, in order to detect lactoferrin in dairy products, researchers have conducted various studies, such as:

[0004] Reference 1 discloses a reliability verification method for lactoferrin detection equipment, which includes steps such as standard preparation, solution preparation, repeated washing, enzyme-labeled antibody incubation, repeated washing, substrate colorimetric reaction, OD value measurement, accuracy, precision, and sensitivity testing of the detection equipment, and comparison with third-party testing. This method provides a reliability verification method for lactoferrin detection equipment by first preparing standards with different concentrations of lactoferrin, then using these standards to compare the accuracy, precision, and sensitivity of the lactoferrin detection equipment to be tested, and finally using a third-party testing institution for comparison. This effectively verifies the reliability of the lactoferrin detection equipment and ensures its detection capability is truly effective. However, in this technical solution, although skim milk powder was used to prepare the lactoferrin standard in skim milk powder when preparing the quality control sample, it still used externally purchased lactoferrin standard samples.

[0005] Reference 2 discloses a quantitative detection kit for bovine lactoferrin and its application. The kit includes a bovine lactoferrin-specific peptide (amino acid sequence: LRPVAAEIYGTK), an isotopically labeled bovine lactoferrin-specific peptide (amino acid sequence: LRPV*AAEI*YGTK, where I* and L* are carbon and nitrogen isotopically labeled amino acids), and an isotopically labeled bovine lactoferrin internal standard (amino acid sequence: GRDPYKLRPV*AAEI*YGTKESPQTHY, where I* and L* are carbon and nitrogen isotopically labeled amino acids). While this kit can accurately quantify bovine lactoferrin in various dairy products, it primarily uses isotopic labeling rather than standard substances for detection.

[0006] In addition, there has been some research on using milk powder as a matrix to prepare standard substances, for example:

[0007] Reference 3 discloses a Staphylococcus aureus standard substance, its preparation method, and its application. The method involves freeze-drying a Staphylococcus aureus bacterial suspension with a freeze-drying agent to obtain Staphylococcus aureus powder, which is then mixed with milk powder to obtain the Staphylococcus aureus standard substance. However, this technical solution clearly fails to meet the relevant requirements of the Certified Reference Material Regulations (JJF1343-2022), as its technical approach only involves the simple mixing of the standard sample and milk powder.

[0008] References:

[0009] Reference 1: CN115165768A;

[0010] Reference 2: CN103267822A;

[0011] Reference 3: CN108690807A. Summary of the Invention

[0012] The problem the invention aims to solve

[0013] To achieve accurate detection of lactoferrin in milk powder, existing technologies have been appropriately studied at multiple levels. At the same time, there has been some research on the preparation methods of other standard substances in milk powder. However, due to the characteristics of lactoferrin itself and the special requirements of standard substance regulations on samples, research on lactoferrin standard substances in formula milk powder is still scarce.

[0014] In particular, lactoferrin is a glycoprotein and is heat-labile. Studies have found that bovine lactoferrin begins to deactivate at pH 6.6 and temperatures of 65–69°C. Furthermore, the Certified Reference Material (CRP) regulations (JJF1343-2022) impose stringent requirements on sample homogeneity, stability, concentration, and storage time. However, achieving high homogeneity of lactoferrin content in traditional milk powder manufacturing processes requires mixing during the ingredient formulation process. To meet the long-term storage requirements of the CRP, the milk ingredients must undergo pasteurization or UHT (ultra-high temperature instantaneous sterilization) followed by high-temperature spray drying to produce milk powder. However, this process severely damages lactoferrin, making it impossible to meet the required content levels. Therefore, the homogeneity of lactoferrin CRP in formula milk powder and the long-term storage requirements of the sample are two completely contradictory factors, presenting significant challenges. Moreover, the RSD (relative standard deviation) of lactoferrin content in formula milk powder prepared using traditional dry-mixing techniques is generally greater than 5%, failing to meet the homogeneity requirements of national certified reference materials.

[0015] In response, this invention proposes a method that combines innovative processes such as low-temperature mixing, membrane filtration cold sterilization, and low-temperature spray drying to prepare lactoferrin standard substances in formula milk powder that fully meet the relevant requirements of national certified reference materials in terms of uniformity, stability, fixed values, and storage time. Furthermore, it enables the preparation of standard samples with different gradients of lactoferrin content under various complex formula milk powder conditions.

[0016] Solution for solving the problem

[0017] To solve the above-mentioned technical problems, the present invention provides the following technical solution.

[0018] [1]. A method for preparing a lactoferrin standard substance in formula milk powder, wherein the method includes:

[0019] The process of milk purification involves purifying raw milk to obtain milk fat and skim milk.

[0020] The steps for preparing lactoferrin are as follows: the skim milk produced in the milk purification step is subjected to ion exchange chromatography and ultrafiltration concentration to obtain an ultrafiltration concentrate, the ultrafiltration concentrate is passed through a sterile membrane, and then optionally freeze-dried to obtain lactoferrin;

[0021] The steps for preparing the ingredients are as follows: the skim milk flow-through obtained during ion exchange chromatography in the lactoferrin preparation step is homogenized with the milk fat obtained in the purifying step to obtain raw milk; the raw milk is pasteurized to obtain pasteurized milk; wet mixing ingredients are added and homogenized in the pasteurized milk; then steam jet sterilization is performed to obtain a mixture; the mixture is mixed with the lactoferrin to obtain mixed milk.

[0022] The drying step involves preheating the mixed milk obtained in the ingredient preparation step and then performing low-temperature spray drying, which includes the following stages: spray drying in a drying tower, first-stage static bed drying, second-stage static bed drying, and pneumatic conveying drying, as well as optional vacuum drying, to obtain lactoferrin standard material in formula milk powder.

