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Effect of pasteurization homogenization and heat treatments on fat droplet size and zeta-potential Introduction Milk is a complex food that has several nutritional benefits because of its protein, vitamin, carbohydrate, fat, and mineral contents (Tunick, et al., 2015). Milk has a short shelf life. Hence, there is a need for preservation using heat treatments that prevent spoilage by microorganisms (Qi, Ren, Xiao, & Tomasula, 2015). There are various heat treatments used, including HTST pasteurization and UHT sterilization. Normally, the milk is homogenized before final heating as a way of preventing creaming. Under this conditions, the native milk fat globules, that are encapsulated in the milk fat globule membrane, often get reduced to around 1μm with homogenization from around 20μm. There are few studies that have explored the effect of heat treatments and homogenization on fat droplet size and zeta-potential. Overview of Milk Fat and MFGT Milk fat is composed of triacylglycerol (TAG) that acculumate in the bilayer of the endoplasmic reticulum of the mammary glands’ epithelial cells. It is from here that they move to the apical membrane. The newly formed TAGs first get assembled into microdroplets of very minute diamemters at the base of the secretory mammary cells. Here, a monolayer made up of proteins and phospholipids surround the TAGs. They then migrate through the cytoplasm as they become bigger due to the effect of calcium and gangrioside mediation. Assisted by GTP binding proteins, they approach the apical surface and bud out of the secretory cells. Due to the increasing number of consumers purchasing raw milk, there is a claim that heat treated milk is not easily digested because the proteins are denatured, and that it is less nutritious. However, heating whole or skim milk beyond the temperature of 65°C causes aggregation of whey proteins, but there is little interaction under HTST pasteurization conditions. When the temperature gets to UHT sterilization, more protein is denatured, causing interactions between whey and casein proteins. Another claim is that there is reduction in lipid size due to homogenization that may affect human health. According to Tunick, et al. (2015), the association of proteins with MFGM is affected by homogenization steps. The impact depends on whether the process is continuous or batch. An emulsion is a system made up of two immiscinle liquids, with one of them dispersed as a droplet throughout the other. MFGM is, therefore, a comblex membrane that surrounds the fat globules in milk (He, Tang, Yi, Ma, & Wang, 2017). The average fat globule diameter for secreted milk is betweeen 0.15 µm and 15 µm, with slight variations. The size of the globule depends on many factors, including the lactation stage and the cow breed. In about 1% of the fat globules, there is an entrapped cytoplasm which forms a signet or crescent. The complex MFGM structure is around ten to 50 nm thick and contains proteins, sphingolipids, and phospholipids. Out of these, the proteins and phospholips comprise of more than 90% of the membrane. According to reviewed literature, the gross composition of MFGM varies depending on the isolation and purification techniques used. Also, it can be affected by enzymatic, chemical, physiological, and mechanical factors (Rybak, 2016). Impact of Pasteurization Homogenization on fat droplet size Homogenization is the ability to produce a uniform size distribution of particles suspended in a liquid, by forcing it under the effect of pressure through a valve. In dairy beverages, there are homogenizers that process fluid matrices at high pressure, leading to High Pressure Homogenization (HPH). HPH is the most effect alternative to traditional methods for preservation of milk through heat treatment. Technically, a homogenizer has a pump and a homogenizing valve. The pump forces the liquid into the valve where the process happens. The fluid is pumped under pressure through small holes between the valve and the seat. The distance between the seat and the valve can be adjusted as a way of controlling the pressure. Such an application causes microbial inactivation, especially when coupled with the correct heat treatment. The main purpose of homogenization is to break down fat molecules so that they can resist separation. If it is not done, there is a possibility of the molecules rising to the top to form a layer of cream. Tunick, et al. (2015) noted that the size of fat globules decrease due to homogenization and heat treatment of whole milk. Combining heat treatment and homogenization leads to the formation of smaller droplet sizes as opposed to when homogenization is done alone. Adhesion of the fragments of casein milcelles is observed on the fat globule membrane after homogenization, although for whey proteins it only occurs after heating. Michalski and Januel (2006) observed that homogenization not only reduces the fat droplet size but also alters the interface composition. These structural changes depend on the sequence of heat treatment and homogenization. According to a study by Meena, Singh, Borad, and Panjagari (2016), homogenization processes such as Ultrafiltration can cause concentration fo fats, proteins, and saits, while at the same time reducing lactose, vitamins, and water soluble minerals. Tunick and Hekken (2017) showed that high heat treatments and homogenization change the stability of milk components. This may in turn affect their digestability. When milk is homogenized at a pressure of 10–25 MPa, the protective MFGM ruptures, causing fat globules to reduce into smaller droplets. As noted by Michalski and Januel (2006), cream and milk are the best examples of oil-in-water emulsions. The size and distribution of fat globules form the main properties. There seems to be a dependence between the contents of raw milk and fat dispersion. The size of the globules in cream and milk is affected by milk processing, more especially homogenization which reduces the size and affects the dispersion. When milk is well homogenized, the particles spread evenly throughout the entire sample. This mechanism protects against creaming. There is a need to determine the fat globule sizes as a way of standardizing the homogenization processes. Traditionally, this has been done microscopically, by observing and measing the diameters. In recent times, there are various instruments that heve deen designed to specifically measure the sizes. In the manufacture of milk products, HPH is often employed due to its ability to minimize droplet sizes. Normally, homogenization proceeds in two stages in the industry. In the first phase, the milk is forced through thr valve at high pressure, decreasing the fat globule diameter considerably. At the second state, the