**1. Introduction**

Infant milk formula (IMF) is intended to serve as a functional substitute for infants under 12 months of age. The World Health Organization (WHO) recommends absolute breastfeeding of infants for the first 6 months of age [1], whereas the American Society of Paediatrics suggests the same for at least 12 months [2]. However, only 38% of infants are being breastfed globally [3], which indicates the common use of IMF, a \$41 billion USD market [4]. IMF is available in three forms—powdered, liquid concentrate, and liquid ready-to-feed (RTF). Among them, RTF, the most convenient form, is currently manufactured as a sterilized product to ensure safety using UHT (Ultra High Temperature, 135–145 ◦C for a short time). However, UHT causes the nutritional profile of RTF formula to deteriorate, especially vitamin A, B, and D, along with protein denaturation, which requires supplementation of micronutrients (e.g., vitamins and minerals) [5]. Although every possible effort is being made to bring IMF closer to human milk (HM), there is still a gap between them nutritionally, which governs neurological, physiological, and immunological growth and development of infants [6–8]. Such a

difference could be explained by the nutritional balance that remains fixed in an IMF, while it varies in HM throughout the lactation period and even between individuals [9].

Moreover, beta-lactoglobulin (β-Lg) represents about 50% of total whey as a major whey protein in cow milk. It is also considered a major allergen to infants despite having numerous functional and nutritional roles in adult human health. However, interestingly, this β-Lg is absent in HM. To address the cow's milk allergy (CMA), researchers and manufacturers introduced partially hydrolyzed formula (pHF) and extensively hydrolyzed formula (eHF), which are also recommended by paediatricians to reduce early allergy manifestation [10]. In pHF and eHF, enzymatic hydrolysis of proteins with digestive enzymes reduces the allergic properties by breaking them into small peptides and free amino acids, which are not allergenic [11]. However, these hydrolyzed formulas often exhibit bitter taste, poor flavour, reduced lipid emulsifiability, and elevated osmolality, which limit their application in general IMF [12].

On the other hand, alpha lactalbumin (α-Lac) is a bioactive protein present in all mammal milk, which is regarded as a component of lactose synthesis with antimicrobial, prebiotic, and Ca-binding capacity [13]. The α-lac content in human milk is 3–4 g/L, while it is only 1 g/L in mature cow milk. It contains a high level of different essential amino acids like tryptophan, lysine, and cysteine, and hence, fortification of IMF with α-Lac is recommended [13,14].

The standard protein content of infant formula is still a matter of controversy since formula production aims to mimic HM [14]. HM contains 9–11 g/L protein [15], while conventional infant formula provides 15 g/L [16]. Burgeoning demand for low-protein infant formula, especially in Asia, resulted mainly from paediatric obesity [17]. Moreover, excess protein also induces unnecessary strain on immature metabolic organs [18,19]. Thus, the possible alternative could be reducing the protein content and adding free amino acid into infant formula, which would be unphysiological since metabolic consequences of free amino acids are mostly unknown [14]. Thereupon, a logical approach would be to modify the protein profile of formula to make it closer to HM by increasing the ratio of α-Lac to β-Lg, which will yield a formula that has lower total protein, but retains the necessary balance of essential amino acids [14,16,20,21]. Moreover, this would also be an imperative strategy because complete removal of allergenic β-Lg would deteriorate the function of milk proteins since it is necessary for whey proteins to be associated with casein micelles [22]. High pressure processing (HPP) defragments casein micelles into smaller particles and splits them into more soluble components like αs1-, αs2-, β-, and k-caseins [23–25]. More than a 50% reduction in the size of casein micelle was observed after HPP at >300 MPa at 40 ◦C [24]. In powdered IMF, reducing the protein content to as low as 9.77 g protein/100 g did not cause a significant impact on physical stability and shelf-life [26]. However, in RTF liquid IMF, a higher ratio of α-Lac to β-Lg reduces the viscosity and induces rapid sedimentation during storage, as observed in UHT-treated products; and hence, the addition of thickener and stabilizer is suggested to manage the viscosity and sedimentation, respectively [27–29]. Furthermore, Crowley et al. [27] reported increased α-Lac reduced heat-induced coagulation in a model whey protein-dominant IMF. They further observed lower protein–protein interactions in the model IMF due to the fortification of IMF with α-Lac. Additionally, Kamarei [30] patented his invention in manufacturing refrigeration-shelf-stable pasteurized IMF with required quantities of nutrients. In this patent, he reported that UHT-treated RTF formula provides a different and unknown amount of degradable micro nutrients due to the high-heat treatment and subsequent storage, which also affects the nutritional value and sensory attributes. These outcomes evidence the possibility of developing an IMF with a higher ratio of α-Lac to β-Lg.

Previously, Huppertz et al. [31] described the mechanism of HPP-induced denaturation of α-Lac and β-Lg at 200–800 MPa/20 ◦C in whole milk and reported α-Lac as a pressure-resistant protein, unlike β-Lg. Furthermore, Mazri et al. [32] also investigated the denaturation kinetics of these two bioactive proteins in skim milk under HPP of 450–700 MPa at 20 ◦C and confirmed the baroresistance of α-Lac in comparison to β-Lg. Recently, HPP has been recommended to preserve human milk, due to its efficient inactivation of microbial pathogens, along with the retention of unique components [33]. Wesolowska et al. [34] also reviewed the effect of HPP to pasteurize human milk and referred to HPP as superior to the conventional holder pasteurization (63 ◦C/30 min) in maintaining bioactivity of protein components. To the best of our knowledge, the synergistic effect of HPP and heat to achieve a higher ratio of α-Lac to β-Lg has not been explored in IMF, which fundamentally pioneered this work.

Therefore, the aim of this work was to achieve a higher ratio of α-Lac to β-Lg through investigating their retention after HPP at different pressure–temperature–time combinations in reconstituted IMF, fortified with α-Lac. For comparison, we also performed high-temperature short-time (HTST) pasteurization at 72 ◦C for 15 and 30 s, and thereafter measured the concentration of α-Lac and β-Lg. The combined effect of pressure and temperature on the kinetics of denaturation of both proteins was also analyzed.
