La educación superior y recursos humanos como factor de estrategia en el crecimiento

As stated, where shearing conditions were sufficient to cause homogenisation of fat droplets during AMC processing, the presence of soluble proteins present within the cheese curd may have adsorbed at the fat interface during production, thereby modifying the surface properties of emulsion droplets and their interaction with the surrounding protein network.

To explore the role of soluble proteins on AMC structure and properties, the serum from non-fat AMC was collected and protein composition was analysed. Non-fat cheeses were

produced by mixing cheese curd, salt and water in RVA at 60 °C using 800 rpm as the

maximum shear speed in the first 15 minutes then followed by 1000 rpm. Three serum samples were collected by squeezing the hot non-fat cheese produced for 7 min, 10 minutes and 30 min, respectively. Protein composition was compared in these three serum samples (Figure 6.16).

The four primary peaks from left to right in figure 6.16 indicate α-lactalbumin, β- lactoglobulin, β-casein and α-casein. The three serums sampled at different time points all showed the presence of whey proteins and caseins. The protein peak heights were smallest in serum extracted after 7 minutes processing, and with peak height increasing with increasing mixing time. Interestingly, after 30 minutes of production time, the peak of β-lactoglobulin appeared much higher than the other three protein peaks, becoming the dominant protein in the serum, while the concentration of β-casein and α-casein obviously decreased comparing to the non-fat cheese made in 10 minutes. The

production temperature of 60 °C was not high enough to denature whey proteins, and

therefore whey proteins from cheese curd continued to dissolve into serum with the increase of residence time. The caseins showed a low concentration in the serum due to strong interactions in cheese protein net-work. The addition of salts in the non-fat cheese production was considered to increase the concentration of dissociated caseins.

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Figure 6.16: The change of protein composition in serum from non-fat cheese. The serums were collected from non-fat cheese produced in 7 minutes (red), 10 minutes (blue) and 30 minutes (green). Skim milk was used as the control sample in the measurement. A: α-lactalbumin; B: β-lactoglobulin; C: β-casein; D: α- casein.

The increase in availability of serum proteins as mixing time increases may have promoted protein adsorption at fat interface, resulting in the transition from inactive to active fat droplets. To verify this idea, high fat cream of ~80 wt.% fat was mixed with the serum phase from non-fat AMC and homogenised by using sonication. Given that active fat fillers were observed after 30 minutes processing of AMC, when produced at 800 -

1000 rpm 60 °C, serum was collected from non-fat cheese based on these parameters.

Mean fat globule size D[4,3] was seen to drop from 5.3 μm to 3.0 μm after sonication, indicating dramatically increased surface area. The fat and protein dispersion was observed on CLSM where samples were diluted 100 times in EDTA solution (no Tween). Aggregated and individual fat globules were observed in the diluted cream samples (Figure 6.17). The fat globules in aggregates appeared smaller relative to individual fat globules and the fat aggregates also showed a degree of associated protein. Although protein signals were found at surface of individual fat globules, the signals did not appear as strong as for the proteins present in the fat aggregates.

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Figure 6.17: CLSM images of cream diluted by the EDTA solution without Tween. The cream was after sonication with the serum from 30 minutes non-fat cheese. The images are shown in two separated photos. Fat membrane is in red on the left photos and proteins are in green on the right photos.

AMC was made for 15 minutes at 800 rpm 60 °C using the cream under sonication with

serum. Because the serum from non-fat cheese contained salt, the salt added for cheese making was reduced to match the same level as other AMCs. The small fat globules in cream after sonication were strongly linked by proteins indicated by the confocal in figure 6.17, but these aggregates of fat and protein were not observed after cheese making (Figure 6.18b).

These proteins covered fat globules would be expected to interact with the surrounding cheese protein matrix becoming homogeneously dispersed in cheese. The cheese confocal image showed a mixture of large and small, matching the findings of increased particle size distribution after cheese producing in figure 6.18a. The fat globule size distribution of cream includes the primary peak at 0.6 - 20 μm and also a small peak at 0.1 - 0.6 μm. After cheese production fat globule size distribution showed a spreading of the upper modal distribution, with droplets up to ~50 μm. The cheese cooling modified particle size distribution showed a trimodal distribution in the ranges 0.1 - 0.6 μm, 0.6 - 10 μm and 10 - 100 μm, respectively. Confocal imaging did indicate some droplets in

close proximity (Figure 6.18b), and the low temperature (4 °C) of cheese storage was

likely still causing partial coalescence of these fat globules, leading to the formation of the upper modal distribution (10 - 100 μm) in figure 6.18a. However, it could also be

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observed that most of fat globules were homogeneously dispersed in the protein matrix, characteristic of active fat fillers and that their particle size was therefore unlikely to be affected by cheese cooling and storage. Thus, the particle size peak of 0.6 - 10 μm was observed to be the primary peak in chilled cheese. The modification from sonication did not appear to have occurred for all fat globules, with the possibility arising that a fraction of fat droplets could be present that comprised a mixed interface coated with both MFGM and protein. The fat globules in this case might therefore be expected to have acted as a combination of active and inactive fillers.

Figure 6.18: Cheeses were produced using the cream after sonication with serum collected from non-fat cheese. The cheese was produced in 15 minutes at 800 rpm 60 °C. (a) Fat globule size distribution is compared in cheese (fresh cheese, ■; 7 days 4 °C stored cheese, ■; and the cream, ■) used for cheese producing; (b) CLSM images were taken using lenses of x 40 (left photo) and x 25 (right photo) on cheese in 7 days storage at 4 °C.

(a)

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β-lactoglobulin was determined as the major protein in serum after 30 minutes producing (Figure 6.16), and might accordingly be expected to be the predominant protein fraction at the fat interface after extended mixing, thus altering the interaction state of droplets from inactive to active. To validate this hypothesis, cream was sonicated with 1% whey protein isolate (WPI 895, 14.2 % α-lactalbumin and 69.2 % β- lactoglobulin indicated from product bulletin) to create whey stabilised droplets prior to AMC preparation. Fat globule size dropped to 2.9 μm after sonication with confocal imaging showing adsorption of protein at the droplet interface. Cheese production was

attempted in 15 minutes up to 800 rpm at 60 °C using the cream sonicated with whey

proteins. However, it was curious to note that not all the cream could be combined into the cheese, with 2 - 3 g cream was left in the canister for 25 g cheese making. The whey protein covered fat globules appeared less effective at being incorporated into the protein matrix, and the exclusion of cream from the cheese structure as a consequence of processing indicated that binding to the cheese protein matrix may not be taking place.

Many publications have demonstrated how fat globules covered by whey proteins behaved as active fillers in a protein matrix (Xiong and Kinsella 1991, Chen and Dickinson 1999, Liu, Stieger et al. 2015), usually as a consequence of covalent bonding between interfacial and continuous phase protein (Walstra, Wouters et al. 2006). However, whey proteins are unlikely to bind to cheese protein matrix by disulphide bonds in AMC production due to a lack of sulphydryl groups within the rennet network that would be able to form covalent bridges with the whey protein stabilised interfacial layer.

More likely, an electrostatic calcium mediated bridging mechanism was able to take place between negatively charged domains between interfacial and matrix protein. In this respect, pH may represent another important factor on molecular interactions. The pH of AMC is 5.3 which is close to the isoelectric point of β-lactoglobulin (pI 5.2) (Walstra, Wouters et al. 2006), and electrostatic interaction is weak between whey proteins and protein matrix. Although α-lactalbumin has some charge at pH 5.3, but its concentration in serum is low. Hydrophobic bonds will expose in heat, and it is probably the primary

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