pressure is considerably lowered so that the viscosity is reduced. Once homogenization is achieved, the disturbed globules reduce their sizes, effectively increasing their specific surface areas. Hence, industrial milk processing often causes physicochemical interactions betweeen whey proteins and MFGM constituents. Effect of Heat Treatments on fat droplet sizes Pasteurization is the process by which milk is heated up then cooled down quickly to eliminate microbes while at the same time preserving its taste. Normally, milk is heated up to 161.6 degrees Fahrenheit for 15 seconds, a process known as High Temperature Short Time (HTST) pasteurization. The milk can then be fresh for up to 14 days. On the other hans, Ultra-Heat Treatment (UHT) is a process of heating milk at 280 degrees Fahrenheit for a minimum of two seconds. This extends the shelf life to up to nine months. Industrial heat treatments have a majoy impact on MFGM. According to Qi et al. (2015), heat is the single most important factor that affects fat droplet sizes. One study showed that when fat globules are heated in a temperature as low as 60oC, large amounts of α-lactalbumin and lactoglobulin associate with the MFGM (Rybak, 2016). Also, there are mechanical treatments that affect the composition of MFGM, including pumping, agitation, and high shear. Ye, Cui, Dalgleish, and Singh (2017) conducted a study on the effect of heat treatment on the behavior of fat globules during gastric digestion of milk. They found out that heat treatment led to the formation of coagula with crumbled and fragmented structures compared to raw whole milk. They observed that the fat globules in the whole milk was more embedded, leading to formation of clots. The number of pores fromed with heated whole milk is greater than those fromed by pepsin hydrolysis. As noted by Pestana, Gennari, Monteiro, Lehn, and Souza (2015), it is important to maintain milk at low temperatures to avoid affecting its nutiritional value. Heat treatments such as HTST and UHT aim to reduce microbial population, to avoid spoilage and pathogens, inactivate enzymes, and minimize physical and chemical changes. Some effects of heating impact on the properties and quality of milk. Temperature plays a critical role in chemical and physical changes of proteins and lipids in milk. As the temperature increases, lactose is degredated, causing a reduction in PH. Although lipids are not denatured by heat, high temperatures cause associated immunoglobulins to be damaged, hence preventing creaming from occuring. UHT milk processing normally uses heat treatment at 130–140 °C for around two minutes, However, it causes fat separation, in addition to protein denaturation. Although other processes such as lipolysis, natural creaming, cooling, and agitation affect MFGM, it is homogenization and heat treatment that produce the most notable changes. As explained above, heat treatment alters MFGM by promoting interaction between its components and native plasma proteins. The interactions could be resulting from direct binding or by displacement of polypeptides. Another common heat treatment in modern industries is the use of multiple-effect evaporator. According to Ye et al (2017), falling-film evaporators cause various effects on the composition of milk, including a reduction in the fat globule sizes. This is because evaporation disrupts the MFGM. Since boiling forms vapor and yet fat is liquid, fat globules get dirupted. However, there is no available data to suggest that boiling leads to coagulation of fat globules. Emulsions are thermodynamically unstable since they consist of two immiscible liquids, with one dispersed within the other. This causes a change in the zeta potential in relation to the MFGM. At high temperatures, lipid particles achieve greater mobility, a factor that affects their stability. Concequently, higher temperature leads to formation of smaller globules. Effect of pasteurization homogenization on Zeta Potential Zeta potential, denoted as ζ-potential, refers to the potential difference that exists across phase boundaries between liquids and solids. It is used to measure electric charge of particles suspended in liquids (Helmenstine, 2019). For colloidal substances, such as milk, zeta potential is the electric potential difference around a charged colloid ion, across the ionic layer. When the measure is high, it means the colloid is stable. According to Sejersen, et al.,(2007), homogenization and heat treatments can affect the zeta potential of milk. In milk, fat is in the form of emulsified globules that are surrounded by yhe membrane made up of proteins, glycolipids, lipolipids, and phospholipids. This membrane is damaged with homogenization of milk. Once this happens, the casein micelles and plasma proteins adsorp to the surface as a result of increased interfacial area. The effect is that the size of the fat droplet decreases, leading to a reduction in the rate of creaming. Therefore, measuring the zeta potential is a good way of determining the impact of homogenization. Fats in milk takes the form of emulsified globules as noted in the preceding discussion. Each globule is envelo-ed by the fat globule membrane which is composed of lipoproteins, enxymes, glycolipis, and phospholipids. During homogenization, the membrane is destroyed, leading to a reduction in size. At the same time, there is an increae in interfacial area which allows plasma proteins and casein micelles to be adsorbed onto thr surface. It becomes ncessary to assess the changes that homogenization brings, and measuing zeta potential is one of the most effective ways. Zeta potential gives a measure of the effects that occurs when plasma proteins are adsorbed onto the surface of the globule during the process of homogenization. There are instruments that have been developed, such as the Beckman Coulter, which analysts use to measure the zeta potential changes in milk samples at different pressures. Conclusion As noted herein, milk is a complex food substance with several benefits. However, it has a short life and so must be preserved using various techniques. Heat treatment and homogenization are some of the most widely used approaches for preventing spoilage. Heat treatment is commonly done using pasteurization and Ultra-heat treatment (UHT). It has been shown that applying heat affects the structure and composition of the MFGM, and can lead to a reduction in the fat particle sizes. Homogenization is a technique that is used for achieving inform distribution of particles in a liquid. Since high pressure is applied through a small pole, the size of particles decreases as they squeeze through the valve. Combining heat treatment and homogenization leads to the formation of smaller droplet sizes as opposed to when homogenization is done alone