[0023] [2]. According to the preparation method described in [1], in the step of preparing lactoferrin, the sterilization membrane is a double-layer microporous filter membrane.

[0024] [3]. According to the preparation method described in [1] or [2], wherein, in the step of mixing the ingredients, the temperature of the mixture is 25 to 50°C.

[0025] [4]. According to any one of [1] to [3], in the drying step, the main air inlet temperature of the drying tower is 73 to 90°C, the main exhaust temperature is 47 to 65°C, the temperature of the first-stage static bed drying is 30 to 45°C, and the temperature of the second-stage static bed drying is 17 to 33°C.

[0026] [5]. The preparation method according to any one of [1] to [4], wherein, in the drying step, the moisture content of the inlet air for the spray drying, the first-stage static bed drying and the second-stage static bed drying is each less than or equal to 1.8 g / kg.

[0027] [6]. The preparation method according to any one of [1] to [5], wherein, in the step of adding the ingredients, when the wet mixture is added, the added ingredients include any one or more of polysaccharide components, protein components, mineral components, vitamin components and lipid components.

[0028] [7]. According to the preparation method described in [6], wherein, in the step of adding the ingredients, the lipid components do not include OPO structured triglycerides when the wet mixture is added.

[0029] [8]. The preparation method according to any one of [1] to [7], wherein, after the drying step, the preparation method further comprises a metering and packaging step.

[0030] [9]. A lactoferrin standard substance in formula milk powder, wherein the lactoferrin standard substance in formula milk powder is prepared according to any one of [1] to [8].

[0031]

[10] . According to the lactoferrin standard substance in formula milk powder described in [9], the lactoferrin content in the lactoferrin standard substance in formula milk powder is 20mg / 100g to 50mg / 100g, based on the total mass of the lactoferrin standard substance in formula milk powder.

[0032] The effects of the invention

[0033] By implementing the above technical solution, the present invention can achieve the following technical effects:

[0034] First, this invention provides a method for preparing lactoferrin standard material in formula milk powder. By adopting innovative processes such as low-temperature mixing, membrane filtration cold sterilization, and low-temperature spray drying, it simultaneously solves the contradiction between the uniformity of lactoferrin standard material in formula milk powder and the long-term storage requirements of the sample. The prepared standard material sample fully meets the requirements of the national certified reference material regulations (JJF1343-2022) regarding the uniformity, stability, value determination, and storage time of the sample.

[0035] Secondly, the method for preparing lactoferrin standard substances in formula milk powder provided by this invention does not involve the external purchase of lactoferrin standard samples. The process starts from the preparation of lactoferrin and can realize the preparation of standard samples with different gradients of lactoferrin content under various complex formula milk powder conditions, which greatly reduces the cost of lactoferrin detection and has a very broad market application prospect. Attached Figure Description

[0036] Figure 1 The production process flow diagram of the method for preparing lactoferrin standard material in formula milk powder provided by the present invention is shown below.

[0037] Figure 2 The lactoferrin freeze-dried powder produced during the production process of the method for preparing lactoferrin standard material in formula milk powder provided by this invention.

[0038] Figure 3 The present invention provides a standard substance for lactoferrin in formula milk powder.

[0039] Figure 4 This invention relates to a brown, sealed glass bottle for packaging lactoferrin standard material in the aforementioned formula milk powder. Detailed Implementation

[0040] 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 and examples are also included in the technical scope of the present invention.

[0041] Unless otherwise stated, the terminology used in this invention has the following meanings:

[0042] In this invention, the range of values ​​represented by "value A to value B" refers to the range that includes the endpoint values ​​A and B.

[0043] In this invention, the numerical range indicated by "above" or "below" refers to the numerical range that includes the stated number.

[0044] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0045] In this invention, the terms "optional" or "optionally" are used to indicate the use or non-use of certain substances, components, execution steps, application conditions, etc.

[0046] 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.

[0047] In this invention, the term "about," "substantially," or "essentially" can mean that a value includes the standard deviation of the error of the apparatus or method used to determine that value. The numerical ranges and parameters used to define this invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains a standard deviation due to the aforementioned testing apparatus or method. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified with "about." Here, "about" generally means that the standard deviation of the actual value from the theoretical model or theoretical data is within 3%, preferably 2%, more preferably 1%.

[0048] In this invention, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described 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 the elements may be combined in any suitable manner in various embodiments.

[0049] In this invention, "raw milk" refers to the liquid obtained directly from the mammary glands.

[0050] In this invention, the term "children" refers to a group of human beings who are older than 3 years and younger than 18 years, or who are older than 18 years but are still in the growth and development stage.

[0051] In this invention, the term "middle-aged and elderly" refers to the human population aged 45 years and older.

[0052] In this invention, the term "infant" refers to the human group under the age of 3 years.

[0053] In this invention, "static bed" refers to a static fluidized bed dryer.

[0054] In addition, 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.

[0055] Preparation method of lactoferrin standard substance in formula milk powder

[0056] This invention provides a method for preparing a lactoferrin standard substance in formula milk powder, comprising:

[0057] The process of milk purification involves purifying raw milk to obtain milk fat and skim milk.

[0058] The steps for preparing lactoferrin are as follows: the skim milk produced in the milk purification step is subjected to ion exchange chromatography and ultrafiltration concentration to obtain an ultrafiltration concentrate, the ultrafiltration concentrate is passed through a sterile membrane, and then optionally freeze-dried to obtain lactoferrin;

[0059] The steps for preparing the ingredients are as follows: the skim milk flow-through obtained during ion exchange chromatography in the lactoferrin preparation step is homogenized with the milk fat obtained in the purifying step to obtain raw milk; the raw milk is pasteurized to obtain pasteurized milk; wet mixing ingredients are added and homogenized in the pasteurized milk; then steam jet sterilization is performed to obtain a mixture; the mixture is mixed with the lactoferrin to obtain mixed milk.

