**3. Industrial applications of MFGM**

#### **3.1 Isolation and production of MFGM**

Several isolation studies on MFGM have been done due to the presence of nutraceutical proteins and their nourishing effect towards the infant. In spite of its uses, these isolates are produced on commercial scale and it have been supplemented in various nutritive formula and functional foods. Since casein and MFGM are same in size and share almost similar isoelectric points, it is a tedious isolation process. The separation techniques used for MFGM, involve many physical processing methods with repeated washings using chemicals to remove milk proteins, lactose and salts which is suitable for lab purposes, but not optimal for commercial production [13]. The molecular size of milk protein casein and MFGM are same, this makes it even more difficult to isolate during membrane separation [14].

The natural extraction of MFGM can be obtained from the by-products such as buttermilk, cream serum and cheese whey while processing butter, cream and cheese respectively (**Figure 2**). These by-products are the raw materials for the production of MFGM. Processing conditions like cooling, heating and physical separation techniques such as churning and phase inversion affects the migration and association of MFGM fragments in dairy products. Chilling or cooling shifts MFGM towards whey and heating causes complex association between whey protein and exterior layer of MFGM. Same pattern takes place in cream serum that MFGM gets concentrated at water-in-oil emulsion (AMF) during cooling. In cheese preparation, the disruption of MFGM during processing of cheese curd results in migration towards the milk serum portion. Condensation of defatted fluid whey into whey protein concentrates and isolates (WPC and WPI) is used for MFGM extraction. Among the by-products, cream serum and buttermilk provides a great source of MFGM. Dehydration and membrane separation of the above described final ingredients are used to produce MFGM enriched powder [7, 15].

*Milk Fat Globular Membrane: Composition, Structure, Isolation, Technological Significance and… DOI: http://dx.doi.org/10.5772/intechopen.106926*

**Figure 2.**

*Production of MFGM enriched powdered product from various processing methods. Adapted from [7].*

During manufacturing of butter, phase inversion occurs by churning that involves in conversion of oil-in-water to water-in-oil, MFGM associated with TG gets drifted into buttermilk due to coalescence. MFGM isolates from buttermilk at lab scale was studied by [16], commercialized buttermilk powders was used to characterize lipids present in the MFGM isolates. The study showed that MFGM fractions had high cholesterol and PUFA content and serves as the best source of bioactive lipids. The lipid profile proved that MFGM had higher concentration of medium molecular weight TGs mainly due to linoleic acid.

Most successfully used method for production of MFGM from buttermilk was reported [13], using microfiltration and multiple diafiltration. Each batch contains 8 to 16 liters of reconstituted buttermilk with total solids content of 8% w/v in water was used. 2% sodium citrate at 1.4%w/w was added to reconstituted buttermilk to disrupt the casein micelles and to increase the PLs content in the buttermilk whey (pH 7.2), then stored for overnight at 6°C. The first step in separation involved in membrane filtration polyvinyl-dilfluoride (PVDF) membrane with 250,000 and 500,000 Da cut-off at 50°C. Then retentate was feed into 2-HP centrifugal pump with the pressure of 1.2 MPa at 50°C by circulating in shell and tube heat exchanger until favorable concentration is reached. Again the retentate goes to multiple diafiltration at 50°C, followed by high speed centrifugation to isolate MFGM fragments.

The authors noticed that, intact of small amount of β- lactoglobulin (30%) in MFGM even after 2 steps of diafiltration, this is due to the complex formation of whey protein and kappa casein with MFGM during heat processing [17]. Absence of sodium citrate resulted in increased level of skim milk proteins in the retentate and caused contamination of non- MFGM material in final retentate. To obtain MFGM isolate in powdered form, the retentate was freeze dried. During SDS-PAGE electrophoresis analysis, the final isolate contained 60% and 35% of protein and lipid (w/w) respectively, among the protein composition was 70%, 24% and 6% of MFGM, whey protein and casein respectively.

