Quantification system for vitamins in different types of commercial milk and curd compared with raw milk and curd

Abstract

A system for quantifying vitamins in various varieties of commercial milk and curd compared to raw milk and curd, the system comprising: a sample preparation unit configured to receive and prepare test samples of raw milk, raw curd, and various varieties of commercial milk and curd; a reagent preparation unit comprising one or more volumetric flasks and diluents for preparing standard and sample solutions; and a chromatographic analysis unit comprising a reversed-phase high-performance liquid chromatography (RP-HPLC) system.

Description

Field of the invention

[0001] The present invention relates to a quantification system for vitamins in different types of commercially available milk and curd in comparison with raw milk and raw curd. Background of the invention

[0002] The references cited in this disclosure do not necessarily represent prior art and therefore do not constitute an admission that these references are prior art in any jurisdiction. All publications, patents, and patent applications contained herein are incorporated by reference to the same extent as if each individual patent application were expressly and individually identified as being incorporated by reference.

[0003] Several patents have been granted for milk compositions, but none of them are related to the present invention, which enables the quantification of vitamins in various types of commercially available milk and curd compared to raw milk and curd using RP-HPLC. For example, AU2019236599B2 describes a system comprising a pumping device with an interface for connection to the breast and an associated actuating device. By actuating the actuating device, negative pressure is applied to the breast via the interface to express milk. The system also includes various sensors for quantifying the properties of the expressed milk, and a unique identifier enables inventory management of the expressed milk.

[0004] Another patent, US8545920B2, relates to human milk enrichment compositions, standardized human milk, and methods for their preparation and use. In one embodiment, a pasteurized human milk composition contains a human protein content of about 35-85 mg / ml, a human fat content of about 60-110 mg / ml, and a human carbohydrate content of about 60-140 mg / ml.

[0005] Another patent, US8278046B2, describes methods for examining breast fluid (including milk) to determine or confirm the identity of the donor. Such methods are useful in milk banking to increase security.

[0006] Another patent, US9453257B2, discloses methods, compositions, and kits for extracting and relatively enriching short base-pair nucleic acids by physical separation or amplification in the presence of a high background of genomic material (e.g., host or maternal nucleic acids).

[0007] The main objective of the present invention is a novel quantification of vitamins in different types of commercial milk and curd in comparison with raw milk and curd using RP-HPLC.

[0008] Another aim of the present invention is to compare raw milk and curd and to estimate using the RP-HPLC parameters whether the obtained retention time, resolution, tailing factor and analytical values ​​are within the limits.

[0009] These and other objects and advantages of the present invention will be readily apparent from the following detailed description. Summary of the invention

[0010] This summary does not represent a comprehensive overview of the disclosure and does not reflect the essential features of the facility or determine its scope. It is intended solely to simplify some of the concepts presented here to facilitate later, more detailed explanations.

[0011] The present invention relates to a novel quantification of vitamins in different types of commercial milk and curd in comparison with raw milk and curd using RP-HPLC.

[0012] In some embodiments of the present invention, numerous experiments were conducted during the selection of chromatographic conditions and the best experiments were selected for the optimized method.

[0013] In some embodiments of the present invention, approximately 0.1 mg of vitamins A, C, K, B1, B2, B3, B6, B12, and folic acid were weighed into a 10-ml volumetric flask. 5 ml of diluent was added, dissolved by ultrasonication, and diluted to the desired volume with the diluent. To identify the peaks in the traces, the vitamins were first injected individually. These are the standard peaks and their calculations.

[0014] In some embodiments of the present invention, the VIT B1 peak was observed at 2.568 min with a peak area of ​​2010307, ​​a tailing factor of 1.06. The FA peak was observed at 3.086 min with a peak area of ​​35530, a tailing factor of 1.34, and a resolution of 2.24. The VIT B3 peak was observed at 4.240 min with a peak area of ​​668110, a tailing factor of 1.07, and a resolution of 3.80. The VIT K peak was observed at 5.903 min with a peak area of ​​1200970, a tailing factor of 1.03, and a resolution of 4.34. The VIT B2 peak was observed at 6.914 min with a peak area of ​​1161443, a tailing factor of 1.00 and a resolution of 2.82.

[0015] In some embodiments of the present invention, the VIT B6 peak was observed at 8.852 min with a peak area of ​​11614, a tailing factor of 1.03, and a resolution of 4.94. The VIT A peak was observed at 9.643 min with a peak area of ​​383245, a tailing factor of 0.89, and a resolution of 2.20. The VIT C peak was observed at 10.532 min with a peak area of ​​24302, a tailing factor of 0.97, and a resolution of 1.46. The VIT B12 peak was observed at 11.925 min with a peak area of ​​1256625, a tailing factor of 1.04, and a resolution of 2.23.

[0016] These and other aspects of the embodiments described herein will become more readily understood in conjunction with the following discussion and the accompanying drawings. While the following descriptions show preferred embodiments and numerous specific details, they are illustrative and not limiting. Numerous changes and modifications are possible within the scope of the embodiments described herein without departing from the spirit thereof. The embodiments described herein are intended to encompass all such modifications. DETAILED DESCRIPTION OF THE INVENTION

[0017] In some embodiments of the present invention, the buffer is prepared as follows: 2.5 g of heptane-1-sulfonic acid is dissolved in 1 liter of water, and the pH is adjusted to 2.5 with orthophosphoric acid. Preparation of the standard solution: Approximately 0.1 mg of vitamins A, C, K, B1, B2, B3, B6, B12, and folic acid are weighed into a 10 ml volumetric flask. 5 ml of diluent is added, dissolved by sonication, and diluted to the desired volume with the diluent.