[0060] The drying step involves preheating the mixed milk obtained in the ingredient preparation step and then performing low-temperature spray drying, which includes the following stages: spray drying in a drying tower, first-stage static bed drying, second-stage static bed drying, and pneumatic conveying drying, as well as optional vacuum drying, to obtain lactoferrin standard material in formula milk powder.

[0061] The process flow diagram of the preparation method of lactoferrin standard substance in formula milk powder provided by the present invention is as follows: Figure 1 As shown.

[0062] The formula milk powder mentioned in this invention refers to milk powder products that have been nutritionally designed according to the needs of different target groups, such as infant formula milk powder, children's formula milk powder, or formula milk powder for middle-aged and elderly people.

[0063] The preparation method provided by this invention can prepare standard samples with different gradient lactoferrin contents under various complex formula milk powder conditions.

[0064] (Steps for breast milk purification)

[0065] In the preparation method provided by the present invention, in order to facilitate the preparation of lactoferrin, it is necessary to purify the raw milk to obtain milk fat and skim milk.

[0066] In some specific implementations, the milk purification is carried out by a separator, the separator speed of which can be 3500 to 5000 r / min, for example, 3500 r / min, 3600 r / min, 4000 r / min, 4600 r / min, 4900 r / min or 5000 r / min, etc., preferably 3600 to 4900 r / min.

[0067] In some specific implementations, the raw milk is preheated before purification, and preheating can be performed using equipment such as plate heat exchangers.

[0068] (Steps for preparing lactoferrin)

[0069] The present invention utilizes the skim milk obtained in the milk purification step to prepare lactoferrin, preferably using ion exchange chromatography to prepare lactoferrin.

[0070] In some specific implementations, the preparation of lactoferrin using ion exchange chromatography includes the following steps:

[0071] a) Pass the skim milk through a cation exchange resin chromatography column;

[0072] b) The cation exchange resin in step a) is subjected to a first set of elutions consisting of at least two elutions using the eluent to obtain the eluent collection;

[0073] c) Ultrafiltration is used to desalinate the eluent collected from at least the second eluent in step b);

[0074] d) Pass the eluent collected by ultrafiltration and desalting in step c) through a cation exchange resin chromatography column;

[0075] e) Elute the cation exchange resin in step d) with the eluent to perform a second set of elutions, which includes at least two elutions, to obtain an eluent collection;

[0076] f) Mix the last eluent collected from the second group of elutions in step e) with the last eluent collected from the first group of elutions in step b), and then remove salt by ultrafiltration to obtain an ultrafiltration concentrate.

[0077] For more specific methods for obtaining ultrafiltration concentrate, please refer to document CN113105542A, the entire contents of which are incorporated herein by reference.

[0078] Furthermore, this invention innovatively uses a sterile membrane to filter and sterilize the ultrafiltration concentrate, thus avoiding the damage to lactoferrin caused by temperature rise during the process.

[0079] In some specific embodiments, the sterilization membrane is a double-layer microporous filter membrane, preferably with pore sizes of 0.2 to 0.45 μm, and more preferably with pore sizes of 0.2 μm and 0.45 μm, respectively.

[0080] In some specific embodiments, the present invention freeze-dries the sterilized ultrafiltration concentrate and grinds it into powder for later use. In some more specific embodiments, the freeze-drying includes three stages: plate pre-freezing, primary drying, and desorption drying, with a cumulative freeze-drying time of 19–24 hours. The freeze-drying can be performed in equipment such as a freeze dryer. The lactoferrin freeze-dried powder prepared by the present invention is as follows: Figure 2 As shown.

[0081] In other specific embodiments, in order to subsequently prepare standard substances with specific lactoferrin contents, this invention performs content detection on the sterilized ultrafiltration concentrate to determine the lactoferrin content, so that it can be used directly without freeze-drying. Content detection can be performed using, for example, a near-infrared online detection device.

[0082] (Ingredient preparation steps)

[0083] In order to obtain lactoferrin standard material in formula milk powder with specific content requirements, the present invention requires mixing the prepared lactoferrin with the other ingredients of formula milk powder.

[0084] First, in some embodiments, the present invention homogenizes the (complete) skim milk flow-through obtained during ion exchange chromatography in the lactoferrin preparation step with the milk fat obtained in the milk purification step to obtain raw milk. In some embodiments, the skim milk flow-through refers to the flow-through collected during a) passing the skim milk through a cation exchange resin chromatography column in the lactoferrin preparation process.

[0085] Furthermore, in some embodiments, the raw milk needs to be pasteurized to obtain pasteurized milk. In some typical embodiments, the pasteurization temperature is 70-90°C, for example, 70°C, 73°C, 75°C, 77°C, 80°C, 83°C, 85°C, 87°C, or 90°C, with a preferred pasteurization temperature of 73-87°C; the pasteurization holding time is 15-30 seconds, for example, 15 seconds, 20 seconds, 25 seconds, or 30 seconds, with a preferred pasteurization time of 15-20 seconds. Simultaneously, the pasteurization can be carried out in equipment such as a plate heat exchanger.

[0086] To prepare different formula milk powders, wet mixing can be performed on the pasteurized milk, and this process can be carried out in equipment such as a wet mixing tank. During this process, the mixing time can be controlled at 40-60 minutes, and the temperature can be controlled at 40-50℃ to avoid the loss of nutrients due to excessively high temperatures.

[0087] The ingredients that can be added include, but are not limited to, any one or more of the following: polysaccharides, proteins, minerals, vitamins, and lipids.

[0088] The polysaccharide components mainly refer to disaccharide additives and dietary fiber polysaccharides. The disaccharide additives include maltose, sucrose, and lactose, preferably lactose. The dietary fiber polysaccharides include one or more of inulin, konjac flour, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin, and soybean fiber.