Another method of isolation was studied by [18], coagulation of native casein protein to obtain specific MFGM isolate cannot be obtained through membrane separation due to skim milk protein contaminants. 40% raw cream was cream separated and skim milk was subjected to continuous butter making process and the resulted buttermilk was coagulated by addition of rennet at 0.03% to hydrolyse the casein micelles at 45°C for 30 min to reach pH of 6.8. The obtained buttermilk whey was passed through microfiltration with average pore size of 80 nm at the constant pressure of 0.1 MPa at 50°C to remove whey protein. To remove further residues of whey protein, the retentate was diafiltered with deionized water (6 diafiltration steps). Interestingly, after 6 diafiltration steps complete absence of whey protein was detected during SDS-PAGE protein analysis.

High amount of protein loss was encountered in this isolation method, however the loss was considerably lesser when compared to the cream washing method [19]. The final isolate contained 70%, 30% and 20% of peripheral protein – periodic Schiff acid (PAS 6/7), XO and butyrophilin (BTN) respectively. Compared to the previously mentioned method of isolation [18], the coagulation method [19] showed neither whey protein nor casein in the final isolate and this method can be used industrially.

Effect of addition of cationic salts like calcium acetate and zinc acetate on selective isolation of MFGM in cheese whey was studied by [20–23]. The studies showed that removal of Ca2+/Mg2+ from cheese whey through diafiltration and addition of 25 m*m* of zinc acetate at pH 4.2 at 30–35°C causes the maximum precipitation of MFGM.

Ethanolic extraction method (90% ethanol at 70°C) was used to solubilize the calcium chloride and acetate in dairy by-products to maximize the extraction of MFGM and phospholipids to produce dairy lecithin [22]. Composition and physicochemical properties of spray-dried and freeze- dried MFGM isolate from cheese whey showed that freeze dried MFGM has higher retention of bioactive components and better oxidative stability than spray dried MFGM [24]. This study supports the findings of [25], concentrated buttermilks from raw milk (RCB) and pasteurized creams from buttermilk (PCB) were produced by condensing cheese buttermilk in falling film evaporator to reach 20% total solids, and then the spray-dried concentrate was used in the study. Unexpectedly, the amount of lipids was higher in PCB at the level of 19.7% vs. RCB 8.29% under the same skimming level. Double the amount of lipid concentration in PCB was stated due to attachment of milk protein to the exterior of MFGM, inhibiting the coalescence of fat globules [26].

Spray drying of buttermilk resulted in major loss of all classes of phospholipids and found that the inner portion of MFGM was exposed to interaction with other components. As already discussed in previous studies, serum protein contaminants were higher in PCB due to interaction with β- lactoglobulin and formation of complex systems. The color of RCB was reddish brown and PCB was yellowish white due to the presence of iron in RCB. Micrographs of MFGM in RCB and PCB revealed that casein is entrapped in the MFGM rather than its attachment to the exterior layer of MFGM. The study does not explain the storage stability and loss of phospholipids after spray drying.

From above stated studies of MFGM, it was clear that membrane separation techniques like microfiltration, diafiltration and ultrafiltration, addition of sodium citrate and cationic salts plays a major role in isolation of MFGM. Heat treatments like pasteurization of cream and spray drying mainly affected the phospholipids concentration and increased the serum protein contaminants in final MFGM isolates. Optimization of separation techniques and processing conditions should focus on minimal damage on functional bioactive components in MFGM fragments to ensure

*Milk Fat Globular Membrane: Composition, Structure, Isolation, Technological Significance and… DOI: http://dx.doi.org/10.5772/intechopen.106926*

the delivery of clinical benefits to the consumers. Most of the studies were conducted only on the characterization of bovine MFGM isolates rather than other species. Characterization of other species's MFGM uncovers the underlying potential health benefits and commercialization of novel MFGM fractions especially in the neonatal nutrition.
