S0022029923000092jra 75..81

Functions of mucin, as the major macromolecular component in saliva or gastric fluids, are drawing increasing attention in the context of understanding the oral processing or digestion of dairy foods at the molecular level. This study was designed to investigate the interactions between β-lactoglobulin (BLG)-lactose, mucin-lactose and BLG-lactose-mucin at the molecular level under different temperature and pH conditions using fluorescence spectroscopy in combination with scanning electron microscopes (SEM). It is the first study of its kind. There was no lactose-dependent quenching on BLG fluorophore in the range of 0–10 mM lactose concentration. On the contrary, there was a continuous increase in the fluorescence intensity of the BLG protein when the lactose concentration increased, especially at 25°C. BLG-lactose complex became thermally unstable at 37 and 45°C. Moreover, BLG exhibited a pH dependent conformational change and had higher fluorescence intensity at pH 3 than pH 6.8. The fluorescence result was in correspondence with SEM images where we observed lactose crystals gathering around and on the BLG molecule, but lactose molecules could not be seen in the presence of mucin. It was anticipated that mucin molecules interacted with BLG-lactose complex via electrostatic attraction and formed an extra protective layer around the BLG molecules to avoid solvent exposure. The milk from dairy cows contains 12–14% solid matter composed of proteins (2.5–4%), fat (3–5%), and lactose (∼5%) (Osorio et al., 2016). Milk is used in the food industry in many different ways. Not only is milk a very nutritious food but also the by-products released during the production of dairy products carry additional nutritional properties. The best example is the whey, which is released as a waste/by-product during cheese processing. 10 kilos of milk are used to produce 1 kilo of cheese, and approximately 9 kilos of whey are released as a result of the process. Whey contains high amounts of protein. Among them β-lactoglobulin (BLG) represents about 50% of the total whey proteins in bovine milk and possesses high levels of secondary, tertiary and, in most cases, quaternary structures (Kinsella and Whitehead, 1989; Edwards et al., 2009). In addition to protein, whey powders include lactose sugar. Whey is often an ingredient of choice as an additive in the food industry due to its multi-purpose functional properties (Huffman, 1996). Before its industrial processing in different food products, the interactions of the ingredients in whey and their response to changing process conditions such as temperature, pH and humidity must be known. As a result of the studies carried out for this purpose, it has been observed that the milk sugar lactose in whey is affected by the temperature and the water content, and it has been stated that possible changes in lactose molecules affect the shelf life of this type of food product (Vuataz, 2002). Similarly, BLG protein is known to be affected by temperature and pH changes of the environment (Aymard et al., 1996; Fang and Dalgleish, 1997). Not only changes in ambient conditions, but also the interaction of BLG protein with other molecules, affect its functional properties and taste (Guichard and Langourieux, 2000). In recent studies, especially on taste and oral processing, it has been reported that BLG interacts with the mucin protein in the saliva (Çelebioğlu et al., 2015) and, as a result of this interaction, the model food products containing BLG change the friction coefficient in the oral environment and create changes in taste perception (Vardhanabhuti et al., 2011; Çelebioğlu et al., 2016; Çelebioğlu et al., 2020). It is stated that BLG protein also interacts with lactose sugar and this interaction continues during the drying process and storage conditions (Thomas et al., 2004a). It has been shown that the BLG-lactose interaction stabilizes lactose crystallization (Costantino et al., 1998) and thus prevents the formation of undesirable properties like particle caking, Maillard reaction, lipid oxidation and so on (Burin et al., 2000; Özkan et al., 2003; Thomas et al., 2004b). Lactose crystallization in products such as milk powders increases the amount of free fat molecules ((Kim et al., 1981) In order to prevent lactose crystallization, the use of high molecular weight molecules such as proteins (Levine and Slade, 1986; Nasirpour et al., 2006) or sugars other than lactose have been recommended in the literature (Nasirpour et al., 2007). At this point, the interaction of lactose with BLG and mucin protein is an interesting subject to investigate. Although there are some studies related to lactose and BLG as mentioned above, there is no literature on the interaction of lactose and mucin protein. Since mucin protein has both a high molecular weight and a highly glycosylated structure (Bansil and Turner, 2006), its interaction with lactose can be used functionally. Mucin, as the major salivary protein, is an important protein related to oral food processing and digestion. Through the various stages of oral processing and digestion, foods continuously mix with saliva and mucins interact with food components (Çelebioğlu et al., 2020). Therefore, the mucin–lactose interaction or the change of BLG–lactose interaction in the presence of mucin may give important clues about the taste and bioavailability properties of a wide range of products such as milk/whey powder containing lactose and BLG. In line with all these motivations, this study aimed to investigate the interactions of BLG–lactose, mucin–lactose and BLG–lactose–mucin at molecular level. Different temperatures and pH values were used to compare the conditions of different processing and digestion environments. The fluorescence spectroscopy technique was chosen as the optimum technique for this research. Fluorescence spectroscopy is based on the presence of intrinsic fluorophores such as tryptophan (Trp, the dominant fluorophore) and tyrosin (Tyr) residues, in which the emission spectra (both intensity and (λmax), the maximum peak positions) are sensitive to the local environment of the Trp residues, and can thus provide useful information on both conformational and structural changes of proteins (Lakowicz, 2006). Recently, the interaction of BLG and mucin was studied with fluorescence, resulting in useful and interesting clues about the way these two proteins interact (Yılmaz et al., 2021). However, there is no integrating study examining the interaction of lactose, BLG and mucin molecules with fluorescence spectroscopy. It is envisaged that explaining the molecular level interactions between them will contribute to food process optimizations to produce functional products with improved sensory properties and bioavailability. Material and methods