[0089] Examples of protein components include whey, whey protein or its hydrolyzed protein components, and immune protein components.

[0090] The mineral composition may include additives of various metallic mineral elements required by the human body, such as calcium citrate, L-calcium lactate, calcium hydrogen phosphate, potassium gluconate, sodium citrate, ferrous gluconate, potassium iodide, zinc gluconate, sodium selenite, copper gluconate, chromium sulfate, manganese gluconate, and magnesium gluconate.

[0091] The vitamin components may include one or more of the following: 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, choline, inositol, and biotin.

[0092] The lipid components mentioned above can include various fatty acids (e.g., DHA, ARA, etc.) or triglycerides of fatty acids. These fatty acid triglycerides can be derived from various vegetable oils, such as sunflower seed oil, olive oil, sesame oil, palm oil, corn oil, coconut oil, soybean oil, flaxseed oil, camellia seed oil, etc.; or, for fatty acid triglycerides, they can also be fatty acid esters (also known as structural mixed esters) mainly containing one or more specific triglycerides. These structural mixed esters can contain appropriate amounts of OPO (1,3-dioleoyl-2-palmitoylglycerol), OPL (1-oleoyl-2-palmitoyl-3-linoleic acid triglyceride), MLCT (fatty acid esters with medium- and long-chain fatty acid triglycerides as the main component), etc. For example, in this invention, the OPO structural mixed ester is a structural mixed ester with OPO triglyceride as the main component, wherein the OPO structural mixed ester may include triglycerides with other structures such as OPL, OOL, OPP, OLO, and OLL in addition to OPO triglycerides. Furthermore, in some specific embodiments, the content of OPO triglycerides in the OPO structured mixed esters of the present invention is 53% by mass or more, preferably 55% by mass or more, more preferably 58% by mass or more, and even more preferably 60% by mass or more. In addition, there is no particular limit to the upper limit of the OPO triglyceride content; from the perspective of economy or availability, this upper limit can be 75% by mass, preferably 70% by mass, and more preferably 60% by mass.

[0093] Meanwhile, the aforementioned food ingredients can be added in the form of compound foods or compound food additives, such as whole milk powder, skim milk powder, compound minerals, compound vitamins, and compound nutrients. These food ingredients can be obtained commercially, such as commercially available whole milk powder, skim milk powder, lactose, whey protein concentrate, and demineralized whey powder.

[0094] To obtain more homogeneous standard substances and to avoid oxidation of unsaturated fatty acids due to prolonged high-temperature treatment during wet mixing, structural mixed esters, such as those with OPO-structured triglycerides as the main component, can be added after mixing with lactoferrin and then homogenized, preferably at a homogenization pressure of 200-250 bar. That is, structural mixed esters, such as those with OPO-structured triglycerides as the main component, can be added after the wet mixing but before homogenization. Furthermore, this invention has found that not all lipid components can be added after the wet mixing, because fat-soluble vitamins are added during the wet mixing process, thus requiring the simultaneous addition of appropriate lipid substances for their protection.

[0095] To avoid the destruction of lactoferrin by high temperatures, sterilization can be performed after adding the ingredients, and then the mixture can be mixed with lactoferrin. In some embodiments, the sterilization is steam injection sterilization, with a preferred sterilization temperature of 80-99°C, more preferably 85-97°C, and a preferred sterilization holding time of 15-30 seconds, more preferably 15-24 seconds.

[0096] After steam sterilization, a mixture containing the ingredients is obtained. This mixture is then mixed with lactoferrin in a specific ratio to obtain a mixed milk. In some embodiments, the amount of lactoferrin added is 0.025% to 0.045% by mass of the total dry matter of the mixture. In some embodiments, when the lactoferrin is a lyophilized powder, the mixing temperature is 40–50°C, and the mixing time is 7–15 minutes. In other embodiments, when the lactoferrin is a sterilized ultrafiltration concentrate with a defined content, the mixing temperature can be reduced to 25–30°C, and the mixing time can be shortened to 3–5 minutes.

[0097] In some embodiments, the solids content in the mixed milk is 18% (w / v) to 32% (w / v).

[0098] (Drying step)

[0099] To obtain a dried, solid powdered lactoferrin standard substance for infant formula, the mixed milk obtained in the ingredient preparation step needs to be dried. To prevent lactoferrin from being destroyed by high temperatures, this invention uses low-temperature spray drying to dry the mixed milk.

[0100] In some specific implementations, the drying step includes preheating the mixed emulsion obtained in the batching step and performing low-temperature spray drying, which includes the following stages: drying tower spray drying, first-stage static bed drying, second-stage static bed drying and pneumatic conveying drying.

[0101] In some specific implementations, the mixed milk can be preheated to 52-65°C, preferably to 55-62°C.

[0102] In some implementations, a high-pressure pump is used to spray the preheated emulsion mixture into a drying tower, preferably at a pressure of 140-220 bar. Under the given high-pressure pump conditions, the emulsion mixture can be rapidly atomized when sprayed into the drying tower.

[0103] In some embodiments, the main inlet air temperature of the drying tower is 73–90°C, for example, 73°C, 75°C, 78°C, 80°C, 83°C, 85°C, 88°C, or 90°C, preferably 75–88°C. In some embodiments, the main exhaust air temperature of the drying tower is 47–65°C, for example, 47°C, 50°C, 52°C, 55°C, 57°C, 60°C, 62°C, or 65°C, preferably 50–62°C.

[0104] In some embodiments, the temperature of the single-bed drying is 30-45°C, for example, 30°C, 32°C, 34°C, 38°C, 40°C, 42°C, 44°C or 45°C, preferably 32-44°C.

