Extraction, identi cation and quanti cation of water buffalo milk proteins using a combination of reverse phase high-performance liquid chromatography coupled to diode-array detector (RP-HPLC-DAD) and near-infrared spectroscopy (NIRS) measurements

Julián Andrés Castillo Vargas (  jcfcav@gmail.com ) Universidade Federal Rural da Amazônia https://orcid.org/0000-0001-5163-5127 Vinicius da Silva Botelho Duarte Gomes Universidade Federal Rural da Amazônia https://orcid.org/0000-0002-2627-4590 Tiago Costa de Araújo Universidade Federal Rural da Amazônia https://orcid.org/0000-0002-7844-3659 Rafael Mezzomo Universidade Federal Rural da Amazônia https://orcid.org/0000-0002-1889-3287 Raylon Pereira Maciel Universidade Federal Rural da Amazônia https://orcid.org/0000-0001-5097-2797


Introduction
The protein genetic variants of caseins (CN) and whey proteins in the milk of ruminants are known for their differential in uence on the technological and nutritional properties of dairy derivative products (Fox and McSweeney, 2003). Therefore, it is necessary to identify and quantify them e ciently in the milk of different ruminants' species. Among ruminants, buffalo has gained worldwide special attention recently, due to its ability to adapt to different geographical conditions, as well as the broad variety of dairy products that can be manufactured from its milk (Oliveira et al., 2020;Lima et al., 2021). In addition, buffalo's milk has shown bene cial effects on human health, compared to bovine milk due to its chemical composition (Patiño et al., 2012), which has increased the demands of this product for human consumption. satisfactory results. However, Mass Spectrometry detector is unavailable in most analytical laboratories due to its high costs. Additionally, protein quanti cation solely using RP-HPLC-DAD requires pure standards, which in the case of buffalo's milk are not commercially available (Bonfatti et al., 2013).
Hence, for quanti cation using calibration curves, these standards need to be isolated and puri ed from buffalo's milk by preparative liquid chromatography (Bonfatti et al., 2013), a complex method that requires specialized steps and equipment not commonly available in routine analytical laboratories.
The use of near-infrared spectroscopy (NIRS) technique to determine the composition of ruminant's milk (Melfsen et al., 2012) has increased recently, because this method provides rapid estimations of milk composition to take fast decisions in the dairy industry (Yakubu et al., 2020). In a typical report of milk chemical analysis by NIRS, the total protein and casein contents can be accurately determined (Tsenkova et al., 2000). Hence, this technique may be easily associated with other analytical techniques to improve the understanding of ruminants' milk protein composition.
Total protein in buffalo's milk consists of around 20% whey proteins with major components αlactalbumin (α-LA), β-lactoglobulin (β-LG), and 80% caseins divided into major sub-classes α-(αS1-and αS2-), β-, and κ-casein (-CN), which are arranged in micelles (Swaisgood, 1982). Considering that RP-HPLC-DAD provides accurate estimates of the proportions of casein variants and whey proteins, as well as NIRS provides an accurate value of total protein and casein contents, the combination of both techniques may enable an easier analysis of buffalo's milk protein fractions than that using only RP-HPLC-DAD, overcoming the aforementioned technical limitations regarding the solely use of this technique. Thus, we developed a method to quantify different casein genetic variants and major whey proteins in the milk of water buffaloes, using the combination of RP-HPLC-DAD and NIRS techniques. -Centrifuge (reaching at least 14,000 x g).
-Automatic pipettes and tips.
-Vials, caps, and septa for chromatography (one set per sample). Note: you can climate the room (25 °C) in which incubation is running by using an air conditioning unit. Standardization of the incubation conditions contributes to low variations across different processing days.
6. Centrifuge at 14200 x g for 5 min.
7. An upper fat layer is formed after centrifugation. Then, carefully remove it using a micro-spatula.

NIRS analysis
Determine the total protein and casein contents in g/100 g milk in the sample using a NIRS equipment. Additionally, determine the milk sample density. Proportion of whey protein variant = Peak area of whey protein variant/sum of peak areas of whey proteins c. Calculation of total whey protein: assuming that total milk protein = casein + whey protein, then: Whey protein content (g/100 g milk) = total milk protein (g/100 g milk) -casein (g/100 g milk).
d. Calculate the concentration of each whey protein variant in g/100 g milk: Concentration of whey protein variant (g/100 g milk) = Whey protein content (g/100 g milk) x Proportion of whey protein variant e. Optional: calculation of the concentration of whey protein variants in g/L milk: Concentration of whey protein variant (g/L milk) = (Concentration of whey protein variant (g/100 g milk)) * (milk density (g/mL)) * 10 Troubleshooting 1. Take special care with homogenous sampling. In some cases, when milk was storage for long periods or milk is thawed-frozen, it tends to form solid particulate materials. If particulate material is formed, shake the milk's container vigorously to take a representative sample. Otherwise, discard the sample.
2. The equilibrium program before each sample run, is strictly necessary to get a successfully milk protein separation. Check the ow maximum limits of your column before use.  Figure 1