[0018] In some embodiments of the present invention, the sample solution is prepared as follows: 0.1 ml of various types of commercially available milk and curd are added to a 10 ml volumetric flask. 5 ml of diluent is added, dissolved by ultrasonication, and diluted to the desired volume with diluent. Procedure: 10 µl of the blank, standard preparations, and sample preparations are injected five times into the chromatography system, the chromatograms are recorded, and the peak responses for vitamins folic acid, A, C, K, B1, B2, B3, B6, and B12 are measured. The content of each vitamin in the various types of commercially available milk and curd is calculated in the %Assay table and compared to the content in raw milk and curd.

[0019] The present invention relates to a novel and advanced system for the quantification of vitamins in various commercially available milk and curd products compared to raw milk and curd. It utilizes reversed-phase high-performance liquid chromatography (RP-HPLC) and enables precise, reproducible, and comparative analysis of vitamin concentrations in various dairy products. This enables quality assessment and the creation of nutritional profiles.

[0020] It consists of three main components: a sample preparation unit, an RP-HPLC-based chromatographic analysis unit, and a data acquisition and comparison unit.

[0021] During the sample preparation phase, representative milk or curd samples—whether raw or commercial—are precisely measured, diluted, and sonicated with a standard diluent to ensure homogeneity. 0.1 ml of each milk or curd sample is transferred to a 10 ml volumetric flask, followed by 5 ml of diluent. The mixture is sonicated to dissolve particles and then diluted to the final volume. In parallel, standard vitamin solutions are prepared by weighing approximately 0.1 mg of each vitamin—e.g., A, C, K, B1, B2, B3, B6, B12, and folic acid—into individual flasks, sonicating, and diluting to create reference standards.

[0022] The prepared samples and standards are injected into the RP-HPLC system. The chromatographic analysis unit includes a column optimized for vitamin separation, a buffer system of heptane-1-sulfonic acid adjusted to pH 2.5 with orthophosphoric acid, and a UV-visible or PDA detector for peak identification. Vitamins are separated based on their polarity and retention time and identified using known reference standards. Each vitamin has a unique retention time and corresponding peak area. Additional parameters such as tailing factor and resolution are used to validate chromatographic efficiency. For example, vitamin B1 appears after approximately 2.568 minutes, while others such as vitamins A, K, and B12 elute later in the chromatogram.

[0023] The system's data acquisition module records the chromatographic results and extracts relevant quantitative data for each vitamin, including peak area, retention time, tailing factor, and resolution. These measurements are used to calculate the concentration of each vitamin in the tested samples. The analytical results of the various commercially available milk and curd varieties are then compared with raw milk and curd samples to identify any processing-related deterioration, enrichment, or deviations. The comparison is statistically validated and can be output as a table or graph for easy interpretation by nutritionists, quality control laboratories, or regulatory authorities.

[0024] Overall, the system provides a robust platform for the nutritional comparison of dairy products through the highly precise and reproducible quantification of essential vitamins. It serves as a useful tool for food quality assurance, regulatory compliance, and promoting consumer health awareness. The combination of standardized sample handling, optimized chromatographic separation, and automated data analysis ensures reliable results suitable for industrial and research applications in dairy science and food safety. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] AU 2019236599B2

[0003] US 8545920B2

[0004] US 8278046B2

[0005] US 9453257B2

[0006]

Claims

[1] A system for the quantification of vitamins in various varieties of commercial milk and curd compared to raw milk and curd, the system comprising: a sample preparation unit configured to receive and prepare test samples of raw milk, raw curd, and various varieties of commercial milk and curd; a reagent preparation unit comprising one or more volumetric flasks and diluents for preparing standard and sample solutions; and a chromatographic analysis unit comprising a reversed-phase high-performance liquid chromatography (RP-HPLC) system. [2] The system of claim 1, wherein the sample preparation unit is adapted to dilute 0.1 ml of sample in a 10 ml volumetric flask using a diluent, followed by ultrasonication. [3] The system of claim 1, wherein the standard preparation comprises weighing 0.1 mg of each vitamin into a 10 ml flask, adding 5 ml of diluent, dissolving by ultrasonication, and diluting to the desired volume. [4] The system of claim 1, wherein the tailing factor and resolution of each vitamin peak are determined and validated to confirm chromatographic accuracy and reproducibility. [5] The system of claim 1, wherein the data analysis unit is configured to generate analysis reports indicating the quantitative content of each vitamin in percent and comparing them with known standards. [6] The system of claim 1, wherein the comparative report highlights nutrient depletion or vitamin enrichment in processed milk and curd compared to raw milk and curd. [7] The system of claim 1, wherein the chromatographic system is further configured to inject blanks, standards, and samples sequentially to avoid cross-contamination and ensure calibration integrity. [8] The system of claim 1, wherein the detection is performed using a photodiode array (PDA) or a UV-visible detector specific for the vitamin absorption properties. [9] The system of claim 1, wherein all results are processed and visualized using a computer interface that supports tabular, graphical and statistical comparison of the vitamin content of multiple milk and curd samples.