[0105] In some embodiments, the temperature of the two-stage static bed drying is 17 to 33°C, for example, it can be 17°C, 20°C, 22°C, 24°C, 28°C, 30°C, 32°C or 33°C, preferably 22 to 30°C.

[0106] In some implementations, the moisture content of the inlet air for the spray drying, the first-stage static bed drying, and the second-stage static bed drying in the drying tower is independently less than or equal to 1.8 g / kg, for example, it can be 0.8 g / kg, 1.0 g / kg, 1.2 g / kg, 1.4 g / kg, 1.6 g / kg, or 1.8 g / kg, etc.

[0107] In some specific implementations, when the moisture content of the inlet air for the spray drying, first-stage static bed drying, and second-stage static bed drying is all greater than or equal to 1.0 g / kg, vacuum drying is also required. The powdered particles after the second-stage static bed drying can be fed into a vacuum dryer via a pneumatic conveying system for further drying. The vacuum drying is preferably performed at a temperature of 50–55°C, with a preferred vacuum level of 2–3 kPaA (absolute pressure), and a preferred drying time of 30–45 minutes. Furthermore, the air used in the pneumatic conveying system can be dehumidified dry air; the process of transporting the powdered particles using dry air also serves a drying function.

[0108] In some other specific implementations, when the moisture content of the inlet air for the spray drying, the first-stage static bed drying, and the second-stage static bed drying is less than 1.0 g / kg, subsequent vacuum drying is unnecessary, which can improve production efficiency.

[0109] In some embodiments, the low-temperature spray drying is performed by a multi-stage continuous drying system, which includes a drying tower, a primary static bed, a secondary static bed, and a pneumatic conveying device. The powdered particles obtained after drying in the drying tower can enter the primary static bed for further drying. The powdered particles processed in the primary static bed can then continue to the secondary static bed for further drying under air pressure. The powdered particles processed in the secondary static bed can then be transported downstream by the pneumatic conveying device. Furthermore, the primary and secondary static beds can be two continuously arranged independent static fluidized bed dryers, or the first and second layers of a multi-layered static fluidized bed dryer, or the first and second chambers of a multi-chambered static fluidized bed dryer.

[0110] (Other steps)

[0111] In some embodiments, the method for preparing lactoferrin standard material in formula milk powder provided by the present invention further includes a step of accepting raw milk before the step of purifying milk.

[0112] In some embodiments, the method for preparing lactoferrin standard material in formula milk powder provided by the present invention further includes, after the drying step, a metering and packaging step. Typically, it can be used as follows: Figure 4 The brown, sealed glass bottle shown is used for packaging.

[0113] In some embodiments, the method for preparing lactoferrin standard material in formula milk powder provided by the present invention further includes a sampling and testing step after the metering and packaging steps.

[0114] Furthermore, in the method for preparing lactoferrin standard material in formula milk powder provided by the present invention, the process can be temporarily stopped at any step, and substances such as the ultrafiltration concentrate, the material after wet mixing and homogenization, and the mixed milk can be temporarily stored.

[0115] Standard substance of lactoferrin in formula milk powder

[0116] This invention provides a lactoferrin standard substance for formula milk powder, which is prepared according to the above preparation method.

[0117] In some embodiments, the lactoferrin standard substance in the formula milk powder is a powdered solid, such as... Figure 3 As shown.

[0118] In some embodiments, the lactoferrin content in the lactoferrin standard substance in the formula milk powder is 20 mg / 100g to 50 mg / 100g, based on the total mass of the lactoferrin standard substance in the formula milk powder.

[0119] The lactoferrin standard material in the formula milk powder provided by this invention fully meets the requirements of the national certified reference material regulations (JJF1343-2022) regarding the uniformity, stability, value determination, and storage time of the samples.

[0120] Example

[0121] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in the art.

[0122] Example 1

[0123] 1. Raw milk acceptance: Raw milk samples are taken after arriving at the factory and inspected and accepted according to the quality control plan;

[0124] 2. Temporary storage of raw milk: temporarily store in a jacketed insulated milk tank at a temperature of 0-6℃;

[0125] 3. Milk purification: Raw milk is pumped into the pasteurization system balance tank through a discharge pump. After preheating by a plate heat exchanger, it is purified by the separator at 3600 r / min. The purification process yields skim milk and milk fat. The skim milk is chromatographically separated to remove lactoferrin and then refluxed with the milk fat for homogenization as raw milk for subsequent steps.

[0126] 4. Pasteurization: The raw milk after purification in step 3 is put back into the plate heat exchanger for sterilization at a temperature of 73°C for 30 seconds.

[0127] 5. Temporary storage of pasteurized milk: After pasteurization, the pasteurized milk is cooled by ice water in a plate heat exchanger and then enters the pasteurization milk storage chamber, where it is temporarily stored at a temperature of 0-6℃.

[0128] 6. Wet Mixing Ingredients Addition and Mixing: Start the milk preparation program, pump pasteurized milk and fixed-volume RO water into the wet mixing tank, start the circulation system, and circulate the wet mixing tank and vacuum mixer. The system starts to automatically extract the metered ingredients from the powder hopper, and the materials enter the mixing circulation. The added ingredients are whole milk powder, skim milk powder, lactose, concentrated whey protein powder, demineralized whey powder, compound minerals, compound vitamins, and compound nutrients. The mixing time is controlled at 40-60 minutes. Pump the materials from the wet mixing circulation system into the wet mixing tank. The solid content of the mixed milk is 18%, and the mixing temperature is 40℃.

[0129] 7. Homogenization and Temporary Storage: Start the homogenization program. The mixed esters (a mixture of palm oil, corn oil, coconut oil and soybean oil) with OPO structure triglycerides are combined with the mixed milk at the front end of the homogenizer and enter the homogenizer. After homogenization, the mixture is cooled by a plate heat exchanger and temporarily stored in a wet mixed storage tank. The homogenization pressure is 200 bar.

[0130] 8. Steam jet sterilization: The material is pumped into the sterilizer and insulation pipe for sterilization. The sterilization temperature is 85℃ and the sterilization holding time is 15s.

[0131] 9. Lactoferrin preparation: Lactoferrin powder was prepared using the skim milk obtained in step 3. The specific steps are shown in Example 4, wherein the cumulative freeze-drying time in the freeze dryer was 24 hours.

[0132] 10. Mixing: Add the mixed milk from step 8 and the lactoferrin powder from step 9 to a high-shear mixer at a certain ratio (lactoferrin accounts for 0.035% of the total dry matter of the mixed milk). The mixing time is controlled at 7 minutes and the mixing temperature is 40°C.

[0133] 11. Preheating and feeding: After mixing, the mixed milk is temporarily stored in a balance tank, preheated to 55°C by a preheater, and then sprayed into a low-temperature drying tower under the pressure of a high-pressure pump at 140 bar.

[0134] 12. Low-temperature drying: The moisture content of the main inlet air of the drying tower is 1.0 g / kg, the temperature is 75℃, the temperature of the main exhaust air is 50℃, the moisture content of the inlet air of the first static bed is 1.0 g / kg, the temperature is 32℃, the moisture content of the inlet air of the second static bed is 1.0 g / kg, the temperature is 22℃, the formula powder collected in the second static bed enters the pneumatic powder conveying system and is carried into the vacuum dryer. The vacuum dryer jacket is heated and kept at 50℃, the vacuum degree is 2-3 kPaA (absolute pressure), and the drying time is 45 min.

[0135] 13. Measuring and Packaging: After vacuum drying, the formula milk powder enters the packaging machine for measuring and packaging, and is packaged in brown sealed glass bottles;

[0136] 14. Sampling and testing: According to the sampling plan, the lactoferrin standard substance in the prepared formula milk powder shall be sampled and tested. It can be released after passing the test.

[0137] Example 2

[0138] 1. Raw milk acceptance: Raw milk samples are taken after arriving at the factory and inspected and accepted according to the quality control plan;

[0139] 2. Temporary storage of raw milk: temporarily store in a jacketed insulated milk tank at a temperature of 0-6℃;

[0140] 3. Milk purification: Raw milk is pumped into the pasteurization system balance tank through a discharge pump. After preheating by a plate heat exchanger, it is purified by the separator at a speed of 4900 r / min. The purification process yields skim milk and milk fat. The skim milk is chromatographically separated to remove lactoferrin and then refluxed with the milk fat for homogenization as raw milk for subsequent steps.

[0141] 4. Pasteurization: The raw milk after purification in step 3 is put back into the plate heat exchanger for heating and sterilization. The sterilization temperature is 87℃ and the sterilization holding time is 15s.

[0142] 5. Temporary storage of pasteurized milk: After pasteurization, the pasteurized milk is cooled by ice water in a plate heat exchanger and then enters the pasteurization milk storage chamber, where it is temporarily stored at a temperature of 0-6℃.

[0143] 6. Wet Mixing Ingredients Addition and Mixing: Start the milk preparation program, pump pasteurized milk and fixed-volume RO water into the wet mixing tank, start the circulation system, and circulate the wet mixing tank and vacuum mixer. The system starts to automatically extract the metered ingredients from the powder hopper, and the materials enter the mixing circulation. The added ingredients are whole milk powder, skim milk powder, lactose, concentrated whey protein powder, demineralized whey powder, compound minerals, compound vitamins, and compound nutrients. The mixing time is controlled at 60 minutes. Pump the materials from the wet mixing circulation system into the wet mixing tank. The solid content of the mixed milk is 32%, and the mixing temperature is 50℃.

[0144] 7. Homogenization and Temporary Storage: Start the homogenization program. The mixed esters (a mixture of palm oil, corn oil, coconut oil and soybean oil) with OPO structure triglycerides are combined with the mixed milk at the front end of the homogenizer and enter the homogenizer. After homogenization, the mixture is cooled by a plate heat exchanger and temporarily stored in a wet mixed storage tank. The homogenization pressure is 250 bar.

[0145] 8. Steam jet sterilization: The material is pumped into the sterilizer and insulation pipe for sterilization. The sterilization temperature is 97℃ and the sterilization holding time is 15s.

[0146] 9. Lactoferrin preparation: Lactoferrin powder was prepared using the skim milk obtained in step 3. The specific steps are shown in Example 4, wherein the cumulative freeze-drying time in the freeze dryer was 19 hours.

[0147] 10. Mixing: Add the mixed milk from step 8 and the lactoferrin powder from step 9 to a high-shear mixer at a certain ratio (lactoferrin accounts for 0.035% of the total dry matter of the mixed milk). The mixing time is controlled at 15 minutes and the mixing temperature is 50°C.

[0148] 11. Preheating and feeding: After mixing, the mixed milk is temporarily stored in a balance tank, preheated to 62°C by a preheater, and then sprayed into a low-temperature drying tower under the pressure of a high-pressure pump at 220 bar.

[0149] 12. Low-temperature drying: The moisture content of the main inlet air of the drying tower is 1.8 g / kg, the temperature is 88℃, the temperature of the main exhaust air is 62℃, the moisture content of the inlet air of the first static bed is 1.8 g / kg, the temperature is 44℃, the moisture content of the inlet air of the second static bed is 1.8 g / kg, the temperature is 30℃, the formula powder collected in the second static bed enters the pneumatic powder conveying system and is carried into the vacuum dryer. The vacuum dryer jacket is heated and kept at 55℃, the vacuum degree is 2-3 kPaA (absolute pressure), and the drying time is 30 min.

[0150] 13. Measuring and Packaging: After vacuum drying, the formula milk powder enters the packaging machine for measuring and packaging, and is packaged in brown sealed glass bottles;

[0151] 14. Sampling and testing: According to the sampling plan, the lactoferrin standard substance in the prepared formula milk powder shall be sampled and tested. It can be released after passing the test.

[0152] Example 3

[0153] 1. Raw milk acceptance: Raw milk samples are taken after arriving at the factory and inspected and accepted according to the quality control plan;

[0154] 2. Temporary storage of raw milk: temporarily store in a jacketed insulated milk tank at a temperature of 0-6℃;

[0155] 3. Milk purification: Raw milk is pumped into the pasteurization system balance tank through a discharge pump. After preheating by a plate heat exchanger, it is purified by the separator at a speed of 4600 r / min. The purification process yields skim milk and milk fat. The skim milk is chromatographically separated to remove lactoferrin and then refluxed with the milk fat for homogenization as raw milk for subsequent steps.

[0156] 4. Pasteurization: The raw milk after purification in step 3 is put back into the plate heat exchanger for heating and sterilization. The sterilization temperature is 75℃ and the sterilization holding time is 15s.

[0157] 5. Temporary storage of pasteurized milk: After pasteurization, the pasteurized milk is cooled by ice water in a plate heat exchanger and then enters the pasteurization milk storage chamber, where it is temporarily stored at a temperature of 0-6℃.

[0158] 6. Wet Mixing Ingredients Addition and Mixing: Start the milk preparation program, pump pasteurized milk and fixed-volume RO water into the wet mixing tank, start the circulation system, and circulate the wet mixing tank and vacuum mixer. The system starts to automatically extract the metered ingredients from the powder hopper, and the materials enter the mixing circulation. The added ingredients are whole milk powder, skim milk powder, lactose, concentrated whey protein powder, demineralized whey powder, compound minerals, compound vitamins, and compound nutrients. The mixing time is controlled at 50 minutes. Pump the materials from the wet mixing circulation system into the wet mixing tank. The solid content of the mixed milk is 22%, and the mixing temperature is 45℃.

[0159] 7. Homogenization and Temporary Storage: Start the homogenization program. The mixed esters (a mixture of palm oil, corn oil, coconut oil and soybean oil) with OPO structure triglycerides are combined with the mixed milk at the front end of the homogenizer and enter the homogenizer. After homogenization, the mixture is cooled by a plate heat exchanger and temporarily stored in a wet mixed storage tank. The homogenization pressure is 240 bar.

[0160] 8. Steam jet sterilization: The material is pumped into the sterilizer and insulation pipe for sterilization. The sterilization temperature is 90℃ and the sterilization holding time is 24s.

[0161] 9. Lactoferrin preparation: Lactoferrin powder was prepared using the skim milk obtained in step 3. The specific steps are shown in Example 4, wherein the cumulative freeze-drying time in the freeze dryer was 24 hours.

[0162] 10. Mixing: Add the mixed milk from step 8 and the lactoferrin powder from step 9 to a high-shear mixer at a certain ratio (lactoferrin accounts for 0.035% of the total dry matter of the mixed milk). The mixing time is controlled at 12 minutes and the mixing temperature is 45°C.

[0163] 11. Preheating and feeding: After mixing, the mixed milk is temporarily stored in a balance tank, preheated to 60°C by a preheater, and then sprayed into a low-temperature drying tower under the pressure of a high-pressure pump at 210 bar.

[0164] 12. Low-temperature drying: The moisture content of the main inlet air of the drying tower is 1.2 g / kg, the temperature is 78℃, the temperature of the main exhaust air is 52℃, the moisture content of the inlet air of the first static bed is 1.2 g / kg, the temperature is 34℃, the moisture content of the inlet air of the second static bed is 1.2 g / kg, the temperature is 24℃, the formula powder collected in the second static bed enters the pneumatic powder conveying system and is carried into the vacuum dryer. The vacuum dryer jacket is heated and kept at 52℃, the vacuum degree is 2-3 kPaA (absolute pressure), and the drying time is 35 min.

[0165] 13. Measuring and Packaging: After vacuum drying, the formula milk powder enters the packaging machine for measuring and packaging, and is packaged in brown sealed glass bottles;

[0166] 14. Sampling and testing: According to the sampling plan, the lactoferrin standard substance in the prepared formula milk powder shall be sampled and tested. It can be released after passing the test.

[0167] Example 4

[0168] The experimental equipment and conditions used in this embodiment are as follows:

[0169] Chromatography equipment: Lisure APPS Process DN500;

[0170] Chromatography columns I and II: Lisure SAC-Bio-500;

[0171] The packing material for chromatography columns I and II: agarose cation exchange resin;

[0172] Column efficiency of chromatography columns I and II: Column packing height 220cm, column volume: 17.3L;

[0173] Sheet membrane: Merck Millipore ultrafiltration membrane Pellico 3 Cassette with Biomax 30KD, model: 3Biomax box membrane pack, membrane area 0.57m2, molecular weight cutoff 30kDa.

[0174] The skim milk obtained in step 3 of the above embodiment is passed through a chromatography column I filled with cation exchange resin at a constant flow rate of 480 L / h, wherein the flow rate results in a residence time of 4 min for the skim milk in the chromatography column, and the temperature of the skim milk and the chromatography column is controlled at 50°C; the skim milk continues to flow through the chromatography column to continuously adsorb lactoferrin in the emulsion for 6 h.

[0175] Elution was performed on column I with 5 column volumes of 0.15 mol / L sodium chloride solution (elution buffer b1') to obtain elution buffer b1'; elution was performed on column I with 1 column volume of 0.18 mol / L sodium chloride solution (elution buffer b2') to obtain elution buffer b2'; elution was performed on column I with 4 column volumes of 0.4 mol / L sodium chloride solution (elution buffer b3') to obtain elution buffer b3'.

[0176] The second elution collection solution b2' was desalted using a flat sheet membrane to obtain collection solution b2'.

[0177] The collected solution b2' was passed through a chromatography column II packed with cation exchange resin at a constant flow rate of 480 L / h.

[0178] Column II was eluted with 5 column volumes of 0.15 mol / L sodium chloride solution (first eluent e1) to obtain the first eluent collection e1; column II was eluted with 4 column volumes of 0.4 mol / L sodium chloride solution (second eluent e2) to obtain the second eluent collection e2.

[0179] The second elution collection e2 and the third elution collection b3' are mixed, and the mixture is desalted using a flat sheet membrane to obtain an ultrafiltration concentrate.

[0180] The ultrafiltration concentrate is cold sterilized through a double-layer sterilization membrane with pore sizes of 0.2μm + 0.45μm, and then freeze-dried in a freeze dryer. It goes through three stages: plate pre-freezing, primary drying, and desorption drying. After freeze-drying, it is ground and pulverized into powder for later use.

[0181] Test case

[0182] I. Traceability and Value Determination Methods

[0183] The standard reference material prepared in this invention is defined using the agricultural industry standard "Determination of Lactoferrin in Milk and Dairy Products by High Performance Liquid Chromatography" (formerly the group standard "Detection of Lactoferrin in Milk and Dairy Products by Liquid Chromatography" (T / TDSTIA006—2019)), jointly developed by the Dairy Innovation Team of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences, the Dairy Product Quality and Safety Risk Assessment Laboratory of the Ministry of Agriculture and Rural Affairs (Beijing), and the Milk and Dairy Products Quality Supervision and Testing Center of the Ministry of Agriculture and Rural Affairs (Beijing). The standard value is the average of the results of three specific embodiments. The technical solution implementation process uses measurement methods and measuring instruments that meet metrological requirements to ensure the traceability of the standard reference material's quality value.

[0184] II. Characteristic values, reference values, and uncertainties

[0185]

[0186] The uncertainty of the standard value comprehensively considers the uncertainty components introduced by the standard substance's set value, homogeneity, and stability.

[0187] III. Homogeneity Test and Stability Study

[0188]

[0189]

[0190] In accordance with the relevant requirements of section 5.4 of the National Metrological Technical Specification JJF1343-2022, randomly selected aliquoted samples were subjected to homogeneity testing using liquid chromatography and stability testing using accelerated testing (ambient storage temperature ≥40℃, relative humidity RH ≥75%). The test results indicate good homogeneity and stability. The validity period of this standard reference material is tentatively set at 2 years from the date of its determination. The invention team will continue to monitor the stability of this standard reference material to strive for a longer validity period.

[0191] Industrial availability

[0192] This invention can be used for the industrial production of lactoferrin standard substances in formula milk powder.

Claims

1. A method for preparing a lactoferrin standard substance in formula milk powder, characterized in that, The method includes: The process of milk purification involves purifying raw milk to obtain milk fat and skim milk. The steps for preparing lactoferrin are as follows: the skim milk produced in the milk purification step is subjected to ion exchange chromatography and ultrafiltration concentration to obtain ultrafiltration concentrate, the ultrafiltration concentrate is passed through a sterile membrane and then freeze-dried to obtain lactoferrin; The steps for preparing the ingredients are as follows: the skim milk flow-through obtained during ion exchange chromatography in the lactoferrin preparation step is homogenized with the milk fat obtained in the purifying step to obtain raw milk; the raw milk is pasteurized to obtain pasteurized milk; wet mixing ingredients are added and homogenized in the pasteurized milk; then steam jet sterilization is performed to obtain a mixture; the mixture is mixed with the lactoferrin to obtain mixed milk. The drying step involves preheating the mixed milk obtained in the ingredient preparation step and then performing low-temperature spray drying, which includes the following stages: spray drying in a drying tower, first-stage static bed drying, second-stage static bed drying, and pneumatic conveying drying, as well as vacuum drying, to obtain lactoferrin standard material in formula milk powder. In the step of preparing lactoferrin, the sterilization membrane is a double-layer microporous filter membrane with pore sizes of 0.2~0.45μm. In the drying step, the main inlet air temperature of the drying tower is 73~90℃, the main exhaust air temperature is 47~65℃, the temperature of the first-stage static bed drying is 30~45℃, and the temperature of the second-stage static bed drying is 17~33℃.

2. The preparation method according to claim 1, characterized in that, In the ingredient preparation step, the temperature during the addition of the wet mixed ingredients is 25~50℃.

3. The preparation method according to claim 1, characterized in that, In the drying step, the moisture content of the inlet air for the spray drying, the first-stage static bed drying, and the second-stage static bed drying in the drying tower is each independently less than or equal to 1.8 g / kg.

4. The preparation method according to any one of claims 1 to 3, characterized in that, In the ingredient preparation step, when the wet mixture is added, the added ingredients include any one or more of polysaccharide components, protein components, mineral components, vitamin components, and lipid components.

5. The preparation method according to claim 4, characterized in that, In the ingredient preparation step, when the wet mixture is added, the lipid components do not include OPO-structured triglycerides.

6. The preparation method according to any one of claims 1 to 3, characterized in that, Following the drying step, the preparation method further includes metering and packaging steps.

7. A lactoferrin standard substance for formula milk powder, characterized in that, The lactoferrin standard substance in the formula milk powder is prepared by the method according to any one of claims 1 to 6.

8. The lactoferrin standard substance in formula milk powder according to claim 7, characterized in that, Based on the total mass of the lactoferrin standard substance in the formula milk powder, the lactoferrin content in the lactoferrin standard substance in the formula milk powder is 20mg / 100g~50mg / 